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University of Veterinary Medicine Hannover Institute of Physiological Chemistry The role of N-butyl deoxynojirimycin in enzyme function, membrane association and targeting of brush border hydrolases THESIS Submitted in partial fulfilment of the requirements for the degree DOCTOR OF PHILOSOPHY (PhD) Awarded by the University of Veterinary Medicine Hannover by Mahdi Amiri Malayer, Iran Hannover, Germany 2014 Supervisor: Prof. Dr. Hassan Y. Naim Institute of Physiological Chemistry University of Veterinary Medicine Hannover Hannover, Germany Supervision group: Prof. Dr. Georg Herrler Institute of Virology University of Veterinary Medicine Hannover Hannover, Germany Prof. Dr. Rita Gerardy-Schahn Institute for Cellular Chemistry Hannover Medical School Hannover, Germany 1st Evaluation: Prof. Dr. Hassan Y. Naim Institute of Physiological Chemistry University of Veterinary Medicine Hannover Hannover, Germany Prof. Dr. Georg Herrler Prof. Dr. Rita Gerardy-Schahn Institute of Virology Institute for Cellular Chemistry University of Veterinary Medicine Hannover Hannover Medical School Hannover, Germany Hannover, Germany 2nd Evaluation: Prof. Dr. Hannelore Daniel Center of Life and Food Sciences Weihenstephan Technical University of Munich Munich, Germany Date of final exam: 4th November 2014 Sponsorship: Mahdi Amiri was partially supported by a scholarship awarded by the German academic exchange service (DAAD). To my Parents and my wife Azam Parts of the thesis that have already been published or submitted for publication: Mahdi Amiri and Hassan Y. Naim Miglustat-induced intestinal carbohydrate malabsorption is due to the inhibition of αglucosidases, but not β-galactosidases Journal of Inherited Metabolic Diseases (2012) 35:949-954 Mahdi Amiri and Hassan Y. Naim Long term differential consequences of miglustat therapy on intestinal disaccharidases. Journal of Inherited Metabolic Diseases (2014), accepted May 2014, doi: 10.1007/s10545-014-9725-4 , Epub Ahead of Print. Contents Chapter 1 .................................................................................................................... 1 1.1. Protein folding, misfolding and the associated disorders .................................. 2 1.2. N-butyl deoxynojirimycin................................................................................. 13 1.3. Carbohydrate digestion in the intestine .......................................................... 18 1.4. Aim of the study.............................................................................................. 22 Chapter 2 .................................................................................................................. 33 Miglustat-induced intestinal carbohydrate malabsorption is due to the inhibition of α-glucosidases, but not β-galactosidases ............................................................. 34 Chapter 3 .................................................................................................................. 47 Long term differential consequences of Miglustat therapy on intestinal disaccharidases..................................................................................................... 48 Chapter 4 .................................................................................................................. 71 Discussion ............................................................................................................. 72 Summary .................................................................................................................. 82 Zusammenfassung ................................................................................................... 84 Affidavit ..................................................................................................................... 87 Acknowledgments .................................................................................................... 88 Chapter 1: Introduction Chapter 1 Introduction 1 Chapter 1: Introduction 1.1. Protein folding, misfolding and the associated disorders Protein folding Proteins are the major macromolecular constituents of the cells which carry out various biological functions: they form the structural backbone of the cells, they serve as energy sources, and they provide the movement machinery for the organisms. The specific ability of the proteins in selective interactions with other molecules enables them to function as catalysts, channels, pumps, receptors, switches or components of the immune system (Alberts et al 2007). The function of each protein is directly imposed by its biomolecular conformation, which is initially a function of its amino acid sequence known as “primary structure”. Concurrently to the synthesis, a stretch of amino acid residues will start to obtain a 3D structure based on the interactions occurring among its residues internally and with the surrounding environment. In the cellular system, this process is engineered towards acquisition of a “native” functional conformation via two approaches: first, administration of non-covalent interacting folding assistants, called chaperones, and second, covalent attachment of certain functional groups to the polypeptide chain or removal/cleavage of specific sites in the amino acid stretch, referred to as “co- and post-translational modifications” (Alberts et al 2007; Varki and Lowe 2009). The overall process is known as “protein folding”. In the following, protein glycosylation as one of the most important protein modifications is discussed in more detail. Glycosylation Glycosylation is the most common modification of the proteins. This phenomenon occurs co- and post-translationally and involves covalent attachment of certain oligosaccharides or “glycans” to the polypeptide chain followed by a sequence of reconstitutions via trimming/addition of sugar residues (Varki and Lowe 2009). In eukaryotic cells, according to the recipient residue, glycosylation is classified as N, C-, or O-glycosylation. In C-glycosylation the glycans are attached through carboncarbon bonds rather than carbon-nitrogen or carbon-oxygen bonds which are found in N- and O-glycosylations (Nicotra 1997; Furmanek and Hofsteenge 2000). The biological function of the C-glycosylation is not fully understood. Here we focus on Nand O-linked glycans in more detail. 2 Chapter 1: Introduction N-glycosylation N-glycosylation starts simultaneously with the synthesis of the nascent polypeptide in the endoplasmic reticulum (ER). This modification includes transport of a preassembled oligomannose glycan from a dolichol phosphate carrier to the amide group of an asparagine occurring in the tripeptide sequence of Asn-X-Ser/Thr (or Asn-X-Cys in seldom cases) in the flexible domains of the polypeptide (Petrescu et al 2004). This glycan is typically composed of two N-acetyl glucosamine, nine mannose and three glucose residues abbreviated as Glc3Man9GlcNAc2 (Fig 1). Following attachment, the N-glycan is subjected to few trimming actions in the ER, initially to remove the two outermost glucose residues by the sequential action of the ER glucosidases I and II (Ruddock and Molinari 2006). The resulting mono-glucosylated glycoprotein then finds the possibility to associate with and get folding assistance by the ER calnexin/calreticulin chaperon system (Helenius and Aebi 2004). Alpha-glucosidase inhibitors such as nojirimycin derivatives and castanospermine can cause ER retention of the glycoprotein by preventing the glucose trimming and thus interfering with the calnexin/calreticulin association of the proteins (Peyrieras et al 1983; Parodi 2000). After release from calnexin/calreticulin cycle, the final glucose is removed by αglucosidase II. When correctly folded, the protein can be transported to the Golgi apparatus to get complex N- and O-glycosylated; otherwise, it is re- monoglucosylated and returned to the calnexin-calreticulin cycle. Ultimately the “misfolded” protein is recognised by the “ER degradation-enhancing α-mannosidaselike protein” (EDEM) (Hoseki et al 2010; Shang 2011) and forwarded to the ERassociated degradation (ERAD) pathway (Maattanen et al 2010). In many cases, a single mannose from the central arm of the N-glycan is removed by the “ER α-mannosidase I” before transport to the Golgi (Ruddock and Molinari 2006). The glycoprotein at this state is denoted as “mannose-rich” form. After leaving the ER, further maturation of the N-glycan is accomplished during transport from cis- to medial- and trans-Golgi cisternae. This process initially involves trimming of up to 3 mannoses from the N-glycan, followed by selective elongation and branching of the oligomannose core glycan to the hybrid and finally to the complex N-glycosylated 3 Chapter 1: Introduction forms. The added residues are typically N-acetyl glucosamine (GlcNAc), galactose and terminal sialic acid sugars (Varki and Lowe 2009). In the experimental approaches various N-glycosylation inhibitors as well as specific endoglycosidases have been administered to determine the N-glycosylation state of a sample protein. Tunicamycin, castanospermine, 1-deoxynojirimycin (dNM) and 1Deoxymannojirimycin (dMM) are among the most popular N-glycosylation inhibitors (Elbein 1987; Elbein 1991; Jacob 1995)(Fig. 1). Fig. 1: Processing of the N-glycan in the ER and the inhibitors of different steps. Endoglycosidase H (endo H) and peptide:N-glycosidase F (PNGase F) are the most commonly used glycosidases to evaluate protein N-glycosylation (Varki and Lowe 2009). Endo H cleaves mannose-rich and some hybrid N-glycans in between the two N-acetyl glucosamine residues of the oligomannose core (Fig. 2). Further processed hybrid forms in the medial-Golgi and the complex N-glycosylated forms are endo H resistant. PNGase F, can cleave the linkage between the innermost GlcNAc and asparagine in all forms of N-glycans except the fucosylated ones (Kim and Leahy 2013)(Fig. 2) 4 Chapter 1: Introduction Fig. 2: sensitivity of different forms of N -glycans towards endo H and PNGase F treatments. O-glycosylation In glycobiology, O-glycosylation refers to attachment of glycans to the protein via the oxygen atom of amino acid residues such as serine (Ser), threonine (Thr), hydroxyproline (OH-Pro) and to a lesser extent hydroxyserine and hydroxythreonine (Varki and Lowe 2009). O-glycans have two major differences to N-glycans: first, unlike N-glycosylation, Oglycosylation does not begin with the transfer of a pre-assembled oligosaccharide from a carrier, but single mono-saccharides are added separately by specific enzymes; second, in contrast to N-glycans which share a similar backbone, Oglycans constitute a very heterogeneous family. O-glycosylation occurs mainly in the Golgi apparatus, but sometimes it is already started in the ER or ER-Golgi intermediate compartment (Varki and Lowe 2009). Based on the reducing terminal sugar, O-glycans are classified into two types: a) mucin type O-glycans and b) non-mucin type O-glycans. Mucin type O-glycans are the most occuring type of O-glycans which are found on many secreted and membrane bound glycoproteins in higher eukaryotes. Here the reducing terminal sugar is an N-acetylgalactosamine (GalNAc) (Van den Steen et al 1998, Bergstrom and Xia 2013). There are 8 types of core glycans identified for mucin O-glycans which are produced by different combinations of galactose, fucose, N-acetyl glucosamine and sialic acid residues attached to the terminal GalNAc (Hanisch 2001; 5 Chapter 1: Introduction Peter-Katalinic 2005)(Fig. 3). The target sites of mucin type O-glycosylation are commonly not marked with any consensus sequences of amino acids, meanwhile these O-glycans are frequently found as clusters in Ser, Thr and Pro rich motifs (Jensen et al 2010). Fig. 3: different cores of the mucin type O-glycans attached to serine/threonine residues of the polypeptide. In most of the identified non-mucin type of O-glycans, the reducing terminus is made of an O-linked fucose, glucose or N-acetylglucosamine (Hanisch 2001; PeterKatalinic 2005). O-fucosylation and O-glucosylation are often occuring in consensus sequences in epidermal growth factor (EGF), EGF-like repeats and thrombospondin type 1 repeats of many plasma membrane glycoproteins and signalling molecules. These O-glycans are found to play a crucial role in the early stages of embryonic development and vital physiological functions (Wopereis et al 2006). 6 Chapter 1: Introduction Protein misfolding and the associated disorders The fact that the functional “native” conformation is only one out of numerous potential conformations for a protein explains the high complexity of the protein folding process in the cell. According to the energy landscape theory, the final protein folding can be achieved via a variety of intermediate structures rather than an exact settled pathway (Bryngelson and Wolynes 1987). The driving force of the protein folding is provided by lowering of the free energy of an unfolded polypeptide structure to more stable secondary and tertiary structures (Hebert and Molinari 2007). Meanwhile the cell should avoid formation of fibrils and amorphous aggregates which can be thermodynamically in a lower energy phase than the native conformation but are functionally inactive or even toxic (Aridor 2007). In the cells this purpose is achieved by orchestrating the intramolecular interactions in the polypeptide through intermolecular interactions, particularly with molecular chaperones (Fink 1999; Broadley and Hartl 2009). Since the free energy of a polypeptide is directly resulting from its primary structure as well as the covalently associated groups such as glycans, alterations in the amino acid sequence or in the complementary modifications can affect local or even global protein folding (Parodi 2000; Hebert and Molinari 2007). In addition, changes in the protein surrounding conditions such as pH, ionic strength, temperature, charge and redox as well as non-enzymatic glycation can also influence protein conformation during the folding process and afterwards (Herczenik and Gebbink 2008). Altered conformation of the misfolded protein can affect the efficiency of the interactions with its target substances and thus cause “loss of function” of the protein. In some cases misfolding is associated with modified or even toxic functionality of the protein (Wyttenbach and Arrigo 2009). These consequences can induce unhealthy conditions in the living organisms referred to as “protein misfolding diseases”. Loss of function Loss of function occurs when the misfolded protein is in part or totally subjected to one or a combination of the following circumstances: - Recognition by the ER quality control machinery and subsequent degradation - Mis-targeting to a cellular region other than the original site of action 7 Chapter 1: Introduction - Functional incompetency in the site of action An example for the first situation is the cystic fibrosis transmembrane conductance regulator (CFTR) protein mutant delta-F508, which is subjected to ER retardation and finally degraded (Nieddu et al 2013; Tosoni et al 2013). Comprehensive studies on different mutants of sucrase isomaltase (SI) are providing further examples of the rationales of the loss of function in the misfolded protein disorders. SI, which is explained in the following section in more detail, is a heavily glycosylated membrane protein of the intestinal brush border membrane (Naim et al 1988d; Naim et al 1988b). Besides proper folding and co-/post-translational modifications, this protein is particularly sorted to the apical surface of the intestinal epithelial cells, where it performs its glycosidase action in the intestinal lumen (Jacob et al 2000). Insufficient SI activity in the intestine is associated with carbohydrate maldigestion and gastrointestinal intolerances (Naim et al 1988a). In many of the naturally occurring missense mutations identified for this protein, such as glycine to aspartic acid at position 1073 (G1073D (Alfalah et al 2009)), leucine to proline at position 620 (L620P (Ritz et al 2003)), cysteine to tyrosine at position 1229 (C1229Y (Sander et al 2006)) and phenylalanine to cysteine at position 1745 (F1745C (Sander et al 2006)), substitution of a single amino acid results in misfolding and retardation of SI in the early secretory pathway (ER or up to cis-Golgi) followed by protein degradation. In few other SI variants, with glutamine to arginine at position 117 (Q117R (Spodsberg et al 2001)) and cysteine to arginine at position 635 (C635R (Keiser et al 2006)) substitutions, the protein remains transport competent but is randomly sorted also to the basolateral membrane, where it has no access to its substrates. Adverse or toxic effects Occurrence of adverse or toxic effects in correlation to protein misfolding can be sorted into two types. The first type is a direct consequence of the “loss of function” and is ensued from accumulation of the specific substrate(s) of the inactive protein (Gregersen et al 2006). In such cases generally the target protein is directly or indirectly included in catalytic or transport mechanisms. Subsequently the unprocessed/untransported substrate might induce harmful effects per se or after incorporation into undesired secondary interactions. Some related disorders of this type are presented in table 1. 