The processing of human collagen type-V chains was studied using anti-peptide polyclonal antibodies raised against peptide sequences at the N-terminal non-triple-helical region of pro-al(V) and pro-a2(V) chains. The anti-peptide polyclonal antibody raised against positions 48 -57 of the N-terminal a2(V) sequence recognized the mature form of the human a2(V) chain extracted without any proteolytic treatment from several tissues in the presence of a mixture of protease inhibitors. It also recognized the pro-a2(V) and pN-a2(V) collagen chains secreted in the cell-culture media of the rhabdomyosarcoma A204 cell line. The pN-a2(V) collagen chain from this cell line migrated during electrophoresis with the a2(V) chain obtained from tissues. This demonstrates that the a2(V) chain in tissues is incompletely processed and is present as the pN-a2(V) collagen chain which lacks the C-propeptide. In comparison, an anti-peptide polyclonal antibody raised against residues at positions 284-299 of the N-terminal al(V) human sequence failed to recognize the mature form of the al(V) chain while it reacted with the pN-al(V) collagen chain form. These results suggest that the al(V) chain undergoes a processing event in the N-terminal region that involves the removal of at least the first 284 residues.Amino acid sequence analysis was performed on cyanogen-bromide-generated or trypsin-generated peptides of the two electrophoretic bands obtained for the tissue form of collagen V. The slower-migrating band corresponding to the intact al(V) chain gave, as expected, only sequences corresponding to the al(V) chain. However, the band previously considered to be the intact a2(V) chain also gave sequences for the al(V) chain in addition to the a2(V) chain. This result indicates the presence in tissue extracts of a further processed form of al(V) chain which migrates with the intact a2(V) chain. On further analysis, we observed that the two bands of the tissue form of collagen V occurred in a 1 : 1 ratio whereas, after the pepsin digestion to remove non-collagenous regions, two bands were observed with an al(V)la2(V) chain ratio of 3 : 1. These results indicate that the a1 (V) chain exists in an additional stoichiometry, different from [al(V)],a2(V). We suggest the existence of two different populations of type-V collagen molecules consisting of an [a1 (V)],a2(V) heterotrimer bearing considerable N-terminal non-triple-helical extensions of both al(V) and a2(V) chains and an [al(V)], homotrimer composed of fully processed al(V) chains.Collagen V is a quantitatively minor constituent of collagen fibrils in the extracellular matrix, It is mainly found in tissues that contain type-I collagen as the major collagen component. Collagen V is heterogeneous and a1 (V)-a3(V)

Fibromodulin belongs to the family of small, leucine-rich proteoglycans which have been reported to interact with collagens and to inhibit type I collagen fibrillogenesis. Decorin and fibromodulin exhibit a noticeable degree of sequence similarity. However, as previously reported [Font, B., Eichenberger, D., Rosenberg, L. M. & van der Rest, M. (1996) Matrix Biol. 15, 341Ϫ348] the domains of these molecules implicated in the interactions with type XII and type XIV collagens are different, these being the dermatan sulphate/chondroitin sulphate chain for decorin and the core protein for fibromodulin. At the present time the fibromodulin domains implicated in the interactions with fibrillar collagens remain unknown. In experiments reported here, we have sought to identify the structural requirements for fibromodulin interaction with collagen and for the control of type I collagen fibrillogenesis. Circular dichroism spectra and fibrillogenesis inhibition studies show that fibromodulin structure and its collagen fibrillogenesis control function are strictly dependent on the presence of intact disulphide bridge(s). In addition, we show that the binding of fibromodulin (or fibromodulin-derived fragments) to type I collagen is not necessarily correlated with fibrillogenesis inhibition. To isolate fibromodulin domains, the native proteoglycan was submitted to mild proteolysis. We have isolated an A-chymotrypsinϪresistant fragment which contains the bulk of the N-terminal and central region of the molecule including the leucine-rich repeats 4 and 6 reported for decorin to be involved in type I collagen binding. This fragment does not bind to type I collagen. Using enzymes with different specificities, a number of large fragments of fibromodulin were obtained, suggesting a compact structure for this molecule which is relatively resistant to proteolysis. None of these N-glycosylated fragments were able to bind to type I collagen in co-sedimentation experiments. Taken together these results suggest that fibromodulin-type I collagen interactions leading to fibrillogenesis inhibition require more than one binding domain. One of these domains could be the Cterminal end of the molecule containing the disulphide loop which is absent in the chymotrypsin-resistant fragment.Keywords : collagen fibrillogenesis; fibromodulin; proteoglycan domain.In connective tissues, proteoglycans play important roles due cysteine residues, with at least one disulphide bridge, (b) a central region with a variable number (10Ϫ12) of asparagineto their interactions with other components of the extracellular matrix [1]. Among proteoglycans, several members of this group containing leucine-rich repeats containing N-linked keratan sulphate (fibromodulin and lumican) or N-linked oligosaccharides are structurally related and constitute the small proteoglycan family, namely biglycan, decorin, fibromodulin and lumican [2]. (biglycan and decorin) distributed among up to five potential sites; and (c) a C-terminal region with a conserved disulphide These molecule...

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