CLINICAL ASPECTS OF BIOCHEMISTRY

PROTEINS AND DISEASE - LECTURES 1 & 2

MIKE WALLIS

Two main groups

(a) keratin, myosin, fibrinogen - mainly alpha helix

(b) collagen, elastin, resilin, silk fibroin - not alpha helix

STRUCTURAL/FIBROUS PROTEINS

X-RAY DIFFRACTION PATTERNS

Myoglobin Silk fibroin ? DNA!

COLLAGEN

Major component of connective tissue: tendons, bone, cartilage etc

~25% of body protein in vertebrates

Relatively little in insects or protozoa

Extracellular - part of the extracellular matrix

Fibres with characteristic striations; strong but not elastic

At least 25 different types (not all form fibres)

COLLAGEN FIBRES

COLLAGEN SECONDARY STRUCTURE

PART OF COLLAGEN SEQUENCE (1)

-Gly-Pro-Met-Gly-Pro-Ser-Gly-Pro-Arg- 13 Gly-Leu-Hyp-Gly-Pro-Hyp-Gly-Ala-Hyp- 22 Gly-Pro-Glu-Gly-Pro-Glu-Gly-Pro-Hyp- 31

TROPOCOLLAGEN

• Soluble form of collagen; the 'subunit' of a collagen fibre

• Yields increased by extracting from lathyrogen-treated animals

• Lathyrogens (e.g. -aminopropionitrile, H2N.CH2,CH2.CN) cause lathyrism in cattle etc.

• Tropocollagen has Mr of ~300,000; dimensions 3000Å x 14Å

• Tropocollagen contains 3 polypeptide chains, each of ~1000 aas

SOME COLLAGEN TYPES

Type I tendon and bone [1(I)]22

Type II cartilage [1(II)]3

Type III cardiovascular system [1(III)]3

fetal dermis

TypeIV basal membranes [1(IV)]3

Modified amino acids in Collagen

Tropocollagen chain structure

polar N-terminal region - (Gly - X - Y)n - polar C-terminal region

Non-helical ~ 95% ofsequence

Non-helical

n = ~330X is often ProY is often hydroxy Pro

Carbohydrate attachment in collagen

NH3+

| CH2

1-2 1 |Glucose - galactose - O - CH | CH2 hydroxylysine | CH2 ____________ |________

COLLAGEN DISEASES

POINT MUTATIONS AND DELETIONS IN TYPE I COLLAGEN LEAD TO OSTEOGENESIS IMPERFECTA (OI)

Especially Gly X mutations

Dominant, variable severity, usually lethal in homozygote

~70% of OI is due to Type I collagen defects

~1100 other mutations of 6 types of collagen cause human diseases, including:

ChondrodysplasiasSome osteoporosis and osteoarthritisSome kidney disease and vascular disease

(transgenic models have been developed for many of these diseases)

Organization of a collagen fibre

Collagen denaturation and gel formation

Thermal stability of Collagen

Crosslinks in Collagen

Histidino-hydroxymerodesmosine

Further crosslinking of collagen (one of several possible routes)

Crosslinks between tropocollagen molecules

Conversion of Procollagen to Collagen

N

ASSEMBLY OF TROPOCOLLAGEN

CHAIN SELECTIONREGISTRATIONNUCLEATION

PROPAGATION

Collagen biosynthesis

FIBROBLAST

1. Procollagen synthesis2. Hydroxylation & glycosylation

3. Secretion PROCOLLAGEN

4. Hydrolytic processing TROPOCOLLAGEN

5. Self assembly COLLAGEN FIBRE

6. Cross linking MATURE FIBRE

HYDROXYLATION OF PROLINE AND LYSINE

• Requires Fe2+, O2, ascorbic acid (vitamin C)

• 2 microsomal enzymes, prolylhydroxylase and lysylhydroxylase

• Hydroxylation of Pro in position Y:

Gly - X - Y - Gly - X - Y -

• Lack of vitamin C leads to scurvy - many symptoms are due to defective collagen hydroxylation

COLLAGENASES

Two types:

1. E.g. Clostridium histolyticum (causes gas gangrene)

X - Gly - Pro - Y - 2. Tissue collagenase "Remodelling"/growth of vertebrate tissues.

Very specific - cleaves at residue ~750 only. Tm of collagen important. Important for metastatic invasion.

COLLAGEN DEFECTS

1. Mutations in collagen genes - Osteogenesis imperfecta etc.

2. Lack of vitamin C - scurvy

3. Defective procollagen conversion: Dermatosparaxis (cattle) Ehlers-Danlos syndrome (human)

4. Defective cross-linking - lathyrism

5. Too much cross-linking - ageing of skin etc

6. Too much collagen - fibrosis