8 Chapter 1: Introduction The second type of adverse effects occurs when the disposal of the misfolded protein cannot be accomplished efficiently. Thereafter accumulation of the unfolded protein structures as aggregates or plaques will induce “unfolded protein response” with successive cell death and tissue damages (Dill et al 2008; Vendruscolo 2012). Many of the degenerative diseases including Alzheimer’s disease, Atherosclerosis, Parkinson’s disease and Huntington’s disease are originated from this type of protein toxicity (Gregersen et al 2006; Herczenik and Gebbink 2008; Jadhav et al 2013). . 9 Table 1: Some examples of relevant disorders that occur in association with detrimental accumulation of unprocessed/untransported substrates as a loss of function of the misfolded proteins. Type of disorder Examples Carbohydrate maldigestion - Congenital sucrase isomaltase deficiency - Congenital maltase deficiency - Congenital lactase deficiency Lysosomal storage disorders - Gaucher disease - Fabry disease - Niemann-Pick disease - Tay Sachs disease - mucopolysaccharidoses Glycogen storage diseases - Glycogen storage diseases type I, III and IV Deficient proteins Detrimental substances Intestinal glycosidases such as sucrase-isomaltase, maltase-glucoamylase and lactase-phlorizin hydrolase The maldigested/unabsorbed carbohydrates are subjected to bacterial fermentation in the colon associated with production of irritative substances Different lysosomal hydrolases/transporters such as glucocerebrosidase, alphagalactosidase, NPC1/2 , etc Interrupted recycling/degradation of the related substances causes accumulation of these material in the lysosomes resulting to lysosomal/cellular dysfunction Enzymes included in the synthesis/breakdown of glycogen such as G6Pase, glycogen branching enzymes, etc Production of glycogen with abnormal structure or accumulation of glycogen in liver and muscles causes malfunction of the organs 10 References (Alfalah et al 2009) (Nichols et al 2002) (Behrendt et al 2009) (Aerts and Boot 2007) (Ikonen and HölttäVuori 2004) (Das et al 2008) (Akram et al 2011) (Chou et al 2010) (Oezen 2007) Chapter 1: Introduction Therapeutic approaches towards protein misfolding disorders As discussed earlier, appearance of protein misfolding disorders is generally based on loss of function, misfolded protein accumulation, unprocessed/untransported substrate accumulation or a combination of these occasions. There has been a variety of therapeutic approaches suggested or applied to overcome these complications. Protein replacement therapy One approach to correct the loss of function consequence of protein misfolding is “protein replacement therapy”; i.e. to compensate the absent function of a misfolded protein by supplementing with an active (analogue) protein produced from an external source. “Enzyme replacement therapy” (ERT) to substitute the nonfunctional lysosomal enzymes as well as delivery of the missing blood-coagulation factors (such as factor VIIa, factor VIII, factor IX and many others reviewed by Gorzelany and de Souza 2013) by infusion are two practiced examples of this method. The concept of enzyme replacement therapy for treatment of lysosomal enzyme deficiencies was proposed by Christian de Duve in 1964 (Deduve 1964) based on the fact that intracellular uptaken substances are most likely to end up with in the lysosomes. Enzyme replacement therapy has been one of the most successful treatments for lysosomal storage disorders; however an optimum delivery of the therapeutic enzyme to some body organs like central nervous system cannot be guaranteed by this method (Andersson et al 2005; Lachmann 2011). Administrations of chemical and pharmacological chaperones are other known therapies to restore the structure and function of the misfolded protein and control the potential subsequent adverse effects. Chemical chaperones Chemical chaperones are small molecules that can facilitate protein folding in the ER by mimicking the function of molecular chaperones (Aridor 2007). These compounds can moderate the ER environment in order to promote and stabilize correct protein folding, recover the partially active protein from the ER or moderate protein aggregation in the cells (Zhao et al 2008; Higaki et al 2013). 11 Chapter 1: Introduction Osmolytes (like glycerol and trimethylamine N-oxide), hydrophobic derivatives that can influence structure and dynamics of the cell membranes or proteins (such as lipid derivatives or dimethyl sulfoxide) and selective inhibitors of the ER quality control machinery (like N-butyl deoxynojirimycin and thapsigargin) represent different classes of chemical chaperones (Perlmutter 2002; Papp and Csermely 2006; Aridor 2007). Pharmacological chaperones Pharmacological chaperones are another class of small molecules that can facilitate protein folding via direct interactions with specific regions within the protein. By resembling a specific ligand these molecules can function as a scaffold to impose folding of the protein binding sites and hence increase the chance of overall improved protein conformation (Lieberman et al 2007; Steet et al 2007; Luan et al 2010). In contrast to the chemical chaperones which influence the ER conditions unspecifically, pharmacological chaperones have the advantage of targeting and acting on the structure of a particular protein without undesired interference with other components (Aridor 2007). Original ligands, ligand analogues and (un)competitive inhibitors have the potency to act as pharmacological chaperones (Naik et al 2012; Boyd et al 2013). An interesting example of this approach is structural and functional recovery of mutated lysosomal glucocerebrosidase in type I Gaucher disease by low concentrations of N-butyl deoxynojirimycin (NB-DNJ), a synthetic analogue of glucose (Sanchez-Olle et al 2009). Here NB-DNJ serves as an indigestible substrate and promotes proper folding of a number of partially active mutants as well as the wild-type enzyme in order to enhance their function and cellular transport (Valenzano et al 2011). Substrate reduction therapy In some cases, where the misfolded protein is a hydrolytic enzyme in a cellular metabolic pathway, functional deficiency can result to accumulation of the unprocessed substrate(s) in the cells. Lysosomal storage disorder and glycogen storage disorders are examples of this type of disorder (Talente et al 1994; Futerman and van Meer 2004; Boustany 2013). Accumulation of the unprocessed substrates 12 Chapter 1: Introduction under these conditions can produce adverse effects and result in loss of cellular homeostasis and tissue perturbations (for a review see (Platt et al 2012)). Besides recovering of the missing activity by ERT or chemical/physiological chaperone therapy, “substrate reduction therapy” (SRT) has been introduced as an alternative therapeutic approach for such disorders (Cox 2005; Jakobkiewicz-Banecka et al 2007; Platt and Jeyakumar 2008). In this method small diffusible inhibitor molecules are utilized to partially decrease the biosynthesis of the parental precursor of the accumulated substrate in the cell. Inhibition of glycosphingolipid synthesis by NB-DNJ for treatment of type I Gaucher disease is the most studied case of this approach (Butters et al 2005; Pastores 2006; Ficicioglu 2008). Similar therapies have been proposed for other lysosomal disorders such as Fabry disease (Ashe et al 2013), Batten’s disease (Gavin et al 2013), and different types of ganglisidoses and mucopolysaccharidoses (Jakobkiewicz-Banecka et al 2011). 1.2. N-butyl deoxynojirimycin N-butyl deoxynojirimycin (NB-DNJ, synonyms: NB-DNJ, Zavesca, OGT918) is a synthetic iminosugar derived from nojirimycin, a natural glucose analogue found in certain species of Streptomyces (Inouye et al 1968; Roston and Rhinebarger 1991). Nojirimycin and its stabilized derivative 1-deoxynojirimycin (dNM) are known in glycobiology for interfering with glycosylation via inhibition of ER alpha-glucosidases (Saunier et al 1982; Fuhrmann et al 1985; Karlsson et al 1993). The additional alkyl group of NB-DNJ has reduced its cytotoxicity and improved its membrane transport in comparison to dNM (Durantel et al 2001; Mellor et al 2002; Mellor et al 2003), enabling this compound to be used as a medication (Fig. 4). 13 Chapter 1: Introduction Fig. 4: Chemical structures of a) nojirimycin, b) 1 -deoxynojirimycin and c) N-butyl deoxynojirimycin NB-DNJ was initially proposed as an antiviral therapeutic to attenuate the human immunodeficiency virus (HIV) infectivity based on interference with glycosylation and folding of the viral envelope glycoproteins (Karpas et al 1988; Dedera et al 1990; Ratner et al 1991). However, since the required antiviral concentration is quite high and cannot be achieved in the serum, the drug could not pass the clinical trials (Ficicioglu 2008). Later studies showed the high potency of this compound in inhibition of glycolipid biosynthesis in the cells, designating that as a therapeutic for offsetting glucosylceramide accumulation in type I Gaucher disease via “substrate reduction therapy” (Platt et al 1994). Following successful clinical trials, NB-DNJ has been confirmed and presented in the market since 2003 for treatment of type I Gaucher and later Niemann-Pick disease type C (Venier and Igdoura 2012). This drug has been also suggested for treatment of similar lysosomal storage disorders such as Fabry disease (Marshall et al 2010; Maalouf et al 2012) and Tay-Sachs disease (Maegawa et al 2009; Shapiro et al 2009) as well as other types of disorders such as acetic acid induced gastric ulcer formation (Nakashita et al 2013), cystic fibrosis (Norez et al 2006; Antigny et al 2008) and lung inflammation (Norez et al 2006; Antigny et al 2008). 14 Chapter 1: Introduction Type I Gaucher disease Gaucher disease is the most common type of inherited lysosomal storage disorders that occurs in association with dysfunction of lysosomal glucocerebrosidase (acid βglucosidase or GCase, EC 3.2.1.45 (Beutler 2006; Dulsat and Mealy 2009)). GCase contributes to glycosphingolipid breakdown in the cells by hydrolytic cleavage of the beta-glucosidic bond in glucosylceramide molecules present in the lysosomes. Dysfunction of this enzyme interferes with the cellular function through interruption of glycosphingolipid metabolism and lipid homeostasis as well as enlarging lysosomal and hence cellular size as a consequence of uncleaved lipid accumulation (Rosenbloom and Weinreb 2013). Many of the classical symptoms of Gaucher disease such as bone marrow infiltration, cytopenia and hepatosplenomegaly are related to accumulation of affected phagocytes in the body organs (Liu et al 2012). Based on the severity of the glucosidase deficiency, three phenotypic manifestations of Gaucher disease are observed. In the presence of partial enzyme activity (type I or non-neuropathic) the symptoms are mostly associated with parenchymal diseases of the liver, spleen and bone marrow, while in the complete absence of the enzyme activity neurological manifestations additionally appear in acute (type II) or chronic (type III) manners (Rosenbloom and Weinreb 2013). Treatment of Gaucher disease Therapeutic approaches practiced so for treatment of Gaucher disease include enzyme replacement therapy (ERT), substrate reduction therapy (SRT) and pharmacological chaperone therapy. In ERT of type I Gaucher disease a recombinant form of glucocerebrosidase called imiglucerase (Cerezyme® by the Genzyme Corporation) is applied to replace the deficient enzyme. After infusion to the blood circulation, the recombinant enzyme is picked up by the cells via endocytosis and transported to its site of function, lysosomes (Charrow 2009). Invasive method of drug delivery (intravenous infusion), limited or no effect on the central nervous system and high costs are the disadvantageous aspects of this approach. SRT with NB-DNJ is used as an alternative or complementary approach for treatment of Gaucher disease (Trapero and Llebaria 2013). Unlike recombinant enzyme in 15 Chapter 1: Introduction ERT, NB-DNJ is a small diffusible molecule with is consumed orally and can pass through the blood-brain barrier to the CNS (Ficicioglu 2008). NB-DNJ reduces the production of the endogenous substrate of the glucocerebrosidase (i.e. glucosylceramide) by competitive and reversible inhibition of glucosylceramide synthase (ceramide glucosyl transferase, EC 2.4.1.80). The latter enzyme is a resident integral membrane protein of the cis/medial-Golgi that catalyses the transfer of glucose from UDP-glucose to ceramide. The product, glucosylceramide, is a common precursor of many glycosphingolipids (Marks et al 1999). Application of this method can successfully stabilize progression of the Gaucher disease, reduce the related symptoms and improve the quality of life in the patients (Cox et al 2003). Using low dosage of NB-DNJ as a chemical/pharmacological chaperone is a more recent approach in treatment of Gaucher disease. In this method, NB-DNJ functions as an undigestable substrate to enhance proper folding mutated glucocerebrosidase on one hand (pharmacological chaperone effect (Abian et al 2011)), and rescue it from ER retardation on the other hand (chemical chaperone effect (Sawkar et al 2002)). In vitro studies have indicated recovery of few variants of this enzyme including N370S, V15M and M123T by this method in association with enhancement of their functional activity (Alfonso et al 2005). However, pharmacological chaperone therapy of Gaucher disease relies of the partial endogenous activity of the folded mutant of glucocerebrosidase enzyme; therefore effectiveness of this method will not apply to variants with severe folding problems, totally absent activity or not efficiently interacting with the NB-DNJ (Yu et al 2007). Niemann-Pick disease type C and treatment with NB-DNJ Niemann-Pick disease type C (NPC) is an autosomal recessive disorder characterized with chronic neurodegenerative symptoms in the adolescence. This disease is caused by functional deficiency of NPC1 (in ~ 95 % of cases) or NPC2 (in ~ 5 % of cases) proteins in the cell which are responsible for cholesterol transport in the lysosomes (Vanier and Millat 2003). As a consequence of impaired cholesterol transport in the endosomal-lysosomal system, unesterified cholesterol molecules are excessively accumulated in many tissues particularly those which are normally enriched in cholesterol like brain (Xie et al 1999). 16 Chapter 1: Introduction Notably, gangliosides GM2 and GM3 as well as sphingolipids have also been shown to be accumulated with cholesterol in NPC (Watanabe et al 1998; Taniguchi et al 2001; Zervas et al 2001). In this respect, there has been an increasing body of evidence that suggest a complex physiological interaction between cholesterol and (glyco-) sphingolipids in the context of lipid homeostasis in the cells. For example: - the chemical similarities between cholesterol and sphingolipids are shown to enable these molecules to displace each other in the lipid rafts, and depletion or accumulation of one can lead to efflux or recruitment of the other one respectively (Rosenbaum and Maxfield 2011). - in the primary fibroblast cultures of patients with different NPC variants, sphingolipids are also found to be stored in the lysosomes (Sun et al 2001). - in some NPC patients with the normal glucocerebrosidase gene, the activity of this enzyme in the lysosome is lowered (Devlin et al 2010). - in the ABCA1 deficient cell model with impaired phosphatidylserine floppase activity and cholesterol accumulation, depletion of Sphingomyelin can counterbalance the floppase deficiency and restore cholesterol efflux (Gulshan et al 2013). Following SRT of Gaucher disease with NB-DNJ, this compound was also approved and applied for treatment of NPC in a similar way (Lyseng-Williamson 2014). According to what discussed earlier, here down regulation of glycosphingolipid synthesis by NB-DNJ seems to partially restore cholesterol efflux in the cells and so improve the associated symptoms. However further investigations at the cellular and molecular levels are required to define these mechanisms. Adverse effects of NB-DNJ therapy The most common adverse effects of NB-DNJ are observed in association with the gastrointestinal system, including diarrhea (80 % -90 %), flatulence and nausea as well as weight loss and tremor (Zimran and Elstein 2003; Weinreb et al 2005; Belmatoug et al 2011). The gastrointestinal symptoms are mostly at mild to moderate severity; however they can affect the efficacy of the treatment or cause an adversion to the therapy (Belmatoug et al 2011), especially in the patients with the background of intestinal intolerances. 