Interaction Properties of the Procollagen C-proteinase Enhancer Protein Shed Light on the Mechanism of Stimulation of BMP-1

Abstract

Procollagen C-proteinase enhancer (PCPE) is an extracellular matrix glycoprotein that binds to the C-propeptide of procollagen I and can enhance the activities of procollagen C-proteinases up to 20-fold. To determine the molecular mechanism of PCPE activity, the interactions of the recombinant protein with the procollagen molecule as well as with its isolated C-propeptide domain were studied using surface plasmon resonance (BIAcore) technology. Binding required the presence of divalent metal cations such as calcium and manganese. By ligand blotting, calcium was found to bind to the C-propeptide domains of procollagens I and III but not to PCPE. By chemical cross-linking, the stoichiometry of the PCPE/C-propeptide interaction was found to be 1:1 in accordance with enzyme kinetic data. The use of a monoclonal antibody directed against the N-terminal region of the C-propeptide suggested that this region is probably not involved in binding to PCPE. Association and dissociation kinetics of the Cpropeptide domains of procollagens I and III on immobilized PCPE were rapid. Extrapolation to saturation equilibrium yielded apparent equilibrium dissociation constants in the range 150 -400 nM. In contrast, the association/dissociation kinetics of intact procollagen molecules on immobilized PCPE were relatively slow, corresponding to a dissociation constant of 1 nM. Finally, pN-collagen (i.e. procollagen devoid of the C-terminal propeptide domain) was also found to bind to immobilized PCPE, suggesting that PCPE binds to sites on either side of the procollagen cleavage site, thereby facilitating the action of procollagen C-proteinases.Bone morphogenetic protein-1 (BMP-1) 1 and other tolloidrelated metalloproteinases (1, 2), also known as procollagen C-proteinases (PCPs), have recently been shown to be involved in the control of a variety of morphogenetic events during development and tissue repair. These include: (i) the deposition of collagen fibrils in the extracellular matrix following the processing of procollagen propeptides (3-6); (ii) dorsoventral patterning (7-10) through the cleavage of the growth factor inhibitors chordin and SOG; (iii) collagen and elastin crosslinking by the processing of the inactive precursors of lysyl oxidases (11, 12); and (iv) adhesion/migration of epithelial cells by the cleavage of laminin 5 chains (13-15). The activities of PCPs on procollagen substrates may be stimulated up to 20-fold by another glycoprotein of the extracellular matrix, procollagen C-proteinase enhancer (PCPE) (16 -19), which lacks intrinsic proteinase activity. Similarly, in the case of chordin and SOG, the protein TSG or its homologues stimulates cleavage by tolloid proteinases (20, 21), thus raising the possibility that PCP processing of different substrates might be specifically regulated by distinct enhancer proteins.Both tolloid proteinases and PCPE are multidomain glycoproteins containing multiple copies of the so-called CUB domain (22), a protein module common to several extracellular and plasma membrane-associated...

Proteomics Discovery of Metalloproteinase Substrates in the Cellular Context by iTRAQ™ Labeling Reveals a Diverse MMP-2 Substrate Degradome

“…3, A and B). Because PCPE binds to procollagen C-propeptide via its N-terminal CUB domain (36) the iTRAQ ratios and peptide mapping are consistent with the shedding of the C-terminal netrin domain of PCPE upon proteolysis with the N-terminal CUB domain remaining bound to the procollagen. We confirmed this using C-terminal FLAGtagged PCPE in culture medium that was cleaved by MMP-2 to release a 22-kDa C-terminal FLAG-tagged fragment encompassing the netrin domain (Fig.…”

“…PCPE stimulates procollagen processing, thereby triggering assembly of collagen fibrils (36). Interestingly the C-terminal netrin domain of PCPE is a weak inhibitor of MMP-2 (41) that might homeostatically regulate the levels of PCPE.…”

Insights into How CUB Domains Can Exert Specific Functions while Sharing a Common Fold

“…5, the binding of PCPE-1 to the immobilized miniprocollagen III was initially fast and then slowed down without reaching a plateau, even when the injection time was doubled. This sensorgram is reminiscent of the sensorgrams previously obtained when the C-propeptides of procollagen I or III were injected over immobilized PCPE-1 (35). The various PCPE-1 mutants were then sequentially injected over the same regenerated mini-procollagen III surface at a fixed concentration of 100 nM, in the absence or presence of 5 mM Ca 2ϩ (Fig.…”

“…We have already shown that the C-propeptide of procollagen III binds calcium (35) and thus, mini-procollagen III like PCPE-1 is expected to bind calcium. To explore further the role played by calcium in PCPE-1, we monitored the effect of calcium binding on the intrinsic fluorescence of PCPE-1.…”

“…Although the mechanism of action of PCPEs is not well understood, this probably involves binding to the substrate, because maximum enhancing activity is found for PCPE: procollagen molar ratio of at least 1:1. BIAcore studies have shown that PCPE-1 binds to both the C-propeptide region as well as elsewhere in the procollagen molecule (35). Recent studies using a miniprocollagen III substrate have shown that enhancing activity is unaffected by removal of essentially all of the triple-helical region using highly purified bacterial collagenase (28).…”