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Characterization of post-translational modifications in full-length human BMP-1 confirms the presence of a rare vicinal disulfide linkage in the catalytic domain and highlights novel features of the EGF domain

Research paper by Chien-Wen Hung, Tomas Koudelka, Cyril Anastasi, Alexander Becker, Catherine Moali, Andreas Tholey

Indexed on: 18 Mar '16Published on: 02 Mar '16Published in: Journal of Proteomics



Abstract

Bone morphogenetic protein 1 (BMP-1) is an essential metalloproteinase to trigger extracellular matrix assembly and organogenesis. Previous structural studies on the refolded catalytic domain of BMP-1 produced in E. coli have suggested the existence of a rare vicinal disulfide linkage near the active site. To confirm that this was not an artifact of the refolding procedure, the full-length human BMP-1 produced in mammalian cells was investigated via sequence-dependent enzyme cleavage under native conditions followed by high mass accuracy and high resolution LC-MS/MS analysis to interrogate the post-translational modifications. Ten disulfide linkages of BMP-1, including the vicinal disulfide linkage C185-C186 could be unambiguously identified. Further, around 50% of this vicinal disulfide bond was found to be modified by N-ethylmaleimide (NEM), a cysteine protease inhibitor supplied when the BMP-1-containing medium was collected, suggesting that this bond was highly unstable. In the absence of NEM, BMP-1 has a higher tendency to form aggregates, but after aggregate removal, C185 and C186 are almost quantitatively engaged in the vicinal disulfide bond and BMP-1 activity remains unchanged. In addition, three consensus N-glycosylation sites at N142, N363, and N599 could be identified together with a previously unknown O-glycosylation site and an Asn-hydroxylation.

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