17 Chapter 1: Introduction The gastrointestinal intolerances caused by NB-DNJ are proposed to be a consequence of suppressing the intestinal carbohydrate digestion (Belmatoug et al 2011). In similar conditions to disaccharidase deficiency disorders, the maldigested/unabsorbed carbohydrates raise osmotic influx of water and electrolytes into the intestine, and are later subjected to bacterial fermentation in the colon in association with production of irritative substances. 1.3. Carbohydrate digestion in the intestine Carbohydrates constitute a main source of energy in our normal daily diet (Caspary 1992; Costill and Hargreaves 1992). Depending on their structural complexity, carbohydrates are classified as monosaccharide, disaccharide, oligosaccharide or polysaccharide groups. Carbohydrates are absorbed in the intestine only in the monosaccharide form; therefore all of the higher structures need to be broken down to the simple sugars prior to absorption (Caspary 1992). Digestion of carbohydrates to disaccharides commences by the action of amylase in the saliva and pancreas and is accomplished in the intestine by a group of enzymes, the disaccharidases, which are located in the intestinal brush border membranes (Sterchi et al 1990; Levin 1994). These enzymes digest the disaccharides to monosaccharides, including glucose, fructose and galactose, which are then ultimately absorbed and transported into the cell interior via specific transporters, such as intestinal glucose transporter 5 (GLUT 5) (Ferraris 2001). The intestine does not express disaccharide transporters and therefore the failure to digest a disaccharide to the corresponding monosaccharides, for example due to absence of an appropriate disaccharidase, results in carbohydrate malabsorption, an intestinal disorder that is characterized by symptoms such as diarrhea, bloating, cramps and vomiting (Hammer et al 1990). Prominent members of the intestinal disaccharidases are sucrase-isomaltase (SI), maltase-glucoamylase (MGA) and lactase-phlorizin hydrolase (LPH) (Naim et al 1987a; Naim et al 1988c; Naim et al 1988e). Sucrase-isomaltase The sucrase-isomaltase enzyme (SI, EC 3.2.1.48-10) is a type II membrane glycoprotein located at the brush border membranen of the intestinal epithelial cells 18 Chapter 1: Introduction (Naim et al 1988d; Naim et al 1988b). This enzyme is composed of two structural subunits, sucrase and isomaltase (Hauser and Semenza 1983). The isomaltase subunit is located at the N-terminus of the complex with 1006 amino acid residues. This subunit starts with a small cytoplasmic tail, a single transmembrane domain containing the signal anchor, followed by a serine/threonine rich and heavily glycosylated stalk region and finally a globular functional domain (Fig 5 (Jacob et al 2002)). The isomaltase active site performs hydrolytic cleavage of α1-6 and to lower extent α1-4 glucosidic bonds (Sim et al 2010; Jones et al 2012). The sucrase subunit constructs the C-terminus of the complex and is composed of 820 amino acid residues. The sucrase active site has broad substrate specificity; it mainly cleaves α1-2 glucosidic bonds, meanwhile contributes to the hydrolysis of α14 and α1-6 glucosidic bonds in the intestine as well (Sim et al 2010; Jones et al 2012). SI is synthesized as a single polypeptide, initially detected as a 210 kDa band in the electrophoresis that represents the immature ER N-glycosylated or “mannose-rich” form. Complex N- and O-glycosylation of SI is accomplished in the Golgi apparatus, leading to generation of the mature form of this protein with an apparent molecular size of about 245 kDa (Naim et al 1988d; Naim et al 1988b). Fig. 5: The molecular structure of sucrase -isomaltase associated with cholesterol sphingolipid rich lipid rafts. 19 Chapter 1: Introduction Intact glycosylation of SI has a crucial role in cellular transport and functional activity of this protein. In the epithelial cells, SI is predominantly transported to the apical cell surface, where it performs its hydrolytic activity in the intestinal lumen. Apical sorting of SI has been shown to occur through association with cholesterol-sphingolipid rich membrane microdomains (lipid rafts, Fig. 5) in the trans-Golgi network (TGN (Alfalah et al 1999; Jacob et al 2000)). Proper O-glycosylation of the SI stalk region which serves as the apical sorting signal is crucial for its integration to the lipid rafts. Lack of SI association with lipid rafts not only impairs its apical targeting but also decreases the functional efficiency of this enzyme (Wetzel et al 2009). The mature SI is cleaved at the apical surface of the epithelial cells by pancreatic trypsin in between sucrase and isomaltase subunits, which still remain attached to each other through strong non-covalent interactions (Naim et al 1988b). Maltase-glucoamylase Similar to SI, maltase-glucoamylase (MGA, EC 3.2.1.20-3) is a type II membrane glycoprotein of the intestinal epithelium (Naim et al 1988f). Identical exon structures and the remarkable homology between SI and MGA suggest a common evolutionary ancestor for both genes (Naim and Zimmer 2008). Accordingly, many of structural and functional features of MGA are similar to SI. MGA is composed of 1875 amino acid residues organized in two functional domains. Both domains of MGA have α1-4 glucosidic activity and are responsible for the major part of maltase activity and exclusive glucoamylase activity in the intestine (Naim et al 1988f; Sim et al 2010). Synthesis and maturation of MGA resembles those of SI, with an immature “mannose-rich” ER form which acquires complex N- and O- glycosylation in the Golgi. MHA is also mostly sorted to the apical surface of the intestinal cells, for which proper O-glycosylation of the stalk region is essential (Nichols et al 1998; Naim and Zimmer 2008). Lactase-phlorizin hydrolase Lactase-phlorizin hydrolase (LPH, EC 3.2.1.23-62) is the only enzyme in the intestinal lumen that cleaves β-glucosidic bonds of carbohydrate and some phenolic compound (Naim et al 1987b; Naim 1994). The mature LPH has two functional domains, one at the C-terminus with a broad specificity towards various dietary β20 Chapter 1: Introduction glucosylceramides and the other with lactase activity to break down lactose, the major sugar of mammalian milk (GudmandHoyer and Skovbjerg 1996). LPH is a type I membrane glycoprotein. The immature form of LPH is composed of 1927 amino acid residues with an apparent molecular size of about 215 kDa. The ectodomain of the nascent LPH contains four homologous domains denoted as I to IV from N- to C- terminal respectively (Naim 2001). Upon N-glycosylation and folding, LPH forms homodimers in the ER and is then transported to the Golgi to achieve complex N- and O-glycosylation. The protein domain I and II, also known as profragment, serve as an intramolecular chaperone to facilitate protein folding in the ER. Later in the Golgi the profragment is cleaved off and subsequently degraded. After complex glycosylation, the mature LPH is about 160 kDa in size which is composed of a small cytoplasmic tail at the C-terminal followed by a transmembrane region and the two functional domains IV and III, respectively executing lactase and phlorizin hydrolase activities (Behrendt et al 2005). Mature LPH is mainly sorted to the apical membrane via association with membrane microdomains with a different composition of those for SI (Jacob and Naim 2001; Jacob and Naim 2002). Carbohydrate intolerance Carbohydrate intolerance results from the inability of the body to properly process a single type or a group of carbohydrates present in the normal daily diet (Sibley 2004). Carbohydrate intolerances can be categorized as primary or secondary types according to their inducing factor (Raithel et al 2013). Primary carbohydrate intolerances are inherited disorders in which usually the patient is intolerant to a specific type of carbohydrate. Two groups are known for this type of disorder. The first group is caused by incomplete digestion of the carbohydrates in the gastrointestinal tract due to insufficient amounts or activity levels of the intestinal glycosidases. Congenital sucrase-isomaltase, maltase-glucoamylase or lactasephlorizin hydrolase deficiencies and adult type of hypolactasia belong to this group. The second group is associated with systemic disorders that affect processing of the simple sugars in the metabolic pathways of the body. Fructose intolerance and galactosemia place in this group (Lloyd-Still et al 1988; Sibley 2004). 21 Chapter 1: Introduction The secondary types of carbohydrate intolerance disorders appear as a consequence of organ pathology or damages to the intestinal tissue. This type is usually associated with overall or multiple carbohydrate intolerances. These disorders might be induced by ingestion of irritative or toxic substances, chemotherapeutic agents, intestinal inflammatory diseases (infections, inflammatory bowel disease, and celiac disease) or other similar damages to the intestinal epithelium (Raithel et al 2013). 1.4. Aim of the study The aim of the current study is to investigate the role of N-butyl deoxynojirimycin in appearance of gastrointestinal intolerances. NB-DNJ is used as an oral medication for patients with Gaucher disease Type I and Niemann-Pick disease Type C. The most common side effects of this medication are diarrhea, bloating, vomiting, and abdominal cramps due to disturbance of the gastrointestinal function; most notably the digestive function of intestinal disaccharidases is affected in the majority of the patients. These symptoms are similar to those observed in carbohydrate malabsorption, strongly suggesting that intestinal disaccharidases are affected. Nevertheless, it is not established whether the function of these enzymes or their folding, intracellular processing, trafficking and ultimately functional efficiency is influenced. The specific aims of the current project are focusing on two points: 1) Elucidation of the immediate effects of NB-DNJ on the enzymatic function of intestinal disaccharidases (e.g. sucrase-isomaltase (SI), lactase-phlorizin hydrolase (LPH) and maltase-glucoamylase (MGA)); 2) Assessment of the persisting long term effects on the intestinal enzymatic function that could be elicited by alterations in the folding, intracellular processing, trafficking and targeting of the disaccharidases due to NB-DNJ. Respectively these objectives and the related achievements are represented in the following chapters. 22 Chapter 1: Introduction References Abian O, Alfonso P, Velazquez-Campoy A, Giraldo P, Pocovi M, Sancho J (2011) Therapeutic strategies for Gaucher disease: miglustat (NB-DNJ) as a pharmacological chaperone for glucocerebrosidase and the different thermostability of velaglucerase alfa and imiglucerase. Molecular Pharmaceutics 8(6): 2390-2397. 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Philosophical Transactions of the Royal Society of London Series B-Biological Sciences 358(1433): 961-966 . 31 Chapter 2: Carbohydrate malabsorption due to Miglustat 32 Chapter 2: Carbohydrate malabsorption due to Miglustat Chapter 2 Miglustat-induced intestinal carbohydrate malabsorption is due to the inhibition of α-glucosidases, but not β-galactosidases Mahdi Amiri and Hassan Y. Naim JOURNAL OF INHERITED METABOLIC DISEASE Volume: 35 Issue: 6 Pages: 949-954 DOI: 10.1007/s10545-012-9523-9 Published: NOV 2012 Mahdi Amiri has planned and performed the experiments, interpreted the results with Hassan Y. Naim and drafted a first version of the manuscript and contributed to writing of the final version of the manuscript. 33 Chapter 2: Carbohydrate malabsorption due to Miglustat Miglustat-induced intestinal carbohydrate malabsorption is due to the inhibition of α-glucosidases, but not β-galactosidases Mahdi Amiri and Hassan Y. Naim Department of Physiological Chemistry, University of Veterinary Medicine Hannover, Hannover, Germany Corresponding author: Hassan Y. Naim, PhD Professor and Chair of Biochemistry Department of Physiological Chemistry University of Veterinary Medicine Hannover Buenteweg 17 D-30559 Hannover Germany Email: [email protected] 34 Chapter 2: Carbohydrate malabsorption due to Miglustat Abstract Miglustat is an oral medication that has approved indication for type I Gaucher disease and Niemann pick disease type C. Usually treatment with Miglustat is associated with occurrence of gastrointestinal side effects similar to carbohydrate maldigestion symptoms. Here, we studied the direct influence of Miglustat on the enzymatic function of the major disaccharidases of the intestinal epithelium. Our findings show that an immediate effect of Miglustat is its interference with carbohydrate digestion in the intestinal lumen via reversible inhibition of disaccharidases that cleave-glycosidically linked carbohydrates. Higher non physiological concentrations of Miglustat can partly affect lactase activity. We further show that the inhibition of the disaccharidases function by Miglustat is mainly competitive and does not occur via alteration of the enzyme folding. Keywords: Miglustat, carbohydrate maldigestion, intestinal disaccharidases, disaccharidase inhibition. Abbreviations: GSL: glycosphingolipid, SI: sucrase-isomaltase, MGA: maltaseglucoamylase, LPH: lactase-phlorizin hydrolase, BBM: brush border membrane. Running head: Carbohydrate malabsorption due to Miglustat 35 Chapter 2: Carbohydrate malabsorption due to Miglustat Introduction Miglustat (N-butyl-deoxynojirimycin) is an oral medication for treatment of type I Gaucher disease and Niemann-Pick disease type C. This iminosugar blocks the primary step in sphingolipid glycosylation by reversible inhibition of glucosylceramide synthase and thus prevents later glycosphingolipids (GSL) accumulation in the lysosomes via substrate reduction therapy (Cox et al. 2000; Rosenbaum and Maxfield 2011). The initial side effects of this drug appear with high incidence in the gastrointestinal tract showing symptoms like diarrhea, bloating, vomiting, and abdominal cramps. These symptoms are very similar to (congenital) carbohydrate intolerance disorders, strongly suggesting that the activities of the disaccharidases in the intestinal lumen are affected (Andersson et al. 2000; Elstein et al. 2007; Belmatoug et al. 2011). Intestinal disaccharidases are integral membrane glycoproteins that are located in the brush border membrane of intestinal epithelial cells. Prominent members of this family of hydrolytic enzymes are sucrase-isomaltase (SI), lactase-phlorizin hydrolase (LPH) and maltase-glucoamylase (MGA) (Naim et al. 1987a; Naim et al. 1987b; Naim et al. 1988). These enzymes digest carbohydrates in the intestinal lumen and reveal preferential affinities towards cleavage of α- or β-glycosidic linkages. For example, the sucrase and isomaltase activities of SI as well as maltase and glucoamylase activities of MGA can hydrolase the α-glycosidic linkages of the major dietary carbohydrates such as starch, glycogen, sucrose and maltose. The resulting monosaccharides are eventually transported across the brush border membrane of epithelial cells into the cell interior. Only few compounds with β-glycosidically linked monosaccharides are known, such as the most essential carbohydrate in mammalian milk, lactose, which constitutes the primary dietary source for the newborn and is specifically cleaved by LPH (Robayo–Torres et al. 2006). Potential implication of Miglustat in eliciting carbohydrate maldigestion in the intestinal lumen could take place via two distinct mechanisms: i) immediate short term interference with the activity of the intestinal disaccharidases and ii) persisting long term effects that interfere with the intra-cellular processes required for maturation and trafficking of these enzymes. In this paper, we focus on the direct influence of Miglustat on the enzymatic function of sucrase, isomaltase, maltase and lactase as the major disaccharidases of the intestinal epithelium, to determine the 36 Chapter 2: Carbohydrate malabsorption due to Miglustat type and strength of the potential interactions which can immediately interfere with their digestive activity. Experimental procedures Enzyme activity measurement Brush Border Membrane (BBM) vesicles were kindly provided by the Department of Biochemistry, University of Bern, Switzerland, where they were prepared from human intestinal tissue according to Schmitz et al. (1973). The tissue was obtained from kidney donors and approved by the ethical committee at the University of Bern. The total protein content of the suspension was estimated by Bradford method (Bio-Rad) using bovine serum albumin (BSA) as a standard. The enzyme assay samples included 20 µl of BBM in PBS, pH 6.5 containing 0.6 µg of total protein, when the activities of sucrase, isomaltase and lactase were measured and 0.12 µg in the case of maltase. Miglustat (Actelion Pharmaceutical GmbH, Basel, Switzerland) was added at different concentrations (1 µM to 500 µM). Then samples were mixed with an equal volume of the relevant substrate (sucrose up to 100 mM, isomaltose up to 30mM, lactose up to 150mM and maltose up to 40 mM final concentrations) and incubated at 37 °C for 60 min. As