Abstract
Bone morphogenetic protein (BMP)-1 is a glycosylated metalloproteinase that is fundamental to the synthesis of a normal extracellular matrix because it cleaves type I procollagen, as well as other precursor proteins. Sequence analysis suggests that BMP-1 has six potential N-linked glycosylation sites (i.e. NXS/T) namely: Asn(91) (prodomain), Asn(142) (metalloproteinase domain), Asn(332) and Asn(363) (CUB1 domain), Asn(599) (CUB3 domain), and Asn(726) in the C-terminal-specific domain. In this study we showed that all these sites are N-glycosylated with complex-type oligosaccharides containing sialic acid, except Asn(726) presumably because proline occurs immediately C-terminal of threonine in the consensus sequence. Recombinant BMP-1 molecules lacking all glycosylation sites or the three CUB-specific sites were not secreted. BMP-1 lacking CUB glycosylation was translocated to the proteasome for degradation. BMP-1 molecules lacking individual glycosylation sites were efficiently secreted and exhibited full procollagen C-proteinase activity, but N332Q and N599Q exhibited a slower rate of cleavage. BMP-1 molecules lacking any one of the CUB-specific glycosylation sites were sensitive to thermal denaturation. The study showed that the glycosylation sites in the CUB domains of BMP-1 are important for secretion and stability of the molecule.
Highlights
Bone morphogenetic protein-1 (BMP-1),1 known as procollagen C-proteinase-1 (PCP-1) was first identified in osteogenic extracts of bone [1]
BMP-1F was stably expressed in HT1080 and 293-EBNA cells and the secreted protein was incubated with Endopeptidase F/N-glycosidase F (endoF/N), which cleaves all N-linked structures, regardless of their complexity, by hydrolyzing the asparagine-oligosaccharide bond, or endoglycosidase H (endoH), which cleaves high mannose-type structures
In this study we have shown that BMP-1 is N-linked glycosylated at five sites within the molecule and that these sites are important for protein secretion and stability
Summary
Bone morphogenetic protein-1 (BMP-1),1 known as procollagen C-proteinase-1 (PCP-1) was first identified in osteogenic extracts of bone [1]. The study showed that the glycosylation sites in the CUB domains of BMP-1 are important for secretion and stability of the molecule.
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