A comprehensive presentation on Biochemical and clinical aspects of Collagen for MBBS, BDS, B Pharm & Biotechnology students to facilitate self- study.

1. BiochemicalandclinicalaspectsofCollagen
Dr. Rohini C Sane
Epidermolysis bullosa
Scurvy
Ehlers-Danlos syndrome

2. Biochemical aspects of Collagen
❖Biochemical aspects of Collagen:
1. Most abundant fibrous protein(major macromolecules) in human body :70 kg
body weight→ 12-14 kg of total protein→5kg of Collagen (1/3 of total protein)
2. Main Component of : connective tissue, skin(70%) ,bone (90%) tendon(85%),
cartilage ,teeth and liver(4%)
3. Synthesized by fibroblast in connective tissue and osteoblasts in bone
4. Made up of small fibrils → tropocollagen( fundamental units ) containing 3
polypeptide chains each of them in left-handed helix with 3 amino acid per turn.
5. rich in Glycine and rare amino acids like hydroxyproline, hydroxylysine
6. Cysteine and Tryptophan absent
7. have a triple helical secondary structure and rich in helix destabilizing amino
acids (Glycine ,Proline and Hydroxyproline). These amino acids prevent the
formation of the usual - helical and - pleated structure. Instead it forms a triple
helical secondary structure.

3. Triple stranded helix Structure of Collagen
• Collagenhas3 polypeptidechainswoundarounditself. Eachpolypeptidechainsubunitis
calledalpha-chains. EachofAlpha-chain is twistedintoleft-handedhelix ofthreeresidues
perturncomparedwith 3.6forright handedalpha-helix.Threeoftheseleft– handedhelices
arethenwoundtoright-handedsuperhelixtoformastiffrodlike molecule(Triplehelical
secondarystructure).
• Itisrich in helixdestabilizingaminoacids(Glycine ,ProlineandHydroxyproline).Theseamino
acids preventtheformationoftheusual- helicaland-pleatedstructure.Instead,it formsa
triplehelicalsecondarystructure.
• Every3rdresidueis Glycineandtheonlyaminoacidthatcanfitintothetriplestrandedhelix.
• QuarterstaggeredtriplestrandedhelixofCollagenis stabilizedbythestericrepulsionof
ringshydroxyproline andhydrogenbondsbetweenthem.
• Triplehelicalsecondarystructureimpartsthetensilestrengthofsteeltocollagen(has
unusualstrength)
❖Types arrangementofcollagenfibril:
a. Parallelbundles:in tendons,cartilage
b. Sheets:layered atmanyanglesin skin

4. Arrangements of collagen fibers in cartilage of bone

5. Types of Collagen
❖19 different Types of Collagen , composed of 30 distinct polypeptide
chains encoded by separate genes.
❖ Numbering for Types of Collagen: Roman numerals I, II, III….XIX
❖Structure of collagen types : in principle , all types of collagen are
triple helical structures . The triple helix may occur throughout the
molecule or only a part of the molecule.
❖Each one suited to performed specialized function in tissue
❖e.g. Collagen Type I →skin, Collagen Type II → bone

6. Structure of collagen Type 1
❖ Structure of collagen Type 1:
1. Triple stranded helical structure present throughout the collagen
molecule
2. Shape : rod-like molecule → 1.4 nm diameter and 300 nm length
3. Number of Amino acid residues : 1000 per for each polypeptide
chain (3000 /molecule)
4. Amino acid contribution : 1/3 rd of amino acids are Glycine (every
third amino acid in collagen is Glycine.
5. Repetitive amino acid sequence : (Gly – X –Y )n ,where X and Y
represent other amino acids
6. Proline and hydroxyproline : 100 per for each polypeptide chain
7. Function of Proline and hydroxyproline : confer rigidity to the
collagen molecule
8. Collagen Fibril formation : Triple helical molecule of collagen
assemble to form elongated fibrils . It occurs by a quarter staggered
alignment i.e. each triple helix is displaced longitudinally from its
neighbor collagen molecule by about one-quarter of its length
9. Collagen Fiber formation : Collagen Fibrils assemble to form rod like
fibers .
10. Strength of Collagen Fiber : contributed by covalent cross linking of
formed between Lysine and hydroxylysine and also between Proline
and hydroxyproline.

7. Collagen molecules in Collagen fibers
Triple helical molecule of
collagen assemble to
form elongated fibrils .
Triple stranded helical structure
present throughout the collagen
molecule
Collagen Fibrils
assemble to form
rod like fibers .
Repetitive amino acid sequence (Gly – X –Y )n
Proline and hydroxyproline confer rigidity to the collagen molecule

8. Arrangement of Tropocollagen molecules in collagen fibril
Heads of Tropocollagen molecules 64 nm Cross striations
Sections of Tropocollagen moleculeCollagen Fibril formation :
Triple helical molecule of collagen
assemble to form elongated fibrils .
It occurs by a quarter staggered
alignment i.e. each triple helix
is displaced longitudinally from
its neighbor collagen molecule
by about one-quarter of its length.