a common product, liberated glucose amounts were determined by adding 200 µl GOD/PAP monoreagent (AXIOM mbH Germany) using microplate reader at 550 nm and glucose as standard. Each sample is compared to a similar zero time sample for the mean activity. The reported unit of activity is µMol.min-1.mg-1. Proteolytic structural analysis BBM suspension was diluted with PBS pH 7.4 containing 0, 5, 10 & 500 µM concentrations of Miglustat and incubated at 37 °C with short-mixing for 20min to ensure proper exposure and binding of the disaccharidases to Miglustat. The mixture was then subjected to partial proteolysis using 50 µg Trypsin (Sigma-Aldrich) for 60min at 37 °C. Volumes equal to 4 µg protein of BBM were loaded on a 6 % polyacrylamide gel followed by immunoblotting for SI, LPH and MGA. The monoclonal antibodies (mAbs) used were: BBM 3/705 antibody for SI (Hauri et al. 37 Chapter 2: Carbohydrate malabsorption due to Miglustat 1985), mlac10 for lactase (Maiuri et al. 1991) and a mixture of lama205 and lama 77 for maltase-glucoamylase (Quezada-Calvillo et al. 2008). Results and discussion Medications like Miglustat are prescribed at certain doses to reach their efficient plasma concentration. But initially by oral consumption, the drug is at once diluted and taken in with a limited volume of diet and thus finds a gastrointestinal concentration probably higher than later on in the body fluids (Treiber et al. 2007). Therefore this elevated local concentration of drug can trigger intestinal side effects which are not detectable in other organs or have a milder occurrence; a fact that may describe the severity of the immediate side effects of Miglustat observed as carbohydrate maldigestion in the intestine. Fig. 1: The effect of Miglustat on the activities of major intestinal disaccharidases. The activities of sucrase, isomaltase, maltase and lactase were measured in BBM in the presence of up to 10 µM concentrations of Miglustat. The statistical significance is shown as: * P< 0.05, ** P< 0.01, *** P<0.001 for n=3 experiments (ttest, pairwise) 38 Chapter 2: Carbohydrate malabsorption due to Miglustat We isolated the intestinal brush border membrane (BBM) vesicles as a rich source of intestinal disaccharidases. The enzymatic function of major intestinal disaccharidases sucrase, isomaltase, maltase and lactase were estimated in the presence of different concentrations of Miglustat. As presented in Fig. 1, concentrations up to 10 µM of Miglustat remained without any notable effect on lactase activity. By contrast, the hydrolysis of sucrose, isomaltose and maltose, i.e. the classical substrates of sucrase, isomaltase and maltase respectively, was substantially inhibited at these conditions. The inhibitory effect of Miglustat is mostly observed with sucrase, followed by isomaltase and maltase. Given that the inhibited disaccharidases cleave exclusively α-glucosidically linked disaccharides, while the non-inhibited lactase is a β-galactosidase, we conclude that Miglustat competes with the original substrates (i.e. sucrose, isomaltose and maltose) for the catalytic sites of these α-glucosidases. Notably, the maximum concentration of Miglustat used in this experiment was 10 µM, which is smaller than the free concentration of this drug after consumption and yet it considerably suppresses intestinal α-glucosidases. At higher non-physiological concentrations of Miglustat (>50 µM) a slight inhibitory effect on lactase can be detected (Fig. 2b). This effect, however, should not interfere with normal lactose digestion at a notable extent. For further investigation of the kinetics of inhibition, we estimated the activity of the disaccharidases in different concentrations of both related-substrates and Miglustat. The data were analyzed by Michaelis–Menten and Lineweaver-Burk plots (Fig. 2) and the Vmax, KM and KI and/or KI’ parameters were determined for each sample. According to the data presented in Table 1, inhibitory function of Miglustat on the sucrase, isomaltase and maltase appears with a reduction in their maximum velocity and an increase of the KM of the substrate, a phenomenon which is assessed as a mixed type of inhibition; i.e. in these reactions, on one hand Miglustat is competing with the original substrate (sucrose, isomaltose and maltose respectively) at the active site (competitive inhibition), and on the other hand, upon attachment of the original substrate to the active site, Miglustat can attenuate conversion of enzymesubstrate complex to enzyme and product through an allosteric interaction and so reduces the catalysis performance (uncompetitive inhibition, Fig. 3). 39 Chapter 2: Carbohydrate malabsorption due to Miglustat a) 40 Chapter 2: Carbohydrate malabsorption due to Miglustat b) Fig. 2: Michaelis-Menten and Lineweaver-Burk plots for brush border disaccharidases. The enzymatic activities of sucrase, isomaltase, maltase (a) and lactase (b) were measured in the presence of different concentrations of both Miglustat and the related substrates. Comparison of the estimated KI and KI’ values can uncover more details of the inhibitory interactions of Miglustat with the tested disaccharidases. Initially, the K I values for sucrase, isomaltase and maltase are notably smaller compared to the KI’ values, showing a higher tendency of Miglustat to the α-glucosidic active site rather than the allosteric site and thus a higher contribution of competitive inhibition in the total inhibition in general. Secondly, at each single concentration of Miglustat sucrase, isomaltase and maltase have the lowest to the highest values of K I respectively, which is in line with a similar trend for their degree of influence from Miglustat discussed in Fig. 1. This indicates a preferential binding of Miglustat primarily to α1-2 and next to α1-6 and α1-4 glucosidic active sites respectively. Finally, there is a significant rise in the KI’ values of isomaltase and maltase by increasing Miglustat concentrations. This behavioural change in these enzymes could be a consequence of reduced abundance of enzyme-substrate complexes as 41 Chapter 2: Carbohydrate malabsorption due to Miglustat the primary material for uncompetitive inhibition after more efficient competitive inhibition at higher drug concentrations, or a representative of a concentrationdependant allosteric interaction of Miglustat with the target enzymes. Also for LPH a very small but significant reduction of both Vmax and KM values occurs at 10 µM of Miglustat, representing a very slight uncompetitive inhibition which may confirm the concentration-dependant allosteric interaction of Miglustat. In any case, however, lactase inhibition only appears at above 50 µM of Miglustat with a competitive nature and a dissociation constant of the inhibitor which is thousands of times higher than the ones for SI (Table1, Fig. 2). Fig. 3: Schematic presentation of the potential inhibitory mode of interactions of Miglustat with sucrase. Isomaltase and maltase have similar types of interactions. The enzymological analyses did not reveal a non-competitive inhibition of these enzymes by Miglustat. It cannot be excluded, however, that an allosteric structural effect of Miglustat still can be detected at the protein level. 42 Chapter 2: Carbohydrate malabsorption due to Miglustat Table 1: The kinetic parameters of a) sucrase, b) isomaltase, c) maltase and d) lactase in the presence of different concentrations of Miglustat. The data are analysed using Lineweaver-Burk plot. a) Sucrase [Miglustat] µM Vmax s.d. KM s.d. KI KI' 0 1 2 5 7 10 4.52 3.59 3.24 1.67 1.67 1.10 0.34 0.59 0.18 0.07 0.46 0.62 15.84 83.82 140.83 168.32 242.39 229.56 0.89 16.74 9.14 6.46 62.33 143.85 0.18 0.18 0.18 0.17 0.17 4.21 5.39 2.94 4.32 3.62 [Miglustat] µM Vmax s.d. KM s.d. KI KI' 0 1 2 5 7 10 0.95 0.60 0.56 0.50 0.46 0.56 0.07 0.08 0.08 0.05 0.08 0.21 9.01 16.80 27.78 47.89 52.94 94.46 0.59 3.40 6.43 1.26 12.75 35.57 0.52 0.49 0.55 0.64 0.60 1.92 3.28 5.45 7.39 25.42 [Miglustat] µM Vmax s.d. KM s.d. KI KI' 0 1 2 5 7 10 10.67 7.34 6.82 5.04 5.39 4.01 0.71 0.42 0.51 0.19 0.92 0.18 1.97 3.12 4.53 7.17 12.29 13.01 0.38 0.30 0.60 0.80 3.15 1.54 0.79 0.78 0.75 0.62 0.60 2.21 3.57 4.51 7.26 6.07 [Miglustat] µM Vmax s.d. KM s.d. KI KI' 0 10* 25 50 250 500 0.74 0.69 0.72 0.75 0.78 0.78 0.06 0.04 0.04 0.03 0.05 0.02 24.81 22.87 24.09 26.91 33.43 43.88 2.06 1.62 1.47 1.05 3.91 3.76 871.52 703.79 136.79 - b) Isomaltase c) Maltase d) Lactase 43 Chapter 2: Carbohydrate malabsorption due to Miglustat * The reduction in V m a x and K M parameters in 10 µM Mig sample is significant compared to control. ̶ : no significant inhibition was detected We hypothesized therefore that the binding of Miglustat to the disaccharidases affects their folding and hence function. To examine the folding pattern of these enzymes after Miglustat addition we treated Miglustat-incubated BBM with trypsin. An altered folding pattern of the disaccharidases would lead to exposure of additional trypsin cleavage sites causing an altered trypsin cleavage pattern or even protein degradation. As shown in Fig. 4, similar band patterns and intensities of sucrase, maltase and lactase were revealed in the presence or absence of Miglustat indicating no gross structural alteration as a function of direct interaction of Miglustat with these enzymes. Fig. 4: Partial proteolysis of the intestinal disaccharidases by trypsin. BBM were exposed to different concentrations of Miglustat and then subjected to partial proteolysis by trypsin. Here, similar electrophoretic pattern for SI, MGA and LPH in the presence or absence of trypsin were obtained indicating that no gross structural alterations of these proteins have occurred after incubation with Miglustat. Currently it is not clear whether the activity of the disaccharidases in the small intestine is initially affected in metabolic diseases such as Gaucher disease and 44 Chapter 2: Carbohydrate malabsorption due to Miglustat Niemann-Pick type C. A possible effect could arise from an impaired targeting of these proteins to the intestinal brush border membrane, their site of action. In fact, the sorting of sucrase-isomaltase (Alfalah et al. 1999) and possibly also maltaseglucoamylase, in intestinal cells is regulated by membrane microdomains (also known as lipid rafts), which are enriched in cholesterol and sphingolipids (Lindner and Naim 2009).The homeostasis of these two lipids is disturbed or altered in Niemann-Pick type C and could be the underlying cause for impaired trafficking and reduced expression these enzymes at the cell surface of enterocytes leading thus to reduced carbohydrate digestion and subsequent malabsorption. We conclude that Miglustat can cause carbohydrate maldigestion in the intestinal lumen particularly via inhibition of the α-glucosidic activity of the intestinal disaccharidases. The observed inhibition is a combination of competitive and uncompetitive forms with a meaningful proportion for each type of the tested enzymes. Lactase as a β-galactosidase is only partly affected at high nonphysiological Miglustat concentrations, but the extent of inhibition is negligible and does not seem to remarkably diminish normal lactose digestion, and so a lactosefree diet would not be essential. Accumulation of maldigested carbohydrates in the intestinal lumen is associated with osmotic influx of water, increased fermentation activity of the commensal bacteria and production of irritating metabolites which lead to the appearance of gastrointestinal intolerances. The daily diet is normally composed of appreciable amounts of starch, sweets and soft drinks which are enriched in α-glycosidically linked sugars. Therefore it is recommended that the carbohydrate containing meals should follow a few hours after drug intake to avoid coincidence with Miglustat in the intestinal lumen. Acknowledgements This study has been supported by Actelion, Pharmaceuticals GmbH and a Ph.D. scholarship provided by the German Academic Exchange Service (DAAD) to M.A. We thank Heshmat Akbari-Borhani for critical discussion. 45 Chapter 2: Carbohydrate malabsorption due to Miglustat References Alfalah M, Jacob R, Preuss U, Zimmer K-P, Naim H, Naim HY (1999) O-linked glycans mediate apical sorting of human intestinal sucrase-isomaltase through association with lipid rafts. Curr Biol 9: 593-596. Andersson U, Butters TD, Dwek RA, Platt FM (2000) N-butyldeoxygalactonojirimycin: a more selective inhibitor of glycosphingolipid biosynthesis than N-butyldeoxynojirimycin, in vitro and in vivo. Biochem Pharmacol 59: 821-829. 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Lindner R, Naim HY (2009) Domains in biological membranes. Exp Cell Res 315: 2871-2878. Maiuri L, Raia V, Potter J, Swallow D, Ho MW, Fiocca R, Finzi G, Cornaggia M, Capella C, Quaroni A, Auricchio S (1991) Mosaic pattern of lactase expression by villous enterocytes in human adulttype hypolactasia. Gastroenterology 100: 359-369. Naim HY, Sterchi EE, Lentze MJ (1987a) Biosynthesis and maturation of lactase phlorizin hydrolase in the human small intestinal epithelial-cells. Biochem J 241: 427-434. Naim HY, Sterchi EE, Lentze MJ (1987b) Kinetics of the processing of newly synthesized sucraseisomaltase (SI) in organ-cultures of human intestinal biopsies. Pediatr Res 22: 111-111. Naim HY, Sterchi EE, Lentze MJ (1988) Structure, biosynthesis, and glycosylation of human small intestinal maltase-glucoamylase. J Biol Chem 263: 19709-19717. Quezada-Calvillo R, Sim L, Ao Z, Hamaker BR, Quaroni A, Brayer GD, Sterchi EE, Robayo-Torres CC, Rose DR, Nichols BL (2008) Luminal starch substrate “brake” on maltase-glucoamylase activity is located within the glucoamylase subunit. J Nutr 138: 685-692. Robayo–Torres CC, Quezada–Calvillo R, Nichols BL (2006) Disaccharide digestion: clinical and molecular aspects. Clin Gastroenterol Hepatol 4: 276-287. Rosenbaum AI, Maxfield FR (2011) Niemann-Pick type C disease: Molecular mechanisms and potential therapeutic approaches. J Neurochem 116: 789-795. Schmitz J, Preiser H, Maestracci D, Ghosh BK, Cerda JJ, Crane RK (1973) Purification of the human intestinal brush border membrane. Biochim Biophys Acta-Biomembr 323: 98-112. Treiber A, Morand O, Clozel M (2007) The pharmacokinetics and tissue distribution of the glucosylceramide synthase inhibitor miglustat in the rat. Xenobiotica 37: 298-314. 46 Chapter 3: Long-term effects of Miglustat on disaccharidases Chapter 3 Long term differential consequences of Miglustat therapy on intestinal disaccharidases Mahdi Amiri and Hassan Y. Naim JOURNAL OF INHERITED METABOLIC DISEASE Epub Ahead of Print DOI: 10.1007/s10545-014-9725-4 Published online: May 2014 Mahdi Amiri has planned and performed the experiments, interpreted the results with Hassan Y. Naim and drafted a first version of the manuscript and contributed to writing of the final version of the manuscript. 