Biochemistry of the Extracellular Matrix

Question 1

What is in the extracellular matrix?

Answer 1

• Elastin fibre
• Fibroblast
• Collagen fibre
• Proteoglycan

Question 2

What is collagen?

Answer 2

This is the major structural protein of bone, skin, cartilage, tendons and arteries, and it accounts for 25-35% of the protein the body

Question 3

What is the structure of collagen?

Answer 3

each polypeptide chain (~1000 amino acids) forms a left-handed helix
3 polypeptide chains are wound together in a right-handed superhelix
there are H-bonds between chains (not within chains, as there are in an -helix)
All collagens contain long stretches of the repeating sequence glycine-X-Y, where X and Y are proline or 4-hydroxyproline, respectively.

Question 4

What is the amino acid composition of collagen?

Answer 4

General sequence: [Gly-X-Y]n
X is often 3-hydroxyproline
Y is often 4-hydroxyproline
33% glycine, 15-20% proline/hydroxyproline
Nutritionally poor- hydroxyproline is unusable deficient in ile, phe, tyr, cys, met
80% water; the remainder is collagen (~2/3) and glycosaminoglycans (~1/3). GAG aggregates are maintained within a mesh of collagen fibrils – see the ‘balloon and string’ model. This confers elasticity and low friction in joints.

Question 5

What are the major types of collagen?

Answer 5

Type I= (a1I)2 a2I = bone, tendon, skin, lung, cornea, arteries
Type II= (a1II)3 = cartilage
Type III= (a1III)3 = skin, lung, uterus, arteries, ligaments
Type IV= (a1-6IV)3 = basement membranes, lens, capillaries
Type V= a1Va2Va3V = hair, placenta, cell-surface

Question 6

What are the minor types of collagen?

Answer 6

Bone - contains collagen types I, V, XII, XIV
Cartilage - contains collagen types II, VII, IX, X, XI
Skin - contains collagen types I, II, III, V, XI

Question 7

What are the collagen genes?

Answer 7

there are more than 40 genes, to encode all of the different types of a-chain.
Each gene contains about 50 exons (each encoding 6 gly-X-Y repeats, so 6 x 3 x 3 = 54 bases) separated by introns: only ~20% of the gene is translated into protein.

Question 8

What are the stages in collagen synthesis and processing?

Answer 8

1. Translation of mRNA on ER-bound ribosomes, with co-translational passage of protein into the ER, and removal of the 25-aminoacid N-terminal signal sequence, like most secreted proteins.
2. Glycosylation of some asparagine residues
3. Chain association and formation of inter-chain disulphide bonds near the C-terminus
4. Oxidation of most prolines in the Y position (~100 per chain) to 4-hydroxproline;
   this reaction requires oxygen, 2-oxoglutarate and ascorbate (vitamin C) – see below.
5. Triple helix formation, starting at the C-terminus (this occurs only after proline oxidation)
6. Passage of assembled chains to the Golgi, then secretion from the cell (constitutive exocytosis).
   Unassembled collagen chains are retained in the ER.
7. Removal of N- and C-propeptides by extracellular proteinases, to form tropocollagen
8. Association of tropocollagen molecules to form fibrils.
9. Crosslinking of fibrils: lysyl oxidase oxidizes some lysine and 5-hydroxylysine sidechains
   to form allysine (or 5-hydroxy-allysine), which then reacts non-enzymically with other lysine
   or histidine residues, to form covalent crosslinks.

Question 9

What is elastin?

Answer 9

another ECM protein, which forms elastic fibres in lungs, arteries, skin and tendons. It contains much gly, pro, ala, but no triple helix. Chains are covalently cross-linked by oxidation of lysine sidechains, followed by their nonenzymic reaction with other lysines and histidines

Question 10

Describe the crosslinking of amino acid sidechains in elastin and the structure of elastin fibres

Answer 10

Lysine and histidine- oxidation and nonenzymic reactions- desmosines
Elastin core surrounded by microfibrils

Question 11

What is the function of a1-antitrypsin?

Answer 11

Neutrophils release proteinases that degrade elastin and other ECM proteins: these proteinases are inhibited by 1-antitrypsin (a proteinase inhibitor secreted by liver). Congenital deficiency of
1-antitrypsin, or its inactivation by smoking (oxidants, peroxynitrite), leads to emphysema.

Question 12

What is the structure of proteoglycans?

Answer 12

Proteoglycans contain GAGs (chondrotin sulphate, keratan sulphate, etc) covalently attached to core proteins through a tetrasaccharide linked to a serine sidechain in the sequence -SerGlyXGly-. These glycoproteins associate noncovalently with hyaluronic acid.
The strong negative charges produce an extended structure, associated with cations - Na+, Mg2+, Ca2+ - and a lot of water.

