COLLAGEN METABOLISM AND DISORDERS

OUT LINES

➢ Introduction

➢ Structure Of Collagen

➢ Types Of Collagen

➢ Collagen Metabolism

➢Collagen Disorders
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INTRODUCTION
▪ Collagen is
▪ the most abundant protein in mammals

▪ accounts for about 1/3 of the total protein content.

▪ It is the main fibrous structural component in connective tissues such as

✓ bone, cartilage, ligament, Muscles and skin, Tendons.

▪ accounts for three-quarters of the dry weight of the skin

▪ It is the most prevalent component of ECM

▪ Miller and Matukas discovered collagen in 1969, since then more than
28 collagen types have been identified.
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• The ECM contains three major classes of biomolecules:

1. Structural proteins - e.g. collagen, elastin, and fibrillin
2. Specialized proteins
• fibronectin, integrins and laminin
• form a mesh of fibers that are embedded in proteoglycans.
3. Proteoglycans
• Functions of ECM
• critical for connecting cells together to form tissues,
• guides cell migration during embryonic development and wound healing
• responsible for the relay of environmental signals to the surfaces of
individual cells
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• Collagen is produced by a variety of cells types but principally by

fibroblasts, muscle cells and epithelial cells

• Functions of collagen
• It acts as a tissue scaffolding e.g. in basement membranes,

• provide tensile strength: have a rigid rod-like structure that resists stretching

• facilitate cell adhesion,

• cell migration and protection and have roles in tissue repair.

• When collagen is heated in water, it gradually gets broken down

✓produces a soluble derived protein called gelatin or animal glue

 Structure of Collagen 6

• All collagens are trimeric proteins - composed of 3 polypeptide

α-chains coiled around one another to form tripe helix.
• The α chains are left handed helices that wrap around each
other into a right handed rope like triple helical rod.
• Interchain hydrogen bond – stabilizes the triple helix

Figure 1: Collagen triple helix

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• There are around 3 amino acids per turn.
• Each α-chain is composed of Gly-X-Y repeats, where X and Y being
frequently proline and hydroxyproline respectively.

• Glycine

✓ is the smallest of all amino acids

✓occupies every 3rd position in the repeating amino acid sequence

✓35% of a collagen monomer is composed of glycine

✓its frequent occurrence in the collagen fibril is essential for the

tight packing of the helical structure.

✓It is the only amino acid small enough to fit into the limited
spaces where the three chains of the helix come together.
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• Proline
• The X positions are often proline & the Y positions are
often hydroxyproline
• accounts for another 20–25%
• Proline and hydroxyproline in the α chains are imino
acids with a rigid cyclical structure.
• facilitates the formation of the helical conformation of
each α-chain because its ring structure causes"kinks"in
the peptide chain.
• Proline and hydroxyproline confer rigidity on the collagen
molecule.
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• Hydroxylation reactions -
• produce hydroxyproline from proline and hydroxyl lysine from lysine
• occur after the protein has been synthesized
• Enzymes involved are prolyl hydroxylase & lysyl hydroxylase
• require vitamin C as coenzyme
• Hydroxyproline residues
• involved in H.bond formation that helps to stabilize the triple helix.
• Hydroxylysine residues –
• Act as a site of attachment of disaccharides (glycosylation)
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Figure 2. Hydroxylation of proline and lysine residue in collagen

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• In the α chain of type I collagen there are

✓338 Gly – X – Y triplets repeated in a sequence and
✓ additional 32 aas flank the long triplet sequence at each end → known
as telopeptides.
✓There are both an amino- and a carboxy terminal telopeptide.

• Stabilization of the triple helix is achieved by

✓the presence of glycine as every third residue
✓a high content of proline and hydroxyproline
✓ Hydroxyproline maximizes formation of inter-chain hydrogen bonds that
stabilize the triple-helical structure
✓ Inter-chain hydrogen bonds
✓ Electrostatic interactions involving lysine and aspartate
 Types Of Collagen 12

• Based on the structure collagens form, they can be classified as

1. Fibril forming (type I, II, III, V and XI)-have the rope-like structure
▪ Type I-is by far the most abundant, accounts 90% of all collagen
in human body. Predominant collagen type in bone and tendon.
▪ Type II – most abundant in cartilages
▪ Type III – predominantly found in extensible/distensible tissues
2. Network forming (Type IV, VIII, X)
• form a 3-D sheet or mesh, but no fibrils
• type IV – is most abundant in basement membranes
3. Fibril associated collagens with interrupted triple helices (FACIT)
• Includes type IX, XII, XIV, XVI…
• Bind to the surface of collagen fibrils, linking them to one another and to other
components of ECM
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4. Beaded filaments (type VI, XXVI)

