Collagen molecules are the principal structural elements of all connective tissues. Collagen fiber organization and ultrastructure varies from one tissue to the next. The objective of this study is to delineate the basic molecular events which are involved in the molecular fibril assembly processes and to determine the factors which regulate these processes. Four specific aspects of assembly are to be examined in detail. 1) Nascent pro Alpha chains register in the appropriate stoichiometry and initiate triple helix formation as the chains near release from the endoplasmic reticulum membrane. Membrane mediated interactions may guide this phase of assembly. A combination of cell-free translation, polysome isolation and completion, and membrane insertion studies will be used to examine the chain assembly mechanism. 2) Interstitial procollagens must be processed at both COOH - and NH2-propeptides before stable fibrils form. The NH2-propeptidase activity is well understood whereas the COOH- propeptidase has not been isolated and the site of its action not determined. Evidence is accumulating that the COOH-propeptide cleavage might be tied in with late stages of intracellular secretory processing and regulation of intracellular degradation. The processing localization of this event, and its enzymology are to be examined in detail. 3) Fibril formation is an event dominated by telopeptide-helix receptor region interactions. The explicit chemistry of this interaction, and the properties of these molecular domains are to be examined by interaction studies between isolated peptides, by physical studies (CD, FT-IR), and by interaction and conformational predictive studies. 4) Type IV basement membrane collagens form filamentous networks rather than fibrils, from molecules which are minimally processed and retain extensive non-triplet sequence chain segments. Anterior lens-capsule Type IV collagen can be obtained with minimal degradation. The self-assembly process will be examined, by thermal self-assembly studies and various electron microscopic techniques including SLS formation and rotary shadowing. From all these studies we should develop better insights into the synthesis and construction of the collagenous framework of the connective tissues. Our studies of the mechanism of molecular assembly and tissue ordering have the ultimate objective of providing insight into the control of the connective tissue disorders and their treatment.
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