Download biochem ch 49 [2-9

BC chap 49
Intro
 Basic components of extracellular matrix – fibrous structural proteins
o Collagens
o Proteoglycans – contain long GAG chains attached to protein backbone covalently
 GAG chains contain repeating disaccharide units that usually contain a hexosamine and a uronic
acid (frequently sulfated)
 Synthesis starts with attachment of sugar to serine, threonine, or asparagine reside of protein –
additional sugars donated by UDP-sugar precursors and add sequentially to nonreducing end of
molecule
 Synthesized in ER and Golgi complex
 GAG chains are degraded by lysosomal enzymes that cleave one sugar at a time from
nonreducing end of chain
 Inability to degrade leads to mucopolysaccharidoses
o Adhesion proteins – link components of matrix to each other and to cells
 Fibronectin and laminin are examples
 Extracellular glycoproteins that contain separate distinct binding domains for proteoglycans,
collagen, and fibrin
 Contain specific binding domains for integrins, which bind to fibronectin on external surface,
span PM of cells, and adhere to proteins, which bind to intracellular actin filaments of
cytoskeleton
 Integrins provide a mechanism for signaling between cells via both internal signals and signals
generated via ECM
 Cell movement within ECM requires remodeling of various components of ECM – accomplished by matrix
metalloproteinases (MMPs) and regulators of MMPs (tissue inhibitors of matrix metalloproteinases (TIMPs))
o Dysregulation of balance of regulators of cell movement allows cancer cells to metastasize
Composition of Extracellular Matrix
 Fibrous proteins – collagen, elastin, and laminin
o Collagen – produced principally by fibroblasts, muscle cells, and epithelial cells
 Type I collagen is most abundant protein in mammals – about 33% glycine and 21% proline and
hydroxyproline
 Procollagen(I) – precursor of collagen(I) – triple helix of 3 pro-α chains twisted around each
other
 Polymerization of collagen(I) molecules forms collagen fibrils, which provide great tensile
strength to connective tissues
 3 polypeptide chains of triple helix linked by interchain hydrogen bonds
 Each turn of the triple helix contains 3 amino acid residues so that every 3rd amino acid (glycine)
is in close contact with other two strands in center of structure (this must be a glycine, which is
the only AA without a side chain) – amino acid before glycine is usually proline or
hydroxyproline
 Procollagen(I) requires extensive posttranslational modification
 Hydroxylation of proline and lysine requires vitamin C as cofactor of enzymes (prolyl
hydroxylase and lysyl hydroxylase)
 Hydroxyproline residues involved in hydrogen bond formation that helps stabilize triple
helix
 Hydroxylysine residues are sites of attachment of disaccharide moieties (galactoseglucose)
 Side chains of lysine residues may be oxidized to form aldehyde (allysine) – aldehyde
residues produce covalent cross-links between collagen molecules – allysine residue on
one collagen molecule reacts with amino group of lysine residue on another molecule,
forming covalent Schiff base that is converted to more stable covalent cross-links – aldol
condensation may also occur, forming lysinonorleucine
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o
In absence of vitamin C (scurvy), melting temperature of collagen drops dramatically because of
lack of interstrand H bond formation, which is caused by lack of hydroxyproline residues
 28 types of collagen and can be fibril-forming, network-forming, those that associate with fibril
surfaces, those that are transmembrane proteins, endostatin-forming, and those that form
periodic beaded filaments
 Can be composed of non-triple helical domains between triple helices
 Fibril-associated collagens with interrupted triple helices (FACITs) associate with fibrillary
collagens without themselves forming fibers
 Endostatin-forming collagens are cleaved at their C-terminus to form endostatin (inhibitor of
angiogenesis by inhibiting endothelial cell migration) – tumor growth dependent on blood
supply, so this could prevent tumor proliferation
 Network-forming collagens form a mesh-like structure because of large noncollagenous domains
at carboxy terminus
 Type XXV collagen has been associated with neuronal plaques that develop during Alzheimer’s
disease
 Type I, II, and III collagens form fibrils that assemble into large insoluble fibers – fibrils
strengthened through covalent cross-links between lysine residues on adjacent fibrils
 Types of collagen that do not form fibrils perform a series of distinct roles
 Fibril-associated collagens bind to surface of collagen fibrils and link them to other
matrix-forming components
 Transmembrane collagens form anchoring fibrils that link components of extracellular
matrix to underlying connective tissue
 Network-forming collagens form flexible collagen that is part of basement membrane
and basal lamina that surround many cells
 Collagen synthesized in ER as preprocollagen – presequence acts as signal sequence for protein