Question 13

What are glycosaminoglycans?

Answer 13

GAGs are unbranched, polydisperse, acidic polysaccharides, composed of repeating disaccharide units with attached sulphate groups

Question 14

What are mucopolysaccharidoses?

Answer 14

rare hereditary diseases caused by defects in the lysosomal enzymes that degrade GAGS. Partially degraded GAGS accumulate in lysosomes, causing skeletal and other deformities, and often mental retardation

Question 15

What is the chemical composition of bone?

Answer 15

• 20% protein (mostly collagen type 1)
• 70% mineral (inorganic salts)
• 10% water

Question 16

What are the stages in bone deposition?

Answer 16

• Formation of the collagen matrix
• Deposition of CaHPO4 at the ends of collagen fibrils
• Conversion of calcium phosphate to hydroxyapatite 3Ca3(PO4)2.Ca(OH)2
• other ions such as Mg2+, F–, CO22- and citrate are also present in the crystal lattice, with the possibility of ion replacement.

Question 17

What are causes of poor mineralization?

Answer 17

• Vitamin D deficiency (affects intestinal absorption of Ca2+)
• Chronic metabolic acidaemia (affects hydroxyapatite formation)

Question 18

Describe scurvy (Vitamin C deficiency)

Answer 18

dietary deficiency of vitamin C results in progressive inactivation of prolyl hydroxylase, with consequent failure to synthesize, secrete and deposit collagen. Symptoms of scurvy are fragility of blood-vessel walls, petechial haemorrhages, gum inflammation, loss of teeth, poor healing of wounds. Stunted growth, hair and tooth loss
Vitamin D deficiency (or defects in vitamin D metabolism) affects bone mineralization, causing rickets.
Effects= glossitis, parafollicular haemorrhage

Question 19

Why are inherited diseases of bones and joints relatively common?

Answer 19

1. The strict requirement for glyXY sequence repeats means that the collagen structure is intolerant of point mutations (particularly in glycine, the smallest amino acid, and in proline).
2. Because of the complexity of collagen genes, splicing errors are common.
3. There are many types of post-translational modification. Errors in these processes (caused by mutations in genes encoding collagen itself or encoding processing enzymes) result in the failure of collagen secretion by fibroblasts.
4. Because 3 collagen chains interact, mutations in one may destabilize the whole molecule – so mutations tend to be dominant. Mutations near the C-terminus are usually lethal

Question 20

What is osteogenesis imperfecta?

Answer 20

(‘brittle bone disease’) is caused by mutations in collagen I.
OI type I - (commonest and least severe): collagen I is deficient but of normal structure. Decreased type 1 levels, blue sclerae, bone deformities, early fractures
OI type II - (perinatal lethal: ‘crumpled bones’ in utero): there is abnormal collagen structure, abnormal type 1, fractures in utero

Question 21

What is Ehlers-Danlos syndrome?

Answer 21

this is a heterogeneous group of diseases (11 types are recognized) in which collagen processing is affected.
EDS type IV is the most serious: in this, deficiency of collagen type III may lead to rupture of arteries or intestines, pneumothorax, and complications of pregnancy.
Other forms of EDS affect lysyl oxidase (type VI), procollagen peptidase (type VII) etc.
Joint hyperextensibility, skin hyper elasticity, cutaneous fragility

Question 22

What are the types of Ehlers-Danlos syndromes?

Answer 22

1V= decreased type 3 levels= arterial, intestinal and uterine rupture, easy bruising
V= decreased crosslinking= skin and joint hyper-extensibility
V1= decreased lysine hydroxylation= poor wound healing, skin and joint hyper-extensibility, skeletal deformities
V11= N-propeptide not removed= hip dislocations, skin and joint hyper-extensibility, easy bruising

Question 23

How is ascorbic acid involved in hydroxylation?

Answer 23

Prolyl residue to 4-hydroxy prolyl residue

2-oxo glutarate to CO2 succinate (O2)

Question 24

What is Fibrillin-1?

Answer 24

350-kDa Ca2+-binding glycoprotein

Major structural component of 10-nm microfibrils, which form a template for the elastin matrix

Question 25

What is Marfan syndrome?

Answer 25

autosomal dominant inherited disease caused by mutations in the gene for fibrillin 1, a glycoprotein that, together with elastin, forms microfibrils in aorta and ligaments
Affect the amount, structure, stability or secretion of fibrillin-1

Question 26

What occurs in Marfan syndrome?

Answer 26

microfibrils bind a growth factor, TGF-b; in Marfan syndrome, increased levels of free TGF-b cause developmental abnormalities that result in symptoms such as disproportionately long extremities (arachnodactyly), craniofacial abnormalities, joint hypermobility; eventually lens dislocation, pneumothorax (obstructive lung disease), or rupture of the aorta may occur