9. Tropocollagen molecule
❖Tropocollagen : Subunits of Collagen
• Shape : rod shaped
• Length : 300nm
• Thickness : 1.4 nm
• Molecular weight : 300,000
• Constituent polypeptides: three helically interwind polypeptides
of equal length (each with 1000 amino acid residues)
• Primary structure of collagen : all 3 or two out of three chains
have identical in amino acid sequence. Rich in Glycine (35%)and
Alanine(11%) , Gly-Pro-X or Gly-Hpr-X or
• Repetitive amino acid sequence : (Gly – X –Y )n ,where X and Y
represent other amino acids
• Secondary structure of collagen : Each of three polypeptide
chains of tropocollagen is itself -helix. Proline and
hydroxyproline form bends in polypeptide chains that they are
not compatible with -helix structure.

10. Collagen fibrils
❖Collagen fibril :
• Triple helical molecules are associated into Collagen fibrils.
• It consists of recurring polypeptide subunits called tropocollagen, arranged
head to tail in parallel bundles . The heads of the tropocollagen molecules are
staggered along the length of fibers ,accounting for the characteristic 64 nm
spacing of the cross striations in most collagens .
• A section of tropocollagen molecule shows the backbone of triple helix . Each
of three polypeptide chains of tropocollagen is itself -helix whose pitch and
spacing is determined by the rigid R group of the numerous Proline and
hydroxyproline residue .
• The gap between the end of one triple helix and the beginning of the next
where there is the deposition of hydroxyapatite crystals in bone formation.

11. Constituent amino acids of triple stranded helix Structure of Collagen
• ScvConstituent amino
acids of Collagen
% of total amino acids
Glycine 33
Proline and hydroxy
proline
21
Lysine and hydroxy
Lysine
3
Alanine 11
Arginine 5
Cysteine and
Tryptophan
absent
Scurvy:vitaminCdeficiency→failure ofhydroxylationofProlineandLysineleadstoreducedhydrogen
bonding→weaknessofcollagen→Brittlebonedisease:mutation→replacementofcentralGlycine

12. Triple helical secondary structure of Collagen

13. Forces stabilizing Triple helical secondary structure of Collagen
❖Forces stabilizing Triple helical secondary structure of Collagen:
1. Hydrogen bonds : three left handed helices are bound together by
interchain hydrogen bonds.
2. Lysinonorleucine bond: covalent cross links both within and
between triple helical units further stabilize Collagen fibers.
3. Electrostatic interactions
4. Hydrophobic interactions

14. Covalent cross-links in Collagen fibers
• Strength of Collagen Fiber : contributed by covalent cross linking formed
between Lysine and hydroxylysine and also between Proline and
hydroxyproline.
• Covalent cross links are formed both within and between triple helical units
further stabilize Collagen fibers.
• The degree of covalent cross-linking in Collagen molecule increases with age .
• In Elder individuals : skin, blood vessels (Collagen containing tissue) become
less elastic and more stiff → health complications

15. Skin :Collagen containing tissue
In Elder individuals , skin, blood vessels (Collagen containing tissue) become less elastic and
more stiff → health complications

16. Collagen and calcific aortic valve stenosis(CAVS)

17. Biosynthesis of collagen
❖Biosynthesis of collagen: collagen is an extracellular protein but synthesized as an
intracellular precursor molecule before becoming a mature collagen fibril.
• Site : fibroblast ,osteoblasts in bones , chondroblasts in cartilage, odontoblasts in teeth
• Cellular location : ribosomes in endoplasmic reticulum (ER)
• Precursor : preprocollagen (a single polypeptide chain) with leader peptide at amino
terminal 20000 MW and carboxy terminal 35000MW.Both are not present in mature
collagen.
• Function of preprocollagen: contains a signal peptide which directs the protein to each
endoplasmic reticulum (ER)
• Synthesis of procollagen : from preprocollagen in (ER) after cleavage of a signal peptide
• Post transcriptional modification of procollagen : hydroxylation, glycosylation and
disulfide formation . Followed by its secretion in extracellular medium by the way of Golgi
complex .
• Synthesis of collagen in extracellular medium : from preprocollagen after action of
aminopeptidase and carboxypeptidase to remove terminal amino acids. This followed by
a spontaneous assembly of polypeptide chains to form triple helical structure (with 1000
amino acids each) of collagen .

18. Synthesis of Collagen

19. Structural modification of Collagen during its Synthesis
Procollagen
Tropocollagen
Collagen
Glycosylationloss of peptide potion from N-terminal and C-terminal
Each of the 3 chains is in a left handed
helix with 3 amino acids per turn.
3 Chains are further twisted in right handed way to give
cable like structure.
Hydroxylation of Proline and Lysine by Lysyl hydroxylase
and Proline hydroxylase in presence of vitamin C→
Cross linking of hydroxy proline and hydroxy lysine
Since vitamin C is required for collagen synthesis ,a connective tissue , there is a delay in
wound healing process in vitamin C deficiency.