and is cleaved, forming procollagen within ER
 Procollagen is transported to Golgi apparatus – 3 procollagen molecules associate
through formation of interstrand and intrastrand disulfide bonds at carboxy terminus
 3 molecules align and form triple helix from carboxy end toward amino end, forming
tropocollagen, which contains a triple helical segment between 2 globular ends (amino
and carboxy terminal extensions)
 Tropocollagen is secreted from cell, extensions are removed using extracellular
proteases, and mature collagen takes its place in ECM
 Individual fibrils of collagen line up in highly ordered fashion to form collagen fiber
Elastin – major protein in elastic fibers – elastic fibers also contain microfibrils (composed of several
acidic glycoproteins, mostly fibrillin-1 and fibrillin-2
 Tropoelastin – precursor of elastin, highly soluble, and synthesized in rER for eventual secretion
 Contains 2 types of alternating domains
o Hydrophilic sequence rich in lysine and alanine residues
o Hydrophobic sequence rich in valine, proline, and glycine, frequently near
repeats of VPGVG or VGGVG
 Upon secretion from cell, tropoelastin is aligned with microfibrils, and lysyl oxidase
initiates reactions that cross-link elastin molecules, using lysine residues within
hydrophilic alternating domains
 2-4 lysine residues are cross-linked to form stable structure – net result is generation of
fibrous mesh that encircles cells
 Supravalvular aortic stenosis (SVAS) caused by insufficiency of elastin in vessel wall, leading to
narrowing of large elastic arteries – levels of elastin in vessel walls may regulate number of
smooth muscle cell rings that encircle the vessel (low levels of elastin cause smooth muscle
hypertrophy, leading to narrowing and stenosis of artery)
 When elastic fibers are stretched, amorphous elastin structure is stretched, exposing repeating
hydrophobic regions of molecule to aqueous environment, leading to a decrease in entropy of
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water because water molecules need to rearrange to form cages around the hydrophobic
domains
 When stretching force is removed, elastin takes on original structure because of
increase in entropy that occurs because water no longer needs to form cages about
hydrophobic domains
o Laminin – second most abundant protein in basal lamina (after type IV collagen) – provides additional
structural support for tissues through ability to bind to type IV collagen, other molecules present in
ECM, and cell surface-associated proteins
 Heterotrimeric protein composed of α, β, and γ-subunits
 Nomenclature for these is expressed as α1-5β1-3γ1-3
 Major feature is the α-helix, which joins the 3 subunits together and forms a rigid rod
 Only α-chain has significant carboxy-terminal extension past rod-like structure
 Laminin extensions allow laminin to bind to other components of ECM and provide stability for
the structure
 Laminin binds collagen, sulfated lipids, and proteoglycans in ECM
 Laminin synthesized with leader sequence that targets three chains to ER
 Chain association occurs in Golgi apparatus before secretion from the cell
 After laminin is secreted by cell, amino-terminal extensions promote self-association as
well as binding to other ECM components
 Disulfide linkages formed to stabilize trimer, but much less posttranslational processing
of laminin than there is of collagen and elastin
 Junctional epidermolysis bullosa (JEB) caused by defects in structures of laminin 5 or laminin 6 –
severe spontaneous blistering of skin and mucous membranes
 Congenital muscular dystrophy (CMD) results from defect in laminin 2, which is component of
bridge that links muscle cell cytoskeleton to ECM – lack of this bridge triggers muscle cell
apoptosis
ECM serves to keep cells from moving to other locations and prevent large molecules and particles, such as
microorganisms, from reaching contiguous and distant cells
o Infections spread, in part, because infections agent alters containing capacity of ECM
o Cancer cells metastasize by altering integrity of ECM
o Rheumatoid arthritis and osteoarthritis involve damage to functional capacity of ECM
o Alterations in structural characteristics of matrix of renal glomerulus may allow proteins to be excreted
into urine (indication of inexorable decline in renal function)
o Ehlers-Dahlos syndrome – caused by mutations that affect specific collagen genes
o Marfan syndrome – defect in fibrillin
o Deficiencies of lysosomal enzymes involved in normal degradation of molecules of matrix result in
diseases such as mucopolysaccharidoses
Proteoglycans – form gels around fibrous structural proteins – consist of repeating units of disaccharides (GAGs)
linked to core protein
o One sugar is either N-acetylglucosamine or N-acetylgalactosamine, and the other is usually acidic (either
glucuronic acid or iduronic acid) – sugars modified by addition of sulfate groups to parent sugar
o Negatively charged carboxylate and sulfate groups on proteoglycan bind positively charged ions and