47 Chapter 3: Long-term effects of Miglustat on disaccharidases Long term differential consequences of Miglustat therapy on intestinal disaccharidases Mahdi Amiri and Hassan Y. Naim Department of Physiological Chemistry, University of Veterinary Medicine Hannover, Hannover, Germany Corresponding author: Hassan Y. Naim, PhD Professor and Chair of Biochemistry Department of Physiological Chemistry University of Veterinary Medicine Hannover Buenteweg 17 D-30559 Hannover Germany Email: [email protected] 48 Chapter 3: Long-term effects of Miglustat on disaccharidases Abstract Miglustat is an oral medication for treatment of lysosomal storage diseases such as Gaucher disease type I and Niemann Pick disease type C. In many cases application of Miglustat is associated with symptoms similar to those observed in intestinal carbohydrate malabsorption. Previously, we have demonstrated that intestinal disaccharidases are inhibited immediately by Miglustat in the intestinal lumen. Nevertheless, the multiple functions of Miglustat hypothesize long term effects of Miglustat on intracellular mechanisms, including glycosylation, maturation and trafficking of the intestinal disaccharidases. Our data show that a major long term effect of Miglustat is its interference with N-glycosylation of the proteins in the ER leading to a delay in the trafficking of sucrase-isomaltase. Also association with lipid rafts and plausibly apical targeting of this protein is partly affected in the presence of Miglustat. More drastic is the effect of Miglustat on lactase-phlorizin hydrolase which is partially blocked intracellularly. The de novo synthesized SI and LPH in the presence of Miglustat show reduced functional efficiencies according to altered posttranslational processing of these proteins. However, at physiological concentrations of Miglustat (≤50 µM) a major part of the activity of these disaccharidases is found to be still preserved, which puts the charge of the observed carbohydrate maldigestion mostly on the direct inhibition of disaccharidases in the intestinal lumen by Miglustat as the immediate side effect. Keywords: Miglustat, gastrointestinal enzyme intolerance, protein glycosylation and trafficking, lipid rafts. Abbreviations: SI: sucrase-isomaltase, MGA: maltase-glucoamylase, LPH: lactasephlorizin hydrolase, BBM: brush border membrane, DRM: detergent-resistant membranes. Running head: Long-term effects of Miglustat on disaccharidases 49 Chapter 3: Long-term effects of Miglustat on disaccharidases Introduction Miglustat (N-butyl deoxynojirimycin, NB-DNJ) is a synthetic analogue of D-glucose which is clinically administered for treatment of lysosomal storage diseases such as Gaucher disease type I and Niemann Pick disease type C (Venier and Igdoura 2012). The most common side effects of this drug target the gastrointestinal functions; most notably the carbohydrate digestion is affected in the majority of the patients, with symptoms similar to those observed in disaccharidases deficiencies (Cox et al 2003; Belmatoug et al 2011). Sucrase-isomaltase (SI), lactase-phlorizin hydrolase (LPH) and maltase-glucoamylase (MGA) are the major disaccharidases of the intestinal epithelium (Naim et al 1987; Naim et al 1988; Naim et al 1988). These enzymes are integral membrane glycoproteins that are synthesized and transported along the constitutive secretory pathway from the ER via the Golgi to the apical membrane in intestinal cells and are subjected to an extensive processing that includes N- and O-linked glycosylation, intracellular and extracellular proteolytic processing and association with lipid rafts (Keiser et al 2006; Behrendt et al 2009). In a previous study we reported that the activity of disaccharidases with specificity towards α-glucosidic linkages (i.e. SI and MGA) could be substantially inhibited in the intestinal lumen by Miglustat immediately upon administration of this drug (Amiri and Naim 2012). Nevertheless, the effects of Miglustat on the intestinal disaccharidases are not limited to direct inhibition at the cell surface. Miglustat at high concentrations can efficiently lower the cellular contents of glycosphingolipids in order to reverse the sphingolipidoses complications appearing in lysosomal storage diseases such as Gaucher disease (Platt et al 1994; Cox et al 2000; Butters et al 2005). On the other hand membrane microdomains or lipid rafts, which constitute platforms for protein trafficking and sorting, particularly in polarized epithelial cells, are enriched in the glycosphingolipids (Jacob et al 2000; Hanzal-Bayer and Hancock 2007; Lindner and Naim 2009). Therefore, it is plausible to assume that alteration in the glycosphingolipids due to the action of Miglustat could interfere with trafficking and polarized sorting of the disaccharidases in the epithelial cells. It should be noted that Miglustat is a derivative of 1-deoxynojorimycin (dNM), which affects N-glycosylation of proteins in the ER via inhibition of α-glucosidases I and II (Saunier et al 1982; Fuhrmann et al 1985). Since the structure, sorting and function of the glycoproteins are highly dependent on intact glycosylation, Miglustat may 50 Chapter 3: Long-term effects of Miglustat on disaccharidases potentially interfere with the intracellular mechanisms required for proper folding, maturation and trafficking of the cellular proteins including the intestinal glycoproteins (Alonzi et al 2008). In the present study, we have investigated in an intestinal cell culture model the long term effects of Miglustat on the structure, function and trafficking of two intestinal glycoproteins with the ultimate goal of mimicking the in vivo effects in the intestinal lumen upon administration of this drug. In these studies we utilized Caco-2 cells as a cellular model. Caco-2 cells differentiate spontaneously to an enterocyte-like cells which are characterized by the formation of a polarized morphology and constitutive expression of small intestinal glycoproteins such as SI, DPPIV, aminopeptidase N, angiotensin converting enzyme and to a lesser extent also LPH (Rousset et al 1985). Due to the low expression levels of LPH in Caco-2 cells we utilized in our studies Madin-Darby canine kidney cells stably expressing LPH (MDCK-LPH) as well as Chinese hamster ovary cells stably expressing LPH (CHO-LPH). Our data demonstrate that long term treatment of Caco-2 or MDCK-LPH cells with Miglustat influences glycoprotein processing. However, the substantial effects of this drug on intestinal disaccharidases are limited to the high non-physiological concentrations. Therefore, the major effects of Miglustat at the conventional physiological doses will only partly affect the structure and function of the intestinal disaccharidases in long terms, and the major extent of the suppressed carbohydrate digestion by this drug is attributed to the transient inhibition of some disaccharidases in the intestine as an immediate effect. 51 Chapter 3: Long-term effects of Miglustat on disaccharidases Materials and methods Antibodies and reagents Monoclonal antibodies against sucrase-isomaltase (HSI2, HBB2/614/88, HBB3/705/60 (Hauri et al 1985; Beaulieu et al 1989)) and lactase-phlorizin hydrolase (HBB1/909/34/74, MLac1 and MLac10 (Maiuri et al 1991; Hauri et al 1994)) were generously provided by Hans-Peter Hauri, University of Basel and Erwin E. Sterchi, University of Bern, Switzerland and Dallas Swallow, University College London, London, UK. Anti-calnexin and anti-flotillin 2 antibodies were purchased from Sigma (Munich, Germany). HRP-coupled secondary antibodies were purchased from Thermo Scientific (IL. USA) and Alexa Fluor® secondary antibodies were from Invitrogen (Life Technologies GmbH). Media, fetal calf serum (FCS) and antibiotics for cell culture were obtained from PAA (Pasching, Austria). All of the cell extracts in this study had a supplement of protease inhibitors including 1 mM phenylmethylsulfonyl fluoride, 1 μg/ml pepstatin, 5 μg/ml leupeptin, 5 μg/ml aprotinin, 1 μg/ml antipain and 50 μg/ml trypsin inhibitor (Sigma). The rest of utilised chemicals and reagents were from analytical grade and were purchased from Sigma (Munich, Germany) except otherwise mentioned Cell culture and biosynthetic labelling Caco-2 colon carcinoma cells were seeded in Dulbecco's Modified Eagle Medium (DMEM) high glucose. Madin-Darby Canine Kidney (MDCK) epithelial cells and Chinese hamster ovary (CHO) cells both stably transfected with human lactasephlorizin hydrolase (LPH) cDNA were grown in DMEM-low glucose and RPMI 1640 media respectively. All of the media were supplemented with 10 % FCS, 100 U/ml penicillin and 100 μg/ml streptomycin. Cells were incubated in humidified air containing 5 % CO2 at 37 °C. For Biosynthetic labelling, the cells were initially starved for 2 hours in methioninefree DMEM medium and then labelled for different periods with [ 35S]-methionine in the similar medium in the presence or absence of Miglustat. In pulse chase experiments, after starvation the cells were pulsed 60 min for LPH with [ 35S]methionine and chased for the indicated time points. DMEM medium containing extra 2.5 mM non-labelled methionine was used for chasing periods in the presence or 52 Chapter 3: Long-term effects of Miglustat on disaccharidases absence of Miglustat. When used, Miglustat was continuously found in the labelling or chase media. Solubilization, immunoprecipitation, analysis and detection of proteins Cells were solubilised in 25 mM Tris-HCl, pH 8.0 buffer containing 0.5 % Triton X100, 0.5 % deoxycholate and 50 mM NaCl. After precipitation of the cell debris, monoclonal antibodies coupled to protein-A Sepharose (PAS) were used for immunoprecipitation of the target proteins from the supernatant followed by two washing steps with buffer I (0.5 % Triton X-100 and 0.05 % deoxycholate in PBS) and buffer II (125 mM Tris-HCl, pH 8.0, 10 mM EDTA, 500 mM NaCl, 0.5 % Triton X100) three times each. For Co-immunoprecipitation experiments, cells were solubilised in 15 mM Tris-HCl, pH 8.0 buffer containing 150 mM NaCl and 1 % ndodecyl β-D-maltoside. Then the immunoprecipitates were washed with a buffer of similar composition containing 0.2 % n-dodecyl β-D-maltoside. For immunoblotting, the samples were resolved by SDS-PAGE and transferred to Roti®-PVDF membrane (Carl Roth GmbH) using Mini Trans-Blot® Cell system (BioRad Laboratories GmbH). After blocking in 5 % milk-PBS solution, the membrane was incubated for 1 hour with the specific primary antibody in Tween-PBS (Tween® 20, Carl Roth GmbH) and subsequently washed with Tween-PBS for 10 min three times. Incubation with the relative secondary HRP-conjugated antibody was performed in the same manner. Band visualization was performed using chemiluminescence substrate (Thermo Scientific IL. USA) and ChemiDoc™ XRS instrument (Bio-Rad Laboratories GmbH). Where indicated, [35S]-labelled immunoprecipitates were analysed for glycosylation modifications by treatment with endo-β-N-acetylglucosaminidase H (endo H) and/or peptide: N-glycosidase F (PNGase F) (both purchased from Roche Diagnostics) treatment. The samples were then resolved on SDS-PAGE, dried on filter papers and the bands were detected by a phosphor plate using phosphorimaging device (BioRad Laboratories GmbH). The indicated band intensities were quantified by Quantity One® 1-D analysis software. 53 Chapter 3: Long-term effects of Miglustat on disaccharidases Isolation of detergent resistant membranes (DRMs) Caco-2 cells were collected in PBS and solubilised with 1 % Triton X-100 on ice, briefly homogenized by pipetting and incubated at 4 °C for 2 h with gentle shaking. The lysates were then centrifuged at 5000 x g for 10 min at 4 °C and the supernatant were subjected to stepwise sucrose gradient ultracentrifugation at 100,000 x g for 18h at 4 °C. The gradient composed of 5 % (1 ml), 30 % (7ml), 40 % (1 ml, 0.5:0.5 of lysate: sucrose 80 %) and 80 % (1 ml) sucrose concentrations. Finally 1 ml fractions were collected from the top and examined by immunoblotting for the target proteins. Enzyme activity measurement and protein estimation For enzymatic activity of sucrase (SI) and lactase (LPH), hydrolysis of sucrose or lactose in different preparations was measured respectively and the liberated glucose as the final product for both reactions was determined by GOD-PAP mono-reagent method (Axiom mbH Germany) in comparison to a standard curve (Amiri and Naim 2012). Concentration of the protein in solutions was quantified by Bradford method (Bio-Rad) using bovine serum albumin as a standard protein. 54 Chapter 3: Long-term effects of Miglustat on disaccharidases Results Miglustat interferes with N-glycosylation and trafficking of intestinal glycoproteins One of the multi functions of Miglustat is its inhibitory effect on the ER-located αglucosidases. These glucosidases cleave three glucoses from the core carbohydrate side chains that are co-translationally added to newly synthesized proteins in the ER. An inhibition of this early carbohydrate trimming step could have implications on the folding and trafficking of glycoproteins in the early secretory pathway. We therefore examined the effects of Miglustat on the intracellular processing of two major intestinal glycoproteins, sucrase isomaltase (SI) which is endogenously expressed in Caco-2 cells as well as lactase-phlorizin hydrolase (LPH) that was stably expressed in transfected MDCK cells. Here, the biosynthesis of these proteins was investigated in the presence of various concentrations of Miglustat. As shown in Fig. 1, a gradual shift in the size of the SI glycoforms over increasing concentrations of Miglustat is observed. These effects were moderate at concentrations that lie within the physiological range detected in the serum of patients after administration of the drug (≤50 µM). In fact, treatment of the immunoprecipitates with endo H and quantification of the digested glycoforms show that the proportions of the mannose-rich endo H-sensitive ER form of SI (SIh) and the endo H-resistant complex glycosylated SIc forms in total SI are subjected to change in the presence of Miglustat. At Miglustat concentrations up to 50 µM the amounts of complex glycosylated SI in addition to total SI are reduced. However, a substantial decrease of SIc is observed at 250 µM (almost 60 %) which occurs with an obvious decrease in its molecular size. At higher concentrations neither an additional change in the proportion of SIc in total SI nor in its size were observed indicating that here Miglustat action was saturated at the 250 µM concentration. These data altogether are concomitant with a gradual delay of SI in the ER and impaired processing starting at 50 µM, rendering 50 µM as a threshold concentration for the solid action of Miglustat. 55 Chapter 3: Long-term effects of Miglustat on disaccharidases a) b) 56 Chapter 3: Long-term effects of Miglustat on disaccharidases c) Fig. 1: Miglustat effects on the N-glycosylation of intestinal glycoproteins. a) SI (Caco-2 cells) and LPH (MDCK-LPH cells) were immunoprecipitated after biosynthetic labelling of the cells in the presence of different Miglustat concentrations. The immunoprecipitates were further analysed by endoglycosidase H and PNGase F treatments. The ratio of high-mannosylated and complex glycosylated forms of these proteins after endo H treatment has been quantified and presented for different concentrations of Miglustat b) Schematic presentation of the altered N-glycoforms produced in the presence of Miglust at in comparison to normal ones (hypothetical model). c) MDCK-LPH cells were pulsed with [ 3 5 S]methionine and chased for the indicated time points in the presence or absence of 50 µM Miglustat. After immunoprecipitation, the samples were treated with endo H for easier discrimination between mannose rich (LPHh) and complex (LPHc) glycoforms. Miglustat seems to delay maturatio n of LPHh to LPHc substantially. Expression of SI transiently in COS-1 cells or stably in MDCK cells (supplementary Fig.1) supported that the biosynthetic forms (mannose-rich/complex glycosylation), indicative of trafficking, are affected in a similar way as observed in Caco-2 cells (and different from LPH in MDCK-cells). The effects of Miglustat on LPH were more substantial than those observed with SI. Already at 10 µM Miglustat the proportion of the complex glycosylated mature endo H-resistant form of LPH decreased markedly and at 50 µM of the drug the mannose57 Chapter 3: Long-term effects of Miglustat on disaccharidases rich ER form of LPH became the most predominant form, suggesting that Miglustat treatment leads to a block of LPH in the ER. Given the significant effects of Miglustat on LPH at moderate concentrations that resemble those observed in the serum of patients put on the drug, we analysed in details in a pulse-chase experiment the progress of this effect and processing of LPH in the presence of 50 µM of Miglustat. As shown in Fig. 1c the complex glycosylated form of LPH appeared within 2 h of chase in the control Miglustat-free sample is supporting previous data (Naim et al 1987) Suppl. Fig.1: The effect of Miglustat on glycosylation and trafficking of SI in different cell line. Transiently transfected COS-1 cells (a) and stably transfected MDCK cells (b) expressing sucrase-isomaltase were treated with Miglustat, labelled with [ 3 5 S]methionine and resolved by SDS-PAGE. The results indicate that glycosylation and trafficking of SI in these cell lines is delayed in a similar way to Caco -2 cells but different from LPH in MDCK-cells; i.e. although trafficking of SI in all of the tested cell lines is delayed in the presence of Miglustat, this protein remains quite transport competent and is not arrested in the ER. By contrast at this time point the major form of LPH was the mannose-rich ER form. At longer chase periods, LPH was processed to a complex glycosylated form and cleaved mature form whilst LPH in the Miglustat-treated sample revealed only low levels of the complex glycosylated LPH protein, concomitant with delayed trafficking 58 Chapter 3: Long-term effects of Miglustat on disaccharidases of LPH from the ER and reduced processing to a complex glycosylated mature protein. The variable effects of Miglustat on the intestinal proteins are related to variations in their binding capacities to the ER chaperone calnexin The intestinal hydrolases that have been analysed are all heavily N-glycosylated rendering them suitable substrates for trimming in the ER by -glucosidases (Maattanen et al. 2010). Nevertheless, only LPH was significantly affected by Miglustat at low concentrations and its trafficking delayed suggesting a block in the ER presumably due to prolonged binding to ER molecular chaperones. We therefore addressed this possibility by investigating the potential interaction of the two enzymes with calnexin, a type 1 membrane protein that functions as an ER chaperone (Bergeron et al 1994; Maattanen et al 2010). One major mode of action of calnexin is binding to nascent N-glycoproteins specifically through recognition of Glc1Man9 glycans. Association of glycoproteins with calnexin then essentially requires removal of the first two out of three glucose residues in the N-glycan structure by ER glucosidases I and II (Helenius and Aebi 2004). Since Miglustat is a potent inhibitor of the ER glucosidases (Maattanen et al 2010), trimming of the two terminal glucoses in the core oligosaccharide chains could be impaired. This in turn may lead to reduction or possibly also abolishment of the binding of calnexin to the newly synthesized protein with subsequent implications on its proper folding. We assessed this interaction by immunoprecipitation of calnexin from cells that were treated or not treated with Miglustat followed by detection of potentially coimmunoprecipitated SI or LPH by immunoblotting. As shown in Fig. 2, SI was not associated with calnexin neither in the control nor in the Miglustat treated samples (the positive control revealed strong band of the protein). By contrast, LPH was coimmunoprecipitated with calnexin in the absence of Miglustat and its intensity was comparable to that of the positive control. At increasing Miglustat concentrations the intensity of LPH that co-immunoprecipitated with calnexin decreased substantially by almost 60 %. These differences in the mode of binding of SI on one hand and LPH on the other to calnexin provide a possible explanation for the different trafficking and processing of SI versus LPH in the presence of Miglustat. 