Post-translational modification of bone morphogenetic protein-1 is required for secretion and stability of the protein.
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.
Garrigue-Antar
Laure
L
Wellcome Trust Centre for Cell-Matrix Research, University of Manchester, School of Biological Sciences, Stopford Building 2.205, Oxford Road, Manchester M13 9PT, United Kingdom. laure.garrigue-antar@man.ac.uk
Hartigan
Nichola
N
Kadler
Karl E
KE
D019943:Amino Acid Substitution; D001483:Base Sequence; D055395:Bone Morphogenetic Protein 1; D019485:Bone Morphogenetic Proteins / Q000737:chemistry / Q000378:metabolism*; D002460:Cell Line; D017931:DNA Primers; D004795:Enzyme Stability; D005354:Fibrosarcoma; D006031:Glycosylation; D006801:Humans; D007668:Kidney; D007700:Kinetics; D008666:Metalloendopeptidases / Q000737:chemistry / Q000378:metabolism*; D008856:Microscopy, Fluorescence; D016297:Mutagenesis, Site-Directed; D016133:Polymerase Chain Reaction; D011499:Protein Processing, Post-Translational; D013379:Substrate Specificity; D014162:Transfection; D014407:Tumor Cells, Cultured
D019485:Bone Morphogenetic Proteins; D017931:DNA Primers; D008666:Metalloendopeptidases; C525705:BMP1 protein, human; D055395:Bone Morphogenetic Protein 1