form hydrogen bonds with trapped water molecules, creating a hydrated gel, which provides flexible
mechanical support for ECM and acts as a filter that allows diffusion of ions, water, and other small
molecules but slows diffusion of proteins and movement of cells
o Gel also acts as lubricant
o Hyaluronan is only GAG that occurs as single long polysaccharide chain and is only GAG not sulfated
o Proteoglycans found in interstitial connective tissues (synovial joints, vitreous humor of eye, arterial
walls, bone, cartilage, and cornea) – major components of ECM in these tissues – interact with collagen,
elastin, fibronectin, and laminin
o Because long, negatively charged GAG chains repel each other, proteoglycans occupy a very large space
and act as molecular sieves, determining which substances enter or leave cells
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Properties give resilience and a degree of flexibility to substances such as cartilage, permitting
compression and re-expansion of molecule to occur
Protein component of proteoglycans synthesized on ER, where it enters the ER lumen for initial
glycosylations
 UDP-sugars serve as precursors that add sugar units one at a time, first to protein and then to
nonreducing end of growing carbohydrate chain
 Glycosylation occurs initially in lumen of ER and subsequently in Golgi complex
 Glycosyltransferases (enzymes that add sugars to chain) are specific for sugar being added, type
of linkage formed, and sugars already present in chain
 Once initial sugars attached to protein, alternating action of 2 glycosyltransferases adds sugars
of repeating disaccharide to growing GAG chain
 Sulfation occurs after addition of the sugar
 2’-phosphoadenosine 5’-phosphosulfate (PAPS) – also called active sulfate – provides sulfate
groups
 Epimerase converts glucuronic acid residues to iduronic acid residues
 After synthesis, proteoglycans are secreted from cell, where they can form large aggregates,
noncovalently attached by a “link” protein to hyaluronic acid
Long polysaccharide chains of proteoglycans in cartilage contain many anionic groups, attracting cations
that create a high osmotic pressure within cartilage, drawing water into its connective tissue and placing
the collagen network under tension
 Resulting tension balances swelling pressure caused by proteoglycans
Lysosomes fuse with endocytotic vesicles, and lysosomal proteases digest protein component –
carbohydrate component degraded by lysosomal glycosidases
 Lysosomes contain both endoglycosidases and exoglycosidases
 Endoglycosidases cleave chains into shorter oligosaccharides
 Exoglycosidases – specific for each type of linkage – remove sugar residues one at a time
from nonreducing ends
 Deficiencies of lysosomal glycosidases cause partially degraded carbs from proteoglycans,
glycoproteins, and glycolipids to accumulate within membrane-enclosed vesicles inside cells
(residual bodies)
 Mucopolysaccharidoses caused by accumulation of partially degraded GAGs – may cause
deformities of skeleton and mental retardation
IOntegrins
Integrins
 Major cellular receptors for ECM proteins and provide link between internal cytoskeleton of cells (primarily actin
microfilament system) and extracellular proteins, such as fibronectin, collagen, and laminin
 Consist of an α and β-subunit – 24 different combinations discovered
 Integrins involved in wide variety of cell signaling options
 Certain integrins, such as those associated with WBCs, normally inactive because white cell must circulate freely
in bloodstream
o If infection occurs, cells located in area of infection release cytokines, which activate integrins on WBCs,
allowing them to bind to vascular endothelial cells (leukocyte adhesion) at site of infection
o Leukocyte adhesion deficiency (LAD) – genetic disorder that results from mutations in β2-integrin such
that leukocytes cannot be recruited to sites of infection
o Drugs can block either β2-integrin or α4-integrins (on lymphocytes) to treat inflammatory and
autoimmune disorders by interfering with WBC response to cytokines
 Can be activated by intracellular signaling events activating the molecule or by a binding event with extracellular
portion of molecule that initiates intracellular signaling events
 Integrins that bind cells to ECM components – activation of specific integrins can result in migration of affected
cell through ECM – operative during growth, during cellular differentiation, and in metastasis of malignant cells
to neighboring tissues
Movement of Tumor Cells

Metastasis through blood or lymph system and colonization of target tissue requires degradation of ECM to
allow for cell movement – accomplished by matrix metalloproteinases (MMPs)
 MMPs degrade specific ECM components (such as collagen or elastin) allowing cells access through this
compartment
 Gelatin zymography assay – assay for determining if MMPs are present in biological sample
o Polyacrylamide gels are prepared and enzyme samples are run through gel in presence of SDS
o After gel is run, enzyme activity is reconstituted by substituting Triton X-100 for SDS