59 Chapter 3: Long-term effects of Miglustat on disaccharidases Fig. 2: Miglustat interferes with the association of LPH with calnexin in the ER. Caco-2 cells (for SI) or MDCK-LPH cells (for LPH) were incubated with/without Miglustat, lysed and calnexin was immunoprecipitated. Association of SI or LPH with calnexin was validated by immunoblotting of the immunoprecipitates. Immunoprecipitated SI or LPH from the same samples were used as positive controls and the protein A-Sepharose beads (unconjugated) incubated with samples were taken as the negative controls. ** p< 0.01, from three indep endent experiments. Apparently, the quality control of SI in the ER does not essentially implicate calnexin or mechanisms that sense alterations in the N-glycan profile as those that occur immediately in the ER via trimming of the core oligosaccharide chain by ERglucosidases I and II. On the other hand, proper folding of LPH implicates binding to calnexin, the inhibition of which through Miglustat may lead to impaired folding and trafficking to and processing in the Golgi. 60 Chapter 3: Long-term effects of Miglustat on disaccharidases Effects of Miglustat on the association of SI with lipid rafts and apical sorting In intestinal epithelial cells polarized sorting of disaccharidases towards the apical or brush border membrane is a major event that occurs and at the same time warrants intact physiological function of these proteins in the final steps of nutrient digestion (Lindner and Naim 2009). Previous data have shown that the targeting of SI to the apical membrane implicates its association with lipid rafts in the trans-Golgi network (Alfalah et al 1999), while LPH follows a lipid-rafts-independent pathway (Jacob and Naim 2001; Jacob et al 2003). Major components of lipid rafts are sphingolipids and cholesterol and can be isolated from cellular membranes by virtue of their insolubility in non-ionic detergents such as Triton X-100 (Chamberlain 2004; Lindner and Naim 2009). Given that Miglustat inhibits the synthesis of glucosylceramides via inhibition of the corresponding enzyme glucosylceramide synthase, sphingolipid levels will be expected to be altered, presumably reduced in cholesterol- and sphingolipidsenriched lipid rafts. Along this an overall imbalance in membrane lipid composition may be expected with potential interference with trafficking and apical sorting of the intestinal glycoproteins. We have therefore addressed the question whether prolonged exposure of enterocytes to Miglustat could affect the targeting of SI. Lipid rafts were isolated from post-confluent Caco-2 cells that were treated or not treated with Miglustat for 3 days. TX-100 extracts of the cells were fractionated on discontinuous sucrose gradient and the lipid rafts recovered in the upper floating fractions (Jacob et al 2000). The abundance of SI in the floating fractions (lipid rafts) was assessed in Western blots. As shown in Fig. 3a, SI was detected in the top three fractions of the gradients, which contain the lipid rafts-associated SI, as well as mainly in the last bottom fractions, which correspond to the TX-100 soluble or non-lipid rafts-associated SI. The overall intensity of the bands in the lipid rafts fractions (Fr 1-3) was almost comparable to that of the non-rafts fraction (Fr 4-9), suggesting an almost equal distribution of SI in different membrane milieus that can be discriminated by detergent-resistance to TX100. In the presence of Miglustat a partial reduction in the level of SI associated with lipid rafts has been observed. These results indicate that Miglustat can also affect the trafficking of SI to the apical membrane due to its reduced association with lipid rafts (Alfalah et al 1999; Jacob et al 2000). 61 Chapter 3: Long-term effects of Miglustat on disaccharidases a) b) Fig. 3. Legend on the next page 62 Chapter 3: Long-term effects of Miglustat on disaccharidases Fig. 3: Miglustat influences the lipid rafts association, cellular levels and functional efficiencies of SI and LPH. a) Caco-2 cell treated with/without Miglustat were solubilized with 1 % Triton X 100 and fractionated by ultracentrifugation on a sucrose gradient. Flotillin as a raft marker as well as SI were detected by immunoblotting. By increasing Miglustat concentration, the ratio of SI within the raft fraction (Fr 1 -3) versus the non-raft fractions (Fr 4-9) is reduced. b) Caco-2 cells (for SI) and CHO-LPH cells (for LPH) were treated with/without different concentrations of Miglustat for 2 -3 days, homogenized and tested for sucrase and lactase specific activities respectively. The cellular levels of SI and LPH were determined by immunoblotting with equal protein amounts of the samples. Increasing Miglustat concentrations show gradually reducing cellular content and ac tivity of SI and LPH. At the drug concentrations marked with the asterisks the protein levels significantly differ from the detected enzymatic level. * p< 0.05, **p<0.01. The activity of de novo synthesized disaccharidases is partially reduced in the presence of Miglustat In a previous study we could show that the association of SI with lipid rafts results in an almost 3-fold increase in its specific activity (Wetzel et al 2009). The results presented here strongly suggest that the reduced levels of SI in the lipid rafts could be associated with an overall reduction in its activity due to Miglustat. To examine the activity of SI and the second disaccharidase, LPH, differentiated Caco-2 cells and the stably transfected CHO-LPH cells were treated with various concentrations of Miglustat (0-1000 µM) for 2-3 days depending on the turnover of SI and LPH to achieve a homogeneous population of de novo synthesized proteins in the presence of Miglustat in parallel to the untreated control samples. By determination of sucrase or lactase activities in the cell homogenates, a gradual loss in the specific activity of these enzymes with an increasing Miglustat concentration was identified. A reduction in the protein levels of the enzymes was also observed by Western blotting, presumably as a result of delayed trafficking and partial degradation of the altered glycoforms of these proteins (Fig. 3b). We further analysed the activity and protein levels of SI and LPH at higher concentrations of Miglustat at or over 250 µM. Here, the sucrase and lactase 63 Chapter 3: Long-term effects of Miglustat on disaccharidases activities are diminished to a higher extent than at lower concentrations. Nevertheless, these reductions did not parallel the cellular contents of the disaccharidases that are rather comparable to the samples of lower drug concentrations, suggesting that the substantial reduction in the activity levels of SI and LPH was caused by the posttranslational events (Fig. 3b). One of these events is the altered glycosylation pattern as shown above. In fact, N- and O-glycosylations play a decisive role in regulation of the activity of SI and LPH. N- and O-glycosylated LPH is almost 4 times more active than solely N-glycosylated LPH (Naim and Lentze 1992).The fact that LPH is blocked intracellular in the ER as a mannose-rich glycosylated protein already starting Miglustat concentrations of 25 µM is compatible with a reduction in its activity. For SI the glycosylation is also essential, since it mediates the association of the protein with lipid rafts, which increase the activity of SI as well as dictate its targeting to the apical membrane (Wetzel et al 2009). It is plausible therefore to impute the reduced activity levels of SI partially to its reduced association with lipid rafts (Fig. 3a). These results provide a strong support to the views described above that the altered glycosylation pattern of SI and LPH are directly implicated in the regulation of their activities. It is clear therefore that the long term consequences of Miglustat therapy, via interfering with N-glycosylation, partially influence the processing of the structurally active disaccharidases at physiological concentrations of this drug. Discussion Miglustat therapy of lysosomal storage diseases raises gastrointestinal intolerances with similar symptoms to disaccharidases deficiencies. In a previous paper we reported on the inhibition of sucrase, isomaltase and maltase activities immediately after appearance of Miglustat in the intestinal lumen (Amiri and Naim 2012). In the present study we focused on the long term consequences of Miglustat therapy that may influence the structural and functional features of the intestinal disaccharidases such as SI and LPH. Our results show that prolonged incubation of cell lines expressing SI and LPH with Miglustat influences partially several cellular mechanisms. The concerted action of these effects together amplifies the overall effects of Miglustat on the digestive capacity of the disaccharidases. 64 Chapter 3: Long-term effects of Miglustat on disaccharidases The initial effect of Miglustat on glycoproteins is its interference with N-glycosylation in the ER, which leads to a delayed trafficking and maturation of LPH and to a lesser extent also SI. The variations in the glycosylation pattern because of Miglustat have direct effects on the folding of these proteins and subsequently on their enzymatic activities. The reduced activity levels of LPH arise from two alterations in the biosynthetic features of LPH. Firstly, the increased proportion of the mannose-rich forms of LPH and its persistence in this form later on at higher Miglustat concentrations explain per se the reduced activity levels. In fact, N- and Oglycosylation of LPH are important events in regulating the activity levels of LPH (Naim and Lentze 1992). Since LPH is mainly mannose-rich glycosylated, at or above 50 µM Miglustat its activity will be expected to be markedly reduced. Secondly, the reduced levels of LPH in the presence of Miglustat are likely due to protein degradation after its block in the ER and translocation into the proteasome for degradation. These two factors act together to elicit an overall causal reduction in the LPH activity. The SI activity is also posttranslationally regulated but at different levels as compared to LPH. By virtue of its multifunctional properties, Miglustat acts at two different stages that together reduce the activity of SI. While the trafficking between the ER and the Golgi is not as affected as with LPH, SI is affected at the stage between the Golgi and the cell surface. Here, Miglustat inhibits the synthesis of sphingolipids which in turn elicit an imbalance in lipid rafts and consequently plays a dual role that eventually affects an overall digestive function of SI: on one hand the lipid rafts are important for increasing the SI activity (Wetzel et al 2009) and secondly, they are important for the correct targeting of SI to the cell surface (Alfalah et al 1999). Thus, the reduced level of SI in lipid rafts is compatible with reduced expression at the cell surface and thus diminished exertion of its enzymatic function. Both of these events together are associated with a substantial reduction of SI digestive function. In essence, our study elucidates the concept of long term effects of Miglustat on the digestive function of intestinal disaccharidases. While the individual posttranslational events of LPH and SI are partially affected by Miglustat and each could lead to a partial reduction in the enzymatic activities, their combinatorial effects are more substantial and could be associated in some cases with severe carbohydrate malabsorption. Together with the immediate 65 effects of Miglustat on the Chapter 3: Long-term effects of Miglustat on disaccharidases disaccharidase function (Amiri and Naim 2012), the mildness or severity of carbohydrate malabsorption elicited by Miglustat depends on whether the patients under Miglustat treatment are heterozygous for mutations in the SI or LPH genes or are lactose-intolerant. In fact, regarding the SI gene, 13 % of the American population is heterozygous for the mutation Gly1073Asp, 9 % for the mutation Phe1745Cys and 6 % for Val577Gly (Sander et al 2006; Alfalah et al 2009; Uhrich et al 2012). All these three mutations generate a malfolded, enzymatically inactive and transport-incompetent SI protein. Along this an individual with two healthy alleles of SI would express substantially more active SI than an individual with one healthy allele, whereby the latter would be therefore more sensitive towards Miglustat and its effects. Similarly, several mutations of LPH have been identified which result in a posttranslational block of LPH in the ER as an inactive protein (Järvelä et al 1998; Behrendt et al 2009). Also most of the human population is lactose intolerant (Kuokkanen et al 2003; Ingram et al 2009). It is therefore advisable that treatment of patients with Miglustat should be preceded by an assessment of the genetic status of SI or LPH. In either case particular precautions can be taken, such as treatment with SI or LPH substitutes, such as sucraid or bacterial ß-galactosidase preparation, and also by changing the dietary habits to low carbohydrate diets and including proper intervals between the drug uptakes and carbohydrate consumption. Acknowledgements During the course of this work M.A. was a recipient of a Ph.D. scholarship from the German Academic Exchange Service (DAAD), Bonn, Germany. 66 Chapter 3: Long-term effects of Miglustat on disaccharidases References Alfalah M, Jacob R, Preuss U, Zimmer KP, Naim H, Naim HY (1999) O-linked glycans mediate apical sorting of human intestinal sucrase-isomaltase through association with lipid rafts. Curr Biol 9(11): 593-596. Alfalah M, Keiser M, Leeb T, Zimmer KP, Naim HY (2009) Compound heterozygous mutations affect protein folding and function in patients with congenital sucrase-isomaltase deficiency. Gastroenterology 136(3): 883-892. 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Hanzal-Bayer MF, Hancock JF (2007) Lipid rafts and membrane traffic. Febs Lett 581(11): 2098-2104. Hauri HP, Roth J, Sterchi EE, Lentze MJ (1985) Transport to cell-surface of intestinal sucraseisomaltase is blocked in the Golgi-apparatus in a patient with congenital sucrase-isomaltase deficiency. Proc Natl Acad Sci USA. 82(13): 4423-4427. Hauri HP, Sander B, Naim H (1994) Induction of lactase biosynthesis in the human intestinal epithelial-cell line Caco-2. European Journal of Biochemistry 219(1-2): 539-546. 