Mutation of a cleavage site adjacent to the mature domain leads to increase in secreted mature BMP-2 with reduced activity

Abstract

Proteolytic cleavage of precursor bone morphogenetic protein (proBMP) is an important step in generating the active mature BMP. ProBMP-2 contains two proprotein convertase (PC) recognition sites (S1 and S2) and is postulated to be cleaved by PCs at those sites. Cell lines expressing proBMP-2, with a silenced S1 site (mS1) that inhibited PC cleavage, secreted the 20-kDa form BMP-2, while cells expressing wild type (wt) BMP-2 secreted 18- and 20-kDa mature BMP-2 N-terminal isoforms. The mS1 cells secreted 15-fold more mature BMP-2 than the wt, despite their similar mRNA levels. Mutant-secreted BMP-2 demonstrated biological activity in vitro; however, its activity was reduced compared with wt. These data demonstrate that proBMP-2 can be cleaved at an alternative cleavage site without prior S1 site cleavage in cell lines overexpressing BMP-2 and more importantly suggest that the presence of the 2-kDa linker peptide can affect activity and secretion of the mature protein.

Keywords::

• Bone morphogenetic protein
• pro-domain
• proprotein convertase
• proteolytic cleavage

Acknowledgements

This research was supported by a grant from McEwen Research Fund (199-03-1570) to CMLC and SAFP. AJZ is a recipient of a Natural Sciences and Engineering Council Post-Graduate Scholarship. The authors are grateful for the cells lines provided by Induce Biologics Inc. We would also like to thank Drs Tara Moriarty and Michelle Visser and for critical reading of several drafts of the manuscript and fruitful discussions.

Declaration of interest: A patent has been filed describing methods that enhance the production of recombinant proteins, which includes mutation of the protein to resist convertase cleavage (Methods for enhancing recombinant protein, PCT/CA2008/000998). This pending patent has been assigned to Induce Biologics Inc. SAFP and CMLC are shareholders in Induce Biologics Inc.

Notes

SAFP and CMLC are shareholders in Induce Biologics Inc.