o Assay buffer is placed over gel and left overnight
o If a lane on the gel contained MMP activity, MMP would be digesting gelatin in area of gel where MMP
resided
o After activity phase is complete, gel is developed with Coomassie stain, which binds to proteins in gel,
including gelatin – positive result would appear as white bands on blue background
 Fluorescence resonance energy transfer (FRET) – more sensitive assay than gelatin zymography assay
o Uses peptide substrate that contains both fluorophore and a quencher in close proximity – when
excited, quencher blocks fluorescence emittance from fluorophore because of close proximity on
peptide
o After MMP treatment, peptide is cleaved between fluorophore and quencher, such that quencher is no
longer in close proximity, resulting in strong fluorescence emittance – intensity will increase as peptide
is cleaved in presence of MMP
Adhesion Proteins
 Found in ECM and link integrins to ECM components
 Large multidomain proteins that allow binding to many different components simultaneously
 Fibronectin has binding sites for integrins, collagen, and GAGs
o Alternative splicing allows many different forms of adhesion protein to be expressed, including soluble
form (versus cell-associated forms) found in plasma
 As integrin molecule is bound to intracellular cytoskeletal proteins, adhesion proteins provide a bridge between
actin cytoskeleton of cell and cells’ position within ECM
 Loss of adhesion protein capability can lead to physiologic or abnormal cell movement
 Lost when tumor cells develop (many tumor cells secrete less than normal amounts of adhesion protein
material, which allows for more movement within ECM) – this increases potential for tumor cells to metastasize
Matrix Metalloproteinases
 Zinc-containing proteases that use zinc to appropriately position water to participate in proteolytic reaction
 Cleave all proteins found in ECM, including collagen and laminin
 Important to allow cell migration and tissue remodeling during growth and differentiation
 Many growth factors bind to ECM components and, as bound components, do not exhibit their normal growthproducing activity
o Destruction of ECM by MMPs releases these growth factors, allowing them to bind to cell surface
receptors to initiate growth of tissues
 Cancer cells that metastasize require extensive ECM remodeling and usually use MMP activity to spread
throughout body
 Propeptide present in newly synthesized MMPs contains critical cysteine residue which binds to zinc atom at
active site of protease and prevents propeptide from exhibiting proteolytic activity
o Removal of propeptide required to activate MMPs
o Once activated, certain MMPs can activate other forms of MMP
 Regulation of MMPs include transcriptional regulation, proteolytic activation, inhibition by circulating protein α2macroglobulin, and regulation by tissue inhibitors of metalloproteinases (TIMPs)
o Synthesis of TIMPs and MMPs must be coordinately regulated because dissociation of their expression
can facilitate various clinical disorders, such as certain forms of cancer and atherosclerosis
Osteogenesis Imperfecta
 Defect in collagen production – 2 types
o Reduction in synthesis of normal collagen (resulting from genetic problem)
o
Synthesis of mutated form of collagen – most mutations have dominant-negative effect, leading to
autosomal dominant mode of transmission
 Many of known mutations involve substitutions of another amino acid for glycine, resulting in unstable collagen
molecule because glycine is only amino acid that can fit between other 2 chains in triple helix of collagen
o If mutation is near carboxy-terminal end, phenotype of disease is usually more severe than if mutation is
near amino-terminal end
o Mutations that replace glycine with either serine or cysteine are more stable because of hydrogen
bonding capabilities of serine and ability of cysteine to form disulfide bonds, preventing the strand from
unwinding
 Can be treated with bisphosphonates, which consist of 2 phosphates linked by carbon or nitrogen bridge
(analogs of pyrophosphate where 2 phosphates are linked by oxygen)
 In OI, bone resorption outpaces bone formation because osteoclast activity is enhanced (reduced levels of
normal collagen present to act as nucleating sites for bone formation)
 Bisphosphonates inhibit osteoclast action with potential to increase bone mass and its tensile strength
 Can occur due to mutations in genes other than collagen
o Mutations in CRTAP or LEPRE1 lead to defective collagen fibers being produced
o CRTAP forms complex with PH3-1 and cyclophilin to hydroxylate specific proline residue in types I and II
collagen – failure to hydroxylate this proline residue leads to unstable collagen and moderate to severe
forms of OI
o Pattern of inheritance for these is autosomal recessive
Other Diseases
 Lupus – alterations in cell matrix components caused by autoimmune induced trigger
 Type 2 diabetes – cell matrix interactions can be altered because of elevated glucose levels and nonenzymatic
glycosylation