67 Chapter 3: Long-term effects of Miglustat on disaccharidases Helenius A, Aebi M (2004) roles of N-linked glycans in the endoplasmic reticulum. Annu Rev Biochem 73(1): 1019-1049. Ingram CJE, Mulcare CA, Itan Y, Thomas MG, Swallow DM (2009) Lactose digestion and the evolutionary genetics of lactase persistence. Hum Genet 124(6): 579-591. Jacob R, Alfalah M, Grunberg J, Obendorf M, Naim HY (2000) Structural determinants required for apical sorting of an intestinal brush-border membrane protein. J Biol Chem 275(9): 6566-6572. Jacob R, Heine M, Alfalah M, Naim HY (2003) Distinct cytoskeletal tracks direct individual vesicle populations to the apical membrane of epithelial cells. Curr Biol 13(7): 607-612. Jacob R, Naim HY (2001) Apical membrane proteins are transported in distinct vesicular carriers. Curr Biol 11(18): 1444-1450. Järvelä I, Sabri Enattah N, Kokkonen J, Varilo T, Savilahti E, Peltonen L (1998) Assignment of the locus for congenital lactase deficiency to 2q21, in the vicinity of but separate from the lactasephlorizin hydrolase gene. Am J Hum Gen 63(4): 1078-1085. Keiser M, Alfalah M, Propsting MJ, Castelletti D, Naim HY (2006) Altered folding, turnover, and polarized sorting act in concert to define a novel pathomechanism of congenital sucraseisomaltase deficiency. J Biol Chem 281(20): 14393-14399. Kuokkanen M, Enattah NS, Oksanen A, Savilahti E, Orpana A, Jarvela I (2003) Transcriptional regulation of the lactase-phlorizin hydrolase gene by polymorphisms associated with adult-type hypolactasia. Gut 52(5): 647-652. Lindner R, Naim HY (2009) Domains in biological membranes. Exp Cell Res 315(17): 2871-2878. Maattanen P, Gehring K, Bergeron JJM, Thomas DY (2010) Protein quality control in the ER: The recognition of misfolded proteins. Sem Cell Dev Biol 21(5): 500-511. Maiuri L, Raia V, Potter J et al (1991) mosaic pattern of lactase expression by villous enterocytes in human adult-type hypolactasia. Gastroenterology 100(2): 359-369. Naim HY, Lentze MJ (1992) impact of O-glycosylation on the function of human intestinal lactasephlorizin hydrolase - characterization of glycoforms varying in enzyme-activity and localization of O-glycoside addition. J Biol Chem 267(35): 25494-25504. Naim HY, Sterchi EE, Lentze MJ (1987) biosynthesis and maturation of lactase phlorizin hydrolase in the human small intestinal epithelial-cells. Biochem J 241(2): 427-434. Naim HY, Sterchi EE, Lentze MJ (1988) Biosynthesis of the human sucrase-isomaltase complex differential O-glycosylation of the sucrase subunit correlates with its position within the enzyme complex. J Biol Chem 263(15): 7242-7253. Naim HY, Sterchi EE, Lentze MJ (1988) Structure, biosynthesis, and glycosylation of human small intestinal maltase-glucoamylase. J of Biol Chem 263(36): 19709-19717. Platt FM, Neises GR, Dwek RA, Butters TD (1994) N-butyldeoxynojirimycin is a novel inhibitor of glycolipid biosynthesis. J Biol Chem 269(11): 8362-8365. Rousset M, Laburthe M, Pinto M et al (1985) Enterocytic differentiation and glucose-utilization in the human-colon tumor-cell line Caco-2 - modulation by forskolin. J Cell Physiol 123(3): 377-385. Sander P, Alfalah M, Keiser M et al (2006) Novel mutations in the human sucrase-isomaltase gene (SI) that cause congenital carbohydrate malabsorption. Hum Mutat 27(1): 119. Saunier B, Kilker RD, Tkacz JS, Quaroni A, Herscovics A (1982) inhibition of N-linked complex oligosaccharide formation by 1-deoxynojirimycin, an inhibitor of processing glucosidases. J Biol Chem 257(23): 4155-4161. 68 Chapter 3: Long-term effects of Miglustat on disaccharidases Uhrich S, Wu Z, Huang J-Y, Scott CR (2012) Four Mutations in the SI gene are responsible for the majority of clinical symptoms of CSID. J Pediatr Gastroenterol Nutr 55: S34-S35 10.1097/1001.mpg.0000421408.0000465257.b0000421405. Venier RE, Igdoura SA (2012) Miglustat as a therapeutic agent: prospects and caveats. J Med Genet 49(9): 591-597. Wetzel G, Heine M, Rohwedder A, Naim HY (2009) Impact of glycosylation and detergent-resistant membranes on the function of intestinal sucrase-isomaltase. Biol Chem 390(7): 545-549 69 Chapter 3: Long-term effects of Miglustat on disaccharidases . 70 Chapter 4: Discussion Chapter 4 Discussion 71 Chapter 4: Discussion Discussion N-butyl deoxynojirimycin (NB-DNJ, Miglustat or Zavesca) is a synthetic N-alkylated derivative of nojirimycin, a glucose analogue found naturally in some species of Streptomyces. NB-DNJ is a water soluble small molecule that can diffuse through cellular membranes and has negligible cytotoxicity (Neises et al 1997). Initially this compound was proposed as an antiviral agent to attenuate the infective potential of HIV and similar viruses via interference with the processing of their envelope glycoproteins in the ER of the host cells (Ficicioglu 2008). Later, according to its suppressive effects on the biosynthesis of glycolipids in the cells, NB-DNJ was suggested as a medication for substrate reduction therapy (SRT) of the lysosomal storage disorder associated with deficiencies in the glycolipid metabolism (Platt et al 1994). In such disorders, hydrolytic cleavage of glycolipids has been interrupted in the lysosomes due to inherited deficiency of the accountable enzyme. Consequently, imbalanced lipid homeostasis as a malfunction of the lysosomal recycling in the cells in addition to the enlarged lysosomal size can interfere with the normal cell functions and result in the functional perturbation of the related body organs (Neufeld 1991). In the case of Gaucher disease, a prototypical glycosphingolipidosis, SRT with NBDNJ was proposed to modulate the cellular levels of the glucosylceramides via repressing the biosynthetic pathway of this lipid, resulting in reduced lysosomal storage and thus appearance of the later consequences (Platt et al 1994). Followed by passing clinical trials, since 2003 NB-DNJ has been confirmed for treatment of Type I Gaucher disease and later also Niemann Pick disease type C (Venier and Igdoura 2012; Lyseng-Williamson 2014). In comparison to the “enzyme replacement therapy” (ERT) of Gaucher disease, treatment with NB-DNJ has the advantages of oral administration, improved handling of the neurological symptoms (based on the ability of the drug to pass through blood-brain barrier) and lower financial costs (Lachmann 2010; Bennett and Mohan 2013). Meanwhile, the majority (80 % to 90 %) of the patients receiving this medication suffer from carbohydrate intolerance in the intestine as a side effect (Belmatoug et al 2011). Inability to digest carbohydrates as the main components of the daily diet can 72 Chapter 4: Discussion cause secondary complications and so reluctancy to continue the therapy. The observed symptoms include diarrhea, flatulence and nausea, similar to those observed for disaccharidases deficiencies such as congenital sucrase-isomaltase deficiency (CSID) and adult type of hypolactasia; suggesting that NB-DNJ interferes with the function of the intestinal disaccharidases. According to the molecular and functional characteristics of NB-DNJ, we proposed two mechanisms behind the appearance of these side effects. Initially, NB-DNJ might directly interact with the disaccharidases in the intestinal lumen and hamper their hydrolytic activity. This effect can then influence carbohydrate digestion transiently upon presence of NB-DNJ in the intestine and so is referred to as the “immediate side effect”. Secondly, NB-DNJ might influence cellular processes required for proper folding, maturation and trafficking of the disaccharidases, resulting to reduced functional efficiency of these proteins in the intestinal lumen. These consequences are denoted as “long term side effects”, since they will appear later on and will be persistent throughout the therapy. Immediate side effects of NB-DNJ Digestion of the various dietary carbohydrates in the gastrointestinal tract is accomplished by two types of hydrolytic activities: an alpha-glycosyl hydrolase activity from the majority of the intestinal glycosidases including salivary and pancreatic amylases, SI, MGA and trehalase; and a beta-glycosyl hydrolase activity exclusively performed by LPH (Naim and Zimmer 2008). In our experiment with disaccharidases prepared from human intestinal brush border membranes, low concentrations (1-10 µM) of NB-DNJ showed substantial inhibitory effects on the hydrolysis of the alpha-glycosidically linked sugars, but not the betaglycosidically linked one. In other words, sucrase, isomaltase and maltase activities were heavily suppressed by NB-DNJ, while lactase was only inhibited partially at very high concentration of this drug (≥250 µM). The selective inhibitory effect of NB-DNJ initially suggested its direct influence on the active sites of these proteins. Determination of the kinetics of inhibition for different concentrations of NB-DNJ revealed a mixed-type of inhibition on the tested alpha-glucosidases. With high analogy to glucose, NB-DNJ functions as a competitive inhibitor that according to its very low KI values can bind and occupy the active sites of sucrase, isomaltase and 73 Chapter 4: Discussion maltase with a much higher affinity than their original substrates (i.e. sucrose, isomaltase and maltose). In addition, the notable values of KI’ determined for these reactions indicate an additional uncompetitive inhibitory function for the NB-DNJ; that means after formation of the enzyme-substrate complexes, this compound can bind to an allosteric site on the enzyme structure and attenuate its catalytic activity. Interestingly, for sucrase, isomaltase and maltase activities, increasing NB-DNJ concentrations are associated with reduced contribution of the uncompetitive type in the overall inhibition. This is quite likely to occur due to the more intensive competitive inhibition at higher doses of NB-DNJ which reduced the amount of the original substrate-enzyme complexes that could be targeted by uncompetitive inhibition (Yon-Kahn and Hervé 2010). As an overall consequence of the immediate side effects of NB-DNJ administration, digestion of alpha-glycosidically linked carbohydrates including starch, glycogen and the oligo-/di-saccharides derived from partial break down of these complex sugars will be suppressed in the intestinal lumen. The maldigested carbohydrate then account for the majority of dietary sugars. However, lactase activity is not notably affected at this level and no lactose-intolerance as an immediate side effect of NBDNJ therapy is expected. Long term side effects NB-DNJ is absorbed in the intestine and transported to different parts of the body via blood circulation. The final concentration of this drug in the body fluids is estimated to remain below 50 µM. The internal milieu of the intestinal epithelial cells is exposed to NB-DNJ initially during intestinal uptake and later from the systemic circulation. Intestinal disaccharidases such as SI, MGA and LPH are membrane bound glycoproteins mostly located at the apical surface of the intestinal epithelial cells. These proteins are highly N- and O-glycosylated in the ER and Golgi and then mainly sorted to the apical surface through specific pathways that include selective incorporation of these enzymes into different membrane microdomains at the transGolgi network (Naim and Zimmer 2008). Because of its physiochemical properties, NB-DNJ has the potency to influence some cellular mechanisms other than the desired inhibitory function on the glucosylceramide synthase. Reduction of the cellular glycosphingolipid levels and 74 Chapter 4: Discussion inhibition of the ER alpha-glucosidases are examples of the contemporary effects of NB-DNJ (Butters et al 2000). The effects of prolonged incubation of NB-DNJ with the cell lines expressing SI and LPH can be readily observed at the early stages of protein glycosylation. Using biosynthetic labelling, we identified interference of NB-DNJ with the N-glycosylation of SI and LPH in the ER in a concentration dependant manner. This effect is likely arising from inhibition of the ER alpha-glucosidases I and II by this compound which prevents removal of the three glucose residues from one of the branches of the Nglycan. The altered glycoforms of LPH and to a lesser extent those of SI show ER retardation and therefore delayed maturation rates. Further experiments suggested that the more severe trafficking failure and the ER arrest of the LPH originate in its indispensable association with the ER chaperone calnexin. Since trimming of the two outermost glucose residues of the N-glycan is a requisite for the association of proteins with calnexin-calreticulin system (Ellgaard and Frickel 2003), the altered N-glycoform of LPH with the extra glucoses fails to associate with calnexin which is then arrested by the ER quality control machinery and finally degraded. However, in a similar assessment no association between normal or altered glycoforms of SI and calnexin was detected. This occasion enables the altered N-glycoform of the SI to successfully pass the ER quality control and be transported to the Golgi, although with a short delay. When the complex glycosylated SI and LPH are subjected to removal of N-glycans by PNGase F treatment, the size difference between the NB-DNJ treated and untreated samples is no more detectable, indicating that the O-glycosylation of SI and LPH in the Golgi (as one of the major post-translational modification for these proteins) is not affected by this compound. Although theoretically the affected branch of the N-glycan is expected to be cleaved by the action of the Golgi endomannosidase and obtain further processing in the normal manner, no such a corrective function was detected for SI and LPH in all of the tested cell lines (Caco-2, MDCK and CHO) and the observed size difference remained persistent throughout all maturation steps. Lack of Golgi endomannosidase in the tested cells according to 75 Chapter 4: Discussion its tissue-specific expression or possible inhibition of this enzyme by NB-DNJ are possible explanations for this observation. Previous studies have shown that in the intestinal epithelial cells apical targeting of the disaccharidases is achieved via association with specific type of membrane microdomains in the trans-Golgi network (Alfalah et al 1999). In the case of sucraseisomaltase, these membrane microdomains or lipid rafts are enriched in cholesterol and sphingolipids and can be isolated as a detergent insoluble membrane fraction by cold Triton-X100 solution (Jacob et al 2000; Jacob and Naim 2002). The process of selection and lipid raft incorporation of SI in the trans-Golgi network is suggested to be mediated by a class of carbohydrate binding proteins, or lectins, which recognize the sugar groups particularly O-glycans attached to the stalk region of this protein. In the meantime, NB-DNJ on one hand reduces the biosynthesis of glycosphingolipid which constitute one of the major ingredients of the lipid rafts, and interfere with the proper glycosylation of SI on the other hand. Therefore, incubation of the Caco-2 cells with NB-DNJ results to a partial decrease in the association levels of SI with the lipid rafts. The optimum functional efficiency of SI and LPH in the intestinal lumen is achieved upon a variety of requirements, including intact glycosylation and folding, normal cellular transport and proper association with their respective membrane microdomains (Jacob et al 2002; Wetzel et al 2009). Our data have clearly illustrated that NB-DNJ can interfere in part with all of the indicated processes in the cells. Therefore, in assessment of the residual activity of the SI and LPH molecules which are de novo synthesized in the presence of different concentrations of NB-DNJ, lowering functional efficiencies of these disaccharidases in association with increasing drug concentrations was observed. In the doses up to 50 µM of the drug which are speculated to represent the in vivo conditions, the main part of the activities and the cellular contents of SI and LPH are still present which should provide sufficient digestive activity of these enzyme in the intestine. However, the concerted action of these long term effects together with the immediate effects amplifies the overall effects of NB-DNJ on the digestive capacity of the disaccharidases. 