Enzymatic cleavage specificity of the Pro{alpha}1(V) chain processing analysed by site-directed mutagenesis
Résumé
The proteolytic processing of procollagen V is complex and depends on the activity of several enzymes among which the BMP-1/tolloid metalloproteinase and the furin-like proprotein convertases. Few of these processing interactions could have been predicted by analyzing the presence of conserved consensus sequences in the proα1(V) chain. In this study we opted for a cell approach that allows a straightforward identification of processing interactions. A construct encompassing the complete N-terminal end of the proα1(V) chain, referred to as Nα1, was recombinantly expressed to be used for enzymatic assays and for antibody production. Structural analysis showed that Nα1 is a monomer composed of a compact globule and an extended tail which correspond respectively to the non-collagenous Nα1 subdomains, TSPN and the variable region. Nα1 was efficiently cleaved by BMP-1 indicating that the triple-helix is not required for enzyme activity. By mutating residues flanking the cleavage site, we showed that the aspartate residue at position P‘2 is essential for BMP-1 activity. BMP-1 activity at the C-terminal end of the procollagen V was assessed by generating a furin double mutant (Arg1584/1585Ala). We showed that, in absence of furin activity, BMP-1 is capable of processing the C-propeptide even though less efficiently than furin. Together, our results provide new relevant information on this complex and poorly understood mechanism of enzymatic processing in procollagen V function.
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