The Interaction of Recombinant Subdomains of the Procollagen C-Proteinase with Procollagen I Provides a Quantitative Explanation for Functional Differences between the Two Splice Variants, Mammalian Tolloid and Bone Morphogenetic Protein 1

Abstract

The procollagen C-proteinase (PCP) is a zinc peptidase of the astacin family and the metzincin superfamily. The enzyme removes the C-terminal propeptides of fibrillar procollagens and activates other matrix proteins. Besides its catalytic protease domain, the procollagen C-proteinase contains several C-terminal CUB modules (named after complement factors C1r and C1s, the sea urchin UEGF protein, and BMP-1) and EGF-like domains. The two major splice forms of the C-proteinase differ in their overall domain composition. The longer variant, termed mammalian tolloid (mTld, i.e., PCP-2), has the protease-CUB1-CUB2-EGF1-CUB3-EGF2-CUB4-CUB5 composition, whereas the shorter form termed bone morphogenetic protein 1 (BMP-1, i.e., PCP-1) ends after the CUB3 domain. Two related genes encode proteases similar to mTld in humans and have been termed mammalian tolloid like-1 and -2 (mTll-1 and mTll-2, respectively). For mTll-1, it has been shown that it has C-proteinase activity. We demonstrate that recombinant EGF1-CUB3, CUB3, CUB3-EGF2, EGF2-CUB4, and CUB4-CUB5 modules of the procollagen C-proteinase can be expressed in bacteria and adopt a functional antiparallel beta-sheet conformation. As shown by surface plasmon resonance analysis, the modules bind to procollagen I in a 1:1 stoichiometry with dissociation constants (K(D)) ranging from 622.0 to 1.0 nM. Their binding to mature collagen I is weaker by at least 1 order of magnitude. Constructs containing EGF domains bind more strongly than those consisting of CUB domains only. This suggests that a combination of CUB and EGF domains serves as the minimal functional unit. The binding affinities of the EGF-containing modules for procollagen increase in the order EGF1-CUB3< CUB3-EGF2 < EGF2-CUB4. In the context of the full length PCP, this implies that a given module has an affinity that continues to increase the more C-terminally the module is located within the PCP. The tightest binding module, EGF2-CUB4 (K(D) = 1.0 nM), is only present in mTld, which might provide a quantitative explanation for the different efficiencies of BMP-1 and mTld in procollagen C-proteinase activity.

Functional and structural insights into astacin metallopeptidases

“…These domains may be involved in calcium-binding and proteinprotein or enzyme-substrate interactions. It has been demonstrated that the C-terminal CUB-and EGF-like domains of procollagen C-peptidase are important for selectivity in substrate recognition (Sieron et al , 2000 ;Garrigue -Antar et al, 2004 ;Hintze et al , 2006 ;Wermter et al , 2007 ). By virtue of their similar, but not identical, domain composition, sea urchin astacins like SPAN and BP10 are related to tolloids (Lepage et al , 1992 ;Reynolds et al , 1992 ).…”

Activity of ulilysin, an archaeal PAPP-A-related gelatinase and IGFBP protease

“…Another example is provided by bone morphogenetic protein-1, a multidomain procollagen C-proteinase of the astacin family of metzincins, which also shows a much broader substrate profile and higher efficiency in cleaving a variety of ECM proteins for its isolated catalytic domain than the full-length protein. This difference was also attributed to additional C-terminal domains present in the latter, which modulate and restrict the substrate specificity through their potential proteinbinding or steric-hindering competence (Hartigan et al, 2003;Garrigue-Antar et al, 2004;Hintze et al, 2006). A similar scenario is conceivable for PAPP-A and other large pappalysins with a multidomain structure, where the proteolytic potential would be kept in check by additional domains via exosite binding and/or steric hindrance of substrate binding.…”

Role of dimerization and substrate exclusion in the regulation of bone morphogenetic protein-1 and mammalian tolloid

“…Recently, Stöcker and coworkers (18) proposed an explanation for the functional difference between BMP-1 and mTLD, suggesting that binding affinity to procollagen increases toward the C terminus of the molecule. Intriguingly, they also reveal that fragments containing EGF domains bind procollagen more strongly than those containing only CUB domains.…”