76 Chapter 4: Discussion Meantime, in order to obtain a comprehensive insight towards the rationales of the NB-DNJ associated carbohydrate maldigestion in the intestine, possible contribution of the inborn defects in lowering the carbohydrate digestion capacity of the intestine – known as primary type of carbohydrate intolerance, see section 1.3 – should be also taken into account. Pre-existing intestinal conditions To our knowledge, the status of the intestinal disaccharidases in the metabolic disorders such as Gaucher- and NPC diseases has not been studied so far. In these disorders, since the lipid homeostasis is affected in the cells, targeting of the disaccharidases to the apical membrane and thus the optimum disaccharidase activity in the intestinal lumen might be readily influenced. Therefore, although NBDNJ therapy is transiently inhibiting intestinal glycosidases as an immediate effect and partially interferes with their functional efficiency as a long term consequence, it might be able to improve the speculated trafficking deficiency of these disaccharidases via re-balancing the lipid homeostasis in the intestinal cells. This positive influence might explain the observation that over a period of NB-DNJ therapy, the gastrointestinal intolerances are moderated in many of the patients. Here, as a primary consequence of the metabolic deficiency, the patients possibly have an impaired apical sorting of the disaccharidases and therefore reduced carbohydrate digestion capacity in the intestinal lumen. In the early onset of the NBDNJ therapy, combination of the inhibitory functions of the drug with the present conditions of the intestine results to a severe failure in carbohydrate digestion. In a while, improved apical trafficking of the disaccharidases as function of stabilised lipid homeostasis in the intestinal cells by NB-DNJ partially overrides the negative side effects of this drug and results to improved digestive capacity of and reduced carbohydrate intolerance in the intestine. Apart from the lipid homeostasis in the intestinal cells, the carbohydrate digestion capacity of the intestine might be also influenced by the inborn deficiencies of the intestinal glycosidases such as SI, MGA and LPH which already increase the risk of or readily cause gastrointestinal intolerances. SI with a broad substrate specificity is a central player in digestion of starch and many other alpha-glucosidically linked carbohydrates in the intestine. For that, 77 Chapter 4: Discussion congenital sucrase isomaltase deficiency (CSID) has been one of the most frequently diagnosed and intensively studied types of inborn disaccharidase deficiencies (Naim and Zimmer 2008). According to the genotypic attribution, the characterized cases of the CSID fall within one of the following groups: a) Homozygote cases with two similar abnormal alleles of SI, identified in up to 0.2 % individuals of the European descent, 3 % to 7 % of Canadian and Alaskan population and 5 % to 10 % of Greenlanders (Gudmandhoyer et al 1987; Treem 1995; Naim et al 2012). b) Heterozygote cases with a single abnormal allele of SI and activity levels below the normal ranges, detected with the frequency of 2 % to 9 % in European Americans (Peterson and Herber 1967; Welsh et al 1978). c) Compound heterozygote cases with two different abnormal alleles of SI, detected in a number of cohort analyses so far (Alfalah et al 2009; Uhrich et al 2012). Besides CSID, adult type of hypolactasia is another inborn intestinal disorder that is characterized by low levels of the lactase activity in the intestine after the weaning period. This disorder is estimated to occur in 95 % of the adults (Robayo-Torres and Nichols 2007). However since in adulthood lactase is neither an essential nor a prominent sugar of the daily diet, the symptoms and consequences of hypolactasia can be much conveniently avoided than CSID. The measured activity of the intestinal disaccharidases in the people recognized “healthy” for digesting of the daily ingested carbohydrates usually varies within a wide range of “normal levels”. This wide range is resulting from different expressional degrees of the disaccharidases and the general physiological conditions of the intestinal epithelium (Naim and Zimmer 2008). Some heterozygote individuals that carry a singles deficient allele for one of the disaccharidases might be also categorized to lie within the normal levels, since the residual activity of the healthy allele in these cases is sufficient to digest the normal amounts of the ingested carbohydrates with no obvious difficulties or symptoms. These individuals are estimated to constitute a notable proportion in different populations, but usually remain undiagnosed according to their digestive competency at normal situations. However, when the digestion capacity of the carbohydrates at stress situations is analysed, individuals at the lower ranges of the normal activity 78 Chapter 4: Discussion levels including the healthy heterozygotes for the intestinal glycosidases should be considered to be more susceptible to the gastrointestinal intolerances. Ingestion of high amounts of carbohydrates or functional inhibition of the disaccharidases by compound like NB-DNJ could be examples of these stress situations. Conclusions In essence, appearance of the carbohydrate intolerances in the patients upon chemotherapy with NB-DNJ (or other similar drugs) can be resolved according to two distinct mechanisms: I) emerging inhibitory functions of the drug on the catalytic activity of the intestinal glycosidases either via direct inhibition (immediate effect) or through interference with the cellular maturation and trafficking of these enzymes (long term effects). These effects resemble the secondary tape of carbohydrate intolerance. II) pre-existing conditions of the intestinal tissue that influence the carbohydrate digestion capacity in the intestinal lumen, including inborn metabolic deficiencies associated with altered cellular trafficking of the disaccharidases as well as genetic defects that result to decrease or loss of activity of these disaccharidases. It is therefore advisable that treatment of patients with NB-DNJ should be preceded by an assessment of the genetic status of major disaccharidases such as SI and LPH. In either case particular precautions can be taken, including treatment with SI or LPH substitutes, such as sucraid or bacterial ß-galactosidase preparation, and also by changing the dietary habits to low carbohydrate diets and including proper intervals between the drug uptakes and carbohydrate consumption. 79 Chapter 4: Discussion References Alfalah M, Jacob R, Preuss U, Zimmer K-P, Naim H, Naim HY (1999) O-linked glycans mediate apical sorting of human intestinal sucrase-isomaltase through association with lipid rafts. Current Biology 9(11): 593-596, S591-S592. Alfalah M, Keiser M, Leeb T, Zimmer KP, Naim HY (2009) Compound heterozygous mutations affect protein folding and function in patients with congenital sucrase-isomaltase deficiency. Gastroenterology 136(3): 883-892. 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Robayo-Torres CC, Nichols BL (2007) Molecular Differentiation of Congenital Lactase Deficiency from Adult-Type Hypolactasia. Nutrition Reviews 65(2): 95-98. Treem WR (1995) CONGENITAL SUCRASE-ISOMALTASE DEFICIENCY. Journal of Pediatric Gastroenterology and Nutrition 21(1): 1-14. Uhrich S, Wu Z, Huang J-Y, Scott CR (2012) Four Mutations in the SI Gene Are Responsible for the Majority of Clinical Symptoms of CSID. Journal of Pediatric Gastroenterology and Nutrition 55: S34-S35 10.1097/1001.mpg.0000421408.0000465257.b0000421405. Venier RE, Igdoura SA (2012) Miglustat as a therapeutic agent: prospects and caveats. Journal of Medical Genetics 49(9): 591-597. Welsh JD, Poley JR, Bhatia M, Stevenson DE (1978) INTESTINAL DISACCHARIDASE ACTIVITIES IN RELATION TO AGE, RACE, AND MUCOSAL DAMAGE. Gastroenterology 75(5): 847-855. Wetzel G, Heine M, Rohwedder A, Naim HY (2009) Impact of glycosylation and detergent-resistant membranes on the function of intestinal sucrase-isomaltase. Biological Chemistry 390(7): 545549. Yon-Kahn J, Hervé G (2010) Kinetics of Enzymatic Reactions with Michaelian Behaviour. Molecular and Cellular Enzymology: Springer Berlin Heidelberg, 103-191. 81 Summary Summary The role of N-butyl deoxynojirimycin in enzyme function, membrane association and targeting of brush border hydrolases Mahdi Amiri N-butyl deoxynojirimycin (NB-DNJ or Miglustat) is an oral medication for treatment of lysosomal storage diseases such as Gaucher disease type I and Niemann Pick disease type C. Gastrointestinal intolerances including diarrhea, flatulence and nausea are the most common adverse effects of NB-DNJ. These symptoms are proposed to be a consequence of suppressing the intestinal carbohydrate digestion by NB-DNJ. In similar conditions to disaccharidase deficiency disorders, the maldigested/unabsorbed carbohydrates raise osmotic influx of water and electrolytes into the intestine, and are later subjected to bacterial fermentation in the colon in association with production of irritative substances. In the current project, we have investigated the role of NB-DNJ in appearance of the gastrointestinal intolerances via two approaches: 1) Elucidation of the immediate effects of NB-DNJ on the enzymatic function of intestinal disaccharidases including sucrase-isomaltase (SI), lactase-phlorizin hydrolase (LPH) and maltase-glucoamylase (MGA). 2) Assessment of the persisting long term effects of NB-DNJ on the intestinal enzymatic function that could be elicited by alterations in the folding, intracellular processing, trafficking and targeting of the disaccharidases due to NB-DNJ. As an immediate effect, we identified that NB-DNJ can cause carbohydrate maldigestion in the intestinal lumen particularly via inhibition of the α-glucosidic activity of the intestinal disaccharidases like SI and MGA. The observed inhibition is a combination of competitive and uncompetitive forms. Lactase as a β-galactosidase is only partly affected at high non-physiological NB-DNJ concentrations, but the extent 82 Summary of inhibition is negligible and does not seem to remarkably diminish normal lactose digestion at this level. Regarding the long term consequences, NB-DNJ was found to interfere with the Nglycosylation of the disaccharidases in the ER, which leads to a delayed trafficking and maturation of LPH and to a lesser extent also SI. The variations in the glycosylation pattern because of NB-DNJ have direct effects on the folding of these proteins and subsequently on their enzymatic activities. NB-DNJ also inhibits the synthesis of glycosphingolipids, a major component of lipid rafts, which in turn results to reduced association of SI with these membrane microdomains. This effect can eventually affects an overall digestive function of SI, since on one hand the lipid rafts are important for increasing the SI activity and secondly, they are important for the correct targeting of SI to the cell surface. Thus, the de novo synthesized SI and LPH in the presence of NB-DNJ presented reduced functional efficiencies according to altered posttranslational processing of these proteins. However, at physiological concentrations of NB-DNJ (≤50 µM) a major part of the activity of these disaccharidases is found to be still preserved, which attributes the observed carbohydrate maldigestion mostly on the direct inhibition of disaccharidases in the intestinal lumen by NB-DNJ as the immediate side effect. In some patients, the immediate- and long term consequences of NB-DNJ might be concerted with pre-existing abnormalities in the intestinal tissue that affect the carbohydrate digestion capacity in the intestinal lumen in parallel, and thus result to an amplified occurrence of the intestinal manifestations. Inborn metabolic deficiencies associated with altered cellular trafficking of the disaccharidases as well as genetic defects that result to decreased or loss of activity of these disaccharidases are examples of these intestinal abnormalities. It is advisable that treatment of patients with NB-DNJ should be preceded by an assessment of the genetic status of major disaccharidases such as SI and LPH. In either case particular precautions can be taken, including treatment with SI or LPH substitutes, such as sucraid or bacterial ß-galactosidase preparation, and also by changing the dietary habits to low carbohydrate diets and including proper intervals between the drug uptakes and 83 carbohydrate consumption. Zusammenfassung Der Einfluss von N-Butyl-Deoxynojirimycin auf die Enzymfunktion, Membranassoziierung und Zielsteuerung von Hydrolasen der Bürstensaummembran Mahdi Amiri N-Butyl-Deoxynojirimycin (NB-DNJ) ist ein oral angewendetes Medikament zur Behandlung lysosomaler Speicherkrankheiten wie das Gaucher-Syndrom und Niemann-Pick Typ C. Gastrointestinale Unverträglichkeiten, zum Beispiel Durchfall, Blähungen und Übelkeit, sind die häufigsten Nebenwirkungen von NB-DNJ. Es wird vermutet, dass diese Symptome eine Folge der Suppression der intestinalen Kohlenhydratverdauung durch NB-DNJ sind. Ähnlich wie bei Erkrankungen mit Disaccharidase Defizienz, erhöhen die unverdauten und nicht absorbierten Kohlenhydrate den osmotischen Einstrom von Wasser und Elektrolyten in den Darm. Die Kohlenhydrate werden anschließend im Colon durch Bakterien fermentiert wobei die die Symptome auslösenden Substanzen entstehen. Im vorliegenden Projekt haben wir die Rolle von NB-DNJ als Auslöser der gastrointestinalen Intoleranzen über zwei Ansätze untersucht: 1) Aufklärung der unmittelbaren Effekte von NB-DNJ auf die enzymatische Funktion der intestinalen Disaccharidasen Sucrase-Isomaltase (SI), Laktase-Phlorizin- Hydrolase (LPH) und Maltase-Glucoamylase (MGA). 2) Analyse der chronischen Langzeiteffekte von NB-DNJ auf die intestinalen Enzymfunktionen, welche durch Veränderungen in der Proteinfaltung, intrazellulären Prozessierung, des Proteintransportes der und der Zielsteuerung der Disaccharidasen durch NB-DNJ hervorgerufen werden. Wir konnten zeigen, dass NB-DNJ, als unmittelbaren Effekt, die Kohlenhydratmaldigestion im intestinalen Lumen besonders durch Inhibition der α- Zusammenfassung glycosidischen Aktivität der intestinalen Disaccharidasen SI und MAG verursacht. Die beobachtete Hemmung ist eine Kombination aus kompetitiver und unkompetitiver Hemmung. Laktase, eine β-Galactosidase, wird nur teilweise bei hohen, nichtphysiologischen Konzentrationen von NB-DNJ beeinflusst. Zudem ist das Ausmaß der Laktaseinhibition vernachlässigbar und scheint die normale Laktoseverdauung nicht deutlich zu beeinträchtigen. Bezüglich der Langzeiteffekte konnten wir zeigen, dass NB-DNJ die N- Glykosylierung der Disaccharidasen im ER beeinträchtigt. Dies führt zu einem verzögerten Transport und einer verzögerten Reifung von LPH und in geringerem Ausmaß auch von SI. Die durch NB-DNJ hervorgerufenen Variationen im Glykosylierungsmuster haben einen direkten Einfluss auf die Faltung dieser Proteine und folglich auf deren enzymatische Aktivitäten. Des Weiteren inhibiert NB-DNJ die Synthese von Glycosphingolipiden, einem Hauptbestandteil von speziellen Lipid Rafts genannten Membranmikrodomänen, und führt so zu einer reduzierten Assoziierung von SI mit Lipid Rafts. Dieser Effekt könnte die generelle Funktion von SI in der Verdauung von Kohlenhydraten beeinflussen, da die Assoziierung von SI mit Lipid Rafts einerseits die Aktivität der SI erhöht und weiterhin für den korrekten Transport von SI an die Zelloberfläche benötigt wird. Dementsprechend besitzen in Gegenwart von ND-DNJ de novo synthetisiertes SI und LPH eine reduzierte funktionelle Effizienz, aufgrund einer veränderten posttranslationalen Prozessierung der Proteine. Trotzdem bleibt bei physiologischer Konzentration von NB-DNJ (≤ 50 µM) ein Hauptanteil der enzymatischen Aktivitäten dieser Disaccharidasen erhalten. Dies lässt darauf schließen, dass die beobachtete Kohlenhydratmaldigestion Disaccharidasen des hauptsächlich intestinalen auf Lumens die direkte durch NB-DNJ Hemmung als der unmittelbare Nebenwirkung zurückzuführen ist. In einige Patienten können die unmittelbaren Effekte und die Langzeiteffekte von NBDNJ von bereits existierenden Abnormalitäten des intestinalen Gewebes begleitet werden. Diese können die Kohlenhydratverdauung parallel beeinflussen und so die Ausprägung der intestinalen Symptome amplifizieren. Angeborene metabolische Defizienzen, welche mit einem veränderten zellulären Transport der Disaccharidasen assoziiert sind, sowie Gendefekte, welche mit einer Reduktion oder einem Verlust 85 Zusammenfassung der enzymatischen Aktivität der Disaccharidasen einhergehen sind Beispiele dieser intestinalen Abnormalitäten. Eine Untersuchung des genetischen Status der beiden Disaccharidasen SI und LPH vor der Behandlung von Patienten mit NB-DNJ ist anzuraten. In jedem Falle können spezielle Vorsorgen getroffen werden, wie die Behandlung mit SI und LPH Substituenten, zum Beispiel Sucraid und Präparaten der bakteriellen β- Galactosidase. Zudem können die Ernährungsgewohnheiten verändert werden. Eine Diät mit geringen Kohlenhydratanteilen sowie das Einhalten längerer Zeitintervalle zwischen der Einnahme von NB-DNJ und kohlenhydrathaltiger Kost sind zu empfehlen. 86 Affidavit I herewith declare that I autonomously carried out the PhD-thesis entitled “The role of N-butyl deoxynojirimycin in enzyme function, membrane association and targeting of brush border hydrolases”. No third party assistance has been used. I did not receive any assistance in return for payment by consulting agencies or any other person. No one received any kind of payment for direct or indirect assistance in correlation to the content of the submitted thesis. I conducted the project at the following institution: Institute of Physiological Chemistry, University of Veterinary Medicine Hannover, Hannover, Germany The thesis has not been submitted elsewhere for an exam, as thesis or for evaluation in a similar context. I hereby affirm the above statements to be complete and true to the best of my knowledge. _______________________________ 87 Acknowledgments First of all, I would like to give my special thanks to Prof. Dr. Hassan Y. Naim for his excellent guidance, encouragement and advice. He taught me a lot on the process of planning and performing the experiments, analysing and presenting the data and writing up scientific manuscripts. I have always enjoyed the fruitful scientific discussions with him. I would like to thank my advisors, Prof. Dr. Georg Herrler and Prof. Dr. Rita Gerardy-Schahn. I have benefited a lot from their guidance through this study and their helpful ideas, comments and discussions. Also, I would like to sincerely thank the German academic exchange service (DAAD) for awarding a PhD scholarship to me and the kind personals of section 445 for their non-stop support. I would like to thank all of the members of the Institute of Physiological Chemistry. The friendly and cooperative atmosphere made my times in the lab very much enjoyable. I would like to thank the members of the HGNI office and the international office of the University of Veterinary Medicine Hannover for their kind support and cooperation throughout my studies. I would like to thank Actelion Pharmaceuticals Ltd for partial support during this study. Last but not least, I would like to thank my parents and my wife Azam, for love, constant support and kind encouragements all through my life. 88