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.
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.
Hintze
Vera
V
Department I, Cell and Matrix Biology, Institute of Zoology, Johannes Gutenberg-University, D-55099 Mainz, Germany.
Höwel
Markus
M
Wermter
Carsten
C
Grosse Berkhoff
Eva
E
Becker-Pauly
Christoph
C
Beermann
Bernd
B
Yiallouros
Irene
I
Stöcker
Walter
W
D000818:Animals; D001483:Base Sequence; D055395:Bone Morphogenetic Protein 1; D019485:Bone Morphogenetic Proteins / Q000378:metabolism*; D002942:Circular Dichroism; D017931:DNA Primers; D004591:Electrophoresis, Polyacrylamide Gel; D008666:Metalloendopeptidases / Q000378:metabolism*; D045726:Metalloproteases / Q000378:metabolism*; D011347:Procollagen / Q000378:metabolism*; D017510:Protein Folding; D012326:RNA Splicing; D011994:Recombinant Proteins / Q000378:metabolism; D013050:Spectrometry, Fluorescence; D020349:Surface Plasmon Resonance; D055394:Tolloid-Like Metalloproteinases
D019485:Bone Morphogenetic Proteins; D017931:DNA Primers; D011347:Procollagen; D011994:Recombinant Proteins; D045726:Metalloproteases; D055394:Tolloid-Like Metalloproteinases; D008666:Metalloendopeptidases; C525705:BMP1 protein, human; D055395:Bone Morphogenetic Protein 1