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, H 2 N.CH 2,CH 2.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)] 2  2 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% of sequence Non-helical n = ~330 X is often Pro Y is often hydroxy Pro

Carbohydrate attachment in collagen NH 3 + | CH | 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: Chondrodysplasias Some osteoporosis and osteoarthritis Some 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 SELECTION REGISTRATION NUCLEATION PROPAGATION

Collagen biosynthesis FIBROBLAST 1.Procollagen synthesis 2.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 Fe 2+, O 2, 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