• consist of long chains of collagen molecules which have a regular
beaded appearance
5. Transmembrane collagens
• have short intracellular N-terminal domains and extracellular domains
with long interrupted triple helices
6. Anchoring fibrils
• collagen VII forms the main part of in epithelial tissues;
7. Multiplexins
• are collagens with multiple triple helix domains and interruptions.
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Figure 3 :Classification of collagen according to the structures they form

FACITs - fibril associated collagens with interrupted triple helices
Multiplexin - multiple triple helix domains and interruptions
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Biosynthesis Of Collagen
• Collagen is synthesized in ribosomes in a precursor form known as
preprocollagen
• These precursor molecules are synthesized in fibroblasts:
• Osteoblasts in bone and
• Chondroblasts in cartilage
• The biosynthesis involves both intracellular & extracellular processes
• The entire process of collagen biosynthesis involves
✓ Intracellular collagen biosynthetic events
✓Transcription
✓Translation
✓post translational events
✓ Extracellular collagen biosynthetic events
✓ Regulation of synthesis
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Order & location of processing of fibrillar collagen precursor
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SYNTHESIS AND SECRETION OF COLLAGEN
• General Steps
1. Formation of pro-α-chains
2. Hydroxylation
3. Glycosylation
4. Assembly and secretion
5. Extracellular cleavage of
procollagen
6. Collagen fibril formation
7. Covalent-cross link formation
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1. Formation of pro-α chains

• The newly synthesized polypeptide precursors of α chains (prepro-α

chains) contain a special amino acid sequence at their N-terminal
ends known as leader or signal sequence (SS)

✓facilitates the binding of ribosomes to rER,

✓directs the passage of prepro-α chain into the lumen of rER.

✓The SS is rapidly cleaved in rER by signal peptidase to yield

pro-α chain
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2. Hydroxylation:
• pro-α chains are processed by a number of enzymatic steps within
the lumen of rER while the polypeptides are still being synthesized.
• Proline and lysine residues found in the Y-position of the –Gly–X–
Y–sequence can be hydroxylated → Hydroxyproline &
hydroxylysine residues
• Catalyzed by prolyl hydroxylase and lysyl hydroxylase
• Require molecular oxygen, Fe2+, and the reducing agent vitamin C
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3. Glycosylation
• Incorporation of carbohydrates(glucose and galactose ) to
some hydroxylysine residues of pro-α-chains
• Initiated by galactosyltransferase, and glycosyl transferase
catalyzes further addition of glucose.
• Occurs before triple helix formation in rER
• The extent of collagen glycosylation probably depends on
• the speed of collagen folding
• the expression levels of glycosyltransferases
• availability of UDP-Galalctose in rER
• Types of collagen:
• type IV is more extensively glycosylated
• thus, glycosylation is more prominent in basement membrane
collagens than in fibrillar collagens
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4. Assembly and secretion
• After hydroxylation and glycosylation, three pro-α chains form
procollagen - a precursor of collagen
✓has a central region of triple helix flanked by two globular ends,
non-helical N- & C-terminal extensions called propeptides
• The formation of procollagen begins with formation of interchain
disulfide bonds between the C-terminal extensions of pro-α chains.
✓ This brings the three α chains into an alignment favorable for
helix formation
✓The formation is from carboxyl end to amino terminal end
• The procollagen molecules move through the Golgi apparatus, where
they are packaged in secretory vesicles.
✓The vesicles fuse with the cell membrane → release of
procollagen molecules into the extracellular space
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5. Extracellular cleavage of procollagen molecules

• After their release, the procollagen molecules are cleaved by

extracellular proteases (amino-and carboxy-procollagen peptidases)

✓ remove the terminal propeptides→ releasing triple-helical

collagen molecules called mature collagen/ tropocollagen

✓ Tropocollagen
✓a single triple helical collagen molecule (monomeric)

✓ basic structural unit of collagen fibrils

✓ Mature collagen takes its place within the ECM.

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6. Formation of collagen fibrils

• Individual tropocollagen molecules spontaneously associate to form
collagen fibrils.
• They form an ordered, overlapping, parallel array, with adjacent
collagen molecules arranged in a staggered pattern, each overlapping
its neighbor by a length approximately three-quarters of a molecule
7. Covalent cross-link formation
✓Finally, the formation of the structural cross-linking occurs.
✓ Cross-links are formed by Lysyl oxidase (via oxidative deamination)
• Lysyl oxidase aka protein-lysine 6-oxidase
✓ is an extracellular Cu2+-dependent enzyme
✓ Menkes disease
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✓ Oxidatively deaminates some of the lysine &hydroxylysine residues

in collagen → reactive aldehydes (allysine & hydroxyallysine)

o can condense with lysyl or hydroxylysyl residues in neighboring

collagen molecules to form covalent cross-links and thus,
mature collagen fibers

✓ essential for the stability of collagen fibers

✓ Covalent cross-links both between and within the tropocollagen

molecules, confering strength and rigidity on the collagen fiber

✓ Aging vs covalent-cross linking ???

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• Beta-amino propionitrile
• A toxin found in plants of the genus lathyrus
• An irreversible inhibitor of Lysyl oxidase
• Cause Lathyrism –
• disease condition which results from excessive consumption of
Lathyrus Sativus/white pea/vetch
• Characterized by spastic paralysis of limbs
• results in defective collagen due to the lack of cross-links
• Types
✓Osteolathyrism
✓Angiolathyrism
✓Neurolathyrism
Summary on collagen synthesis 27
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Figure : formation of collagen fibers

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Degradation of collagen
• Normal collagens are highly stable molecules
✓ having half-lives as long as several years.
• Its breakdown is increased during starvation and various
inflammatory states.
• Breakdown of collagen fibers is dependent on the proteolytic
action of collagenases
✓It’s part of a large family of matrix
metalloproteinases/MMP
• Two mechanisms for the degradation of collagen
1. Intracellular degradative pathway – collagenase independent
2. Extracellular degradative pathway – collagenase dependent
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1. INTRACELLULAR PATHWAY
Steps
▪ Recognition of collagen fibers
▪ Cleavage of fibrils
▪ Phagocytosis of cleaved fibrils
▪ Formation of phagolysosome
▪ Intracellular digestion by
lysosomal enzyme like
Cathepsin
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2. EXTRACELLULAR PATHWAY
• It is achieved by Matrix metalloproteinases (MMP) secreted by
fibroblasts and other cells
• The MMP family is a group of structurally related proteolytic
enzymes that share several conserved protein features like
• a catalytic domain and a C-terminal hemopexin domain
• Degrade collagen and other matrix molecules into smaller peptides
• 23 identified members of MMP so far
• type I and III collagens
• preferentially cleaved by MMP-1(Collagenase-1)
• type II collagen
• the preferential cleaved by MMP-13 (Collagenase-3).
• Other MMPs, such as MMP-3 and MMP-10, are able to degrade
only collagen type III, and not collagen type I or II
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Collagen disorders: Collagenopathies

• Defects in any one of the many steps in collagen fiber synthesis can result
in a genetic disease involving

✓inability of collagen to form fibers properly

✓ inability to provide tissues tensile strength provided by collagen.

✓Collagen containing mutant chains is not secreted, and is either

degraded or accumulated to high level in intracellular compartments.

• Without the structural support of collagen, blood vessel, tendon and skin
become fragile.
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1. Osteogenesis Imperfecta (brittle bone disease)

• result from certain mutations in gene encoding collagen type I chains.
• Eg., mutation of glycine to almost any other amino acid is
deleterious, disrupting the triple-helical structure and function of
the whole molecule.
2. Alport’s syndrome
• Mutation in the C-terminal globular domain of type IV chains
• This type of collagen is found in the glomerular basement membrane of
kidney and are associated with
• progressive renal failure
• sensorineural hearing loss
• ocular abnormalities
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3. Good pasture’s syndrome

• A relatively rare autoimmune disease, where
✓ the autoantibodies bind to type IV collagen that is
found in glomerular basement membrane and lungs,
✓ results in renal failure and pulmonary hemorrhage
4. Bruck Syndrome
• mutation in the lysyl hydroxylase gene.
• hydroxylation of lysine residue in type I collagen can not occur
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5. Ehlers-Danlos syndrome
• Mutation in the amino acid sequence of collagen type I, III and V.
• Clinically important mutation are found in Type III.
• Marked by extremely loose joints, and hyperelastic skin that bruises
easily, and easily damaged blood vessels.

Figure . Stretchy skin of Ehlers-Danlos syndrome

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6. Scurvy
• Post translation modifications of pro-α chain are crucial for
formation of mature collagen & their assembly into fibrils.
• vitamin C is an essential cofactor for hydroxylases responsible for
adding hydroxyl groups to proline and lysine residues in pro α-chain
• Defects in this modification have serious consequences.
• Vitamin C deficiency (Scurvy)
• lack of proline & lysine hydroxylation
• the pro-α-chains are not hydroxylated
• interchain H-bond formation is impaired
• the formation of a stable triple helix finally impaired
• often show bruises on the limbs/gum as a result of subcutaneous extravasation
(leakage) of blood due to capillary fragility
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