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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
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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.
Belmatoug N, Burlina A, Giraldo P, Hendriksz CJ, Kuter DJ, Mengel E, Pastores GM (2011)
Gastrointestinal disturbances and their management in miglustat-treated patients. J Inherit
Metab Dis 34: 991-1001.
Cox T, Lachmann R, Hollak C, Aerts J, van Weely S, Hrebicek M, Platt F, Butters T, Dwek R, Moyses
C, Gow I, Elstein D, Zimran A (2000) Novel oral treatment of Gaucher's disease with Nbutyldeoxynojirimycin (OGT 918) to decrease substrate biosynthesis. Lancet 355: 1481-1485.
Elstein D, Dweck A, Attias D, Hadas-Halpern I, Zevin S, Altarescu G, Aerts J, van Weely S, Zimran A
(2007) Oral maintenance clinical trial with miglustat for type I Gaucher disease: switch from or
combination with intravenous enzyme replacement. Blood 110: 2296-2301.
Hauri HP, Sterchi EE, Bienz D, Fransen JA, Marxer A (1985) Expression and intracellular transport of
microvillus membrane hydrolases in human intestinal epithelial cells. J Cell Biol 101: 838-851.
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
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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
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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