Stepwise proteolytic activation of type?I procollagen to collagen within the secretory pathway of tendon fibroblasts in situ

摘要

Proteolytic cleavage of procollagen I to collagen I is essential for the formation of collagen fibrils in the extracellular matrix of vertebrate tissues. Procollagen is cleaved by the procollagen N- and C-proteinases, which remove the respective N- and C-propeptides from procollagen. Procollagen processing is initiated within the secretory pathway in tendon fibroblasts, which are adept in assembling an ordered extracellular matrix of collagen fibrils in vivo . It was thought that intracellular processing was restricted to the TGN ( trans -Golgi network). In the present study, brefeldin A treatment of tendon explant cultures showed that N-proteinase activity is present in the resulting fused ER (endoplasmic reticulum)–Golgi compartment, but that C-proteinase activity is restricted to the TGN in embryonic chick tendon fibroblasts. In late embryonic and postnatal rat tail and postnatal mouse tail tendon, C-proteinase activity was detected in TGN and pre-TGN compartments. Preventing activation of the procollagen N- and C-proteinases with the furin inhibitor Dec-RVKR-CMK (decanoyl-Arg-Val-Lys-Arg-chloromethylketone) indicated that only a fraction of intracellular procollagen cleavage was mediated by newly activated proteinases. In conclusion, the N-propeptides are removed earlier in the secretory pathway than the C-propeptides. The removal of the C-propeptides in post-Golgi compartments most probably indicates preparation of collagen molecules for fibril formation at the cell–matrix interface.Abbreviations: ADAMTS, a disintegrin and metalloprotease with thrombospondin motifs; BFA, brefeldin A; BMP1, bone morphogenetic protein 1; *collagen, collagen intermediate (a collective term for all procollagen, pCcollagen, pNcollagen and collagen molecules); COPI, coatomer protein 1; Dec-RVKR-CMK, decanoyl-Arg-Val-Lys-Arg-chloromethylketone; E, embryonic day; ER, endoplasmic reticulum; ERGIC, ER–Golgi intermediate compartment; mTLD, mammalian tolloid; MT1-MMP, membrane type?1 matrix metalloproteinase; NP40, Nonidet P40; PBS-T, PBS with 0.1% Tween; pCcollagen, procollagen lacking the N-propeptides but retaining the C-propeptides; pNcollagen, procollagen lacking the C-propeptides but retaining the N-propeptides; QL, quantum level; TEM, transmission electron microscopy; TGFβ, transforming growth factor β; TGN, trans-Golgi network; TLL, tolloid-like