Medline/PubMed Citation | Related Articles in PubMed | Download to Citation Manager

Biochem. J. (2007) 405 (299–306) (Printed in Great Britain)

Enzymatic cleavage specificity of the proa1(V) chain processing analysed by site-directed mutagenesis

Christelle BONOD-BIDAUD*†‡, Mickaël BERAUD*†‡, Elisabeth VAGANAY*†‡, Frédéric DELACOUX*†‡1, Bernard FONT*†‡, David J. S. HULMES*†‡ and Florence RUGGIERO*†‡2

*Université de Lyon, Université Lyon 1, Lyon France, †Institut de Biologie et Chimie des Protéines, UMR 5086 CNRS – Université Lyon 1, 7 passage du Vercors, 69367 Lyon Cedex 07, France, and ‡IFR 128 BioSciences Lyon-Gerland, 7 Passage du Vercors, 69367 Lyon Cedex 07, France

The proteolytic processing of procollagen V is complex and depends on the activity of several enzymes among which the BMP-1 (bone morphogenetic protein-1)/tolloid metalloproteinase and the furin-like proprotein convertases. Few of these processing interactions could have been predicted by analysing the presence of conserved consensus sequences in the proa1(V) chain. In the present 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 proa1(V) chain, referred to as Na1, was recombinantly expressed to be used for enzymatic assays and for antibody production. Structural analysis showed that Na1 is a monomer composed of a compact globule and an extended tail, which correspond respectively to the non-collagenous Na1 subdomains, TSPN-1 (thrombospondin-1 N-terminal domain-like) and the variable region. Na1 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 P2´ 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 (R1584A/R1585A). We showed that, in absence of furin activity, BMP-1 is capable of processing the C-propeptide even though less efficiently than furin. Altogether, our results provide new relevant information on this complex and poorly understood mechanism of enzymatic processing in procollagen V function.

Key words: extracellular matrix, metalloproteinase, procollagen, proteolytic processing, site-directed mutagenesis, transient cell transfection.

Abbreviations used: ADAMTS, a disintegrin and metalloproteinase domain with thrombospondin type 1 repeats; BMP-1, bone morphogenetic protein-1; DMEM, Dulbecco's modified Eagle's medium; HEK-293 cell, human embryonic kidney cell; mAb, monoclonal antibody; PCPE, procollagen C-proteinase enhancer; TSPN-1, thrombospondin-1 N-terminal domain-like; a1TH, a1 triple helix domain.

¹Present address: Université de Reims Champagne-Ardenne, UFR de Sciences Exactes et Naturelles, CNRS UMR 6198, Laboratoire de Biochimie, Moulin de la Housse BP1039, F-51687 Reims Cedex 2, France.

²To whom correspondence should be addressed (email f.ruggiero@ibcp.fr).

Received 8 January 2007/21 March 2007; accepted 4 April 2007

Published as BJ Immediate Publication 4 April 2007, doi:10.1042/BJ20070051

© The Authors Journal compilation © 2007 Biochemical Society

Enhanced Full Text

PDF

Legacy HTML