20. Functions of Collagen
❖Functions of Collagen : triple helical molecules are associated into fibrils. There is
gap between the end of one triple helix and the beginning of the next where there
is deposition of hydroxyapatite crystals in bone formation.
1. Gives tensile strength, support and shape to tissue . To break a collagen fiber of 1
mm in diameter, a load of 10-40 kg is needed. In disease status tensile strength is
reduced.
2. Contributes to proper alignment of cells ,which in turn help in cell proliferation
and their differentiation to different tissue and organs .
3. Collagen which is exposed in blood vessels contributes to thrombus formation.
❖Collagen can be converted to
a. gelatin by boiling by splitting off some amino acids .Gelatin is highly soluble and
easily digestible. It forms gel on cooling and is provided as diet for convalescents
and invalids. But it lacks essential amino acid Tryptophan.
b. a tough hard substance on treatment with tannic acid (tannic process)

21. Genetic aspects of Collagen Synthesis
❖Genetic aspects of Collagen Synthesis :
1. Complex process
2. Involves at least 30 genes in human
3. about 8 post –transcriptional modifications
4. Inherited diseases due to gene mutations linked with collagen synthesis:
a. Ehlers-Danlos syndrome
b. Alport syndrome
c. Osteogenesis imperfecta
d. Epidermolysis bullosa

22. Abnormalities associated with collagen synthesis
Disease Abnormalities associated with collagen synthesis
Ehlers-Danlos
syndrome
Inherited disorders characterized by hyperextensibility of skin and
abnormal tissue fragility
Alport syndrome Defect in formation of type IV collagen fibers found in the basement
membrane of renal glomeruli→ hematuria and renal disease
Osteogenesis
imperfecta
Characterized by abnormal bone fragility due to deceased synthesis of
collagen
Epidermolysis bullosa due to alteration in in the structure of type VII collagen fibers→ skin
breaks and blister formation even with minor trauma
Scurvy Deficiency of vitamin C→ defective post translational modification of
collagen→ bleeding gums ,poor wound healing, subcutaneous
hemorrhage
Lathyrism (disease of
bone deformities )
CausedbyconsumptionofLathyrussativa(kesaridal)containingtoxiccompound
BetaOxalylAminoAlanine(BOAA).BOAAinhibitsenzymeLysyloxidaseand
interfereswiththecrosslinkingoflysineaminoacidresiduesincollagen.

23. Types of Ehlers-Danlos syndrome
❖Types of Ehlers Danlos syndrome :
• Ehlers-Danlos syndrome type V: inherited deficiency of Lysyl oxidase(copper
requiring enzymes)→prevents cross-linking of collagen→ arterio-vascular and
skeletal changes.
• Ehlers-Danlos syndrome type VI: inherited deficiency of Lysyl hydroxylase
→abnormalities of the eye ,severe scoliosis (abnormal vertebral curvature)
and hyperextensibility of skin and joints.
• Ehlers-Danlos syndrome type VII: non-serving of procollagen as a substrate for
the procollagen amino protease →hip dislocation , increased skin elasticity
and short stature.

24. Ehlers-Danlos syndrome : Clinical manifestations
Hyperextensibility of skin and joints
severescoliosis
(abnormalvertebralcurvature)
Ehlers-Danlos syndrome

25. Alport syndrome
Alport syndrome :Defectinformationof typeIVcollagenfibersfoundinthebasementmembraneof
renalglomeruli→ hematuriaandrenaldisease

26. Alport syndrome :Clinical manifestations
Visual abnormality Deafness
Glomerular Nephritis

27. Epidermolysis bullosa
Epidermolysisbullosa:duetoalterationininthestructureoftypeVIIcollagenfibers→skinbreaksandblister
formationevenwithminortrauma
Epidermolysis bullosa

28. Osteogenesis imperfecta : Clinical manifestations
Osteogenesisimperfecta:Characterizedby abnormalbonefragilityduetodeceasedsynthesisofcollagen

29. Scurvy
Scurvy: Deficiency of vitamin C→ defective post translational modification of collagen→
bleeding gums ,poor wound healing, subcutaneous hemorrhage
Scurvy

30. RoleofLysyloxidasecrosslinkingoflysineaminoacidresiduesincollagen

31. Lathyrism
Lathyrism :CausedbyconsumptionofLathyrussativa(kesaridal)containingtoxiccompoundBetaOxalyl
AminoAlanine(BOAA).BOAAinhibitsenzymeLysyloxidaseandinterfereswiththecrosslinkingoflysine
aminoacidresiduesincollagen.
Lathyrussativa

32. Lathyrism