Lecture 3.docx

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University of Toronto St. George
Cell and Systems Biology
Maurice Ringuette

Lecture 3 – Supramolecular Assembly and Cross-linking of T1C Molecules, Dematosparaxis Extracellular Cross-linking Collagen Molecules - Cross-links formed between modified lysine side chains w/in a collagen fibril o Most of the cross-links are from within the non-helical telopeptide domains forming inter-molecular cross-links, while intramolecular cross-linkages occur within helical telopeptide domains o Collagen molecules stagger by about 1/4 of their length - Side by side interactions bw collagen molecules is stabilized by aldol cross-linkages bw two lysine side chains - Lysine and HO-lysine residues undergo deamination via extracellular lysyl oxidase and form highly reactive aldehyde derivatives which spontaneously cross-link o Reactive aldehydes can react /w each other or /w other Lys or HO-lysines o Lysyl oxidase requires BMP (a matrix metalloproteinase, MMP) to cleave off its pro- domain to become active o Glycosylation specific hydroxylysine residues may prevent their oxidative deamination to aldehydes - Main idea: after collagen has been processed in PM by a metalloproteinase (removal of Pro N and Pro C, via ADAMTS-2, -3 and BMP1 respectively) solubility of collagen molecules are capable of self-assembly and align by ¼; once in registry LO sees lysine/HO-Lys residues and cross-links them into a bond formation that is highly stable - Biosynthetic route collagen fibres o T1C gene is transcribed /w synthesis of PP occuring in ER, signal sequence will be cleaved off via Signal Peptidases o Protocollagen strand has its N-/C- terminal propeptides, which prevent its self aggregation, and many enzymes are used process protocollagen into a triple helix i.e. P4H, P3H, lysyl hydroxylase, protein disulfide isomerise (PDI, to ensure correct a-chain associations); Triple helix formation occurs @ c-terminal and creates a registry using zipper formation + PDI o Once triple helix is formed pro-sequences are cleaved via procollagen N- and C- proteinases (ADAMTS-2,-3 +BMP1) which face extracellular space and change solubility of the molecule i.e. now it is a tropocollagen triple helix /w non-helical telopeptide ends o Tropocollagen N- and C-terminal non-helical telopeptides undergo self-assembly to form staggered collagen microfibrils o After collagen microfibrils are made Lysyl oxidase crosslinks lys/HO-lys residues and form a collagen fibre  Lysyl oxidase is dysregulated in tissue fibrosis, ESPECIALLY in cancer o Oxidation of lysine side chains can lead to spontaneous formation of hydroxylysyl pyridinoline and lysyl pyridinoline cross-links - Periodic Striations o Repeated banding pattern of collagen fiber = due to striated nature o Structure of Fibrillar Collagens  T1C  fibrils  fibers o Adjacent molecules are staggered by 65-67nm = 1D-period = quarter staggered form = overlap gap domain, D-period of fibrillar collagen = region of overlap b/w adjacent molecules, 1 D-period = 1 hole zone (0.6nm) + 1 overlap zone (0.4nm) o Small 40nm gap bw head of one collagen molecule and tail of the next is imp (becomes a target for specific events) o Collagen molecule is 300nm i.e 4.4D - Electron micrographs of native collagen fibrils stained with either +/- stain o +ve stains binds to regions rich in polar AA, -ve stain becomes strapped in “holes zones” o EM shows repeating pattern of hole zones and overlap zones o More staining in –ve than +ve bc of hole zone which takes up more electron dense dye o Overlap zones show little no binding of +ve and
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