Hung, C. W., Koudelka, T., Anastasi, C., Becker, A., Moali, C. and Tholey, Andreas (2016) 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. Journal of Proteomics, 138 . pp. 136-145. DOI 10.1016/j.jprot.2016.02.031.

Full text not available from this repository.

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 C-185-C-186 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, C-185 and C-186 are almost quantitatively engaged in the vicinal disulfide bond and BMP-1 activity remains unchanged. In addition, three consensus N-glycosylation sites at N-142, N-363, and N-599 could be identified together with a previously unknown O-glycosylation site and an Asn-hydroxylation. Significance: An in-depth characterization of post-translational modifications of the full-length human BMP-1 produced in mammalian cells by MS was performed. A rare vicinal disulfide bond in the catalytic domain could be confirmed for the first time by mass spectrometry along with nine other proposed disulfide linkages of mature BMP-1. This vicinal disulfide bond can transiently open to form covalent adducts with the cysteine protease inhibitor (NEM) supplied in cell medium during protein harvesting. Further, we report a previously unknown O-glycosylation site and Asn-hydroxylation site, indicating a novel feature of BMP-1 in the EGF domain. The study clearly outlines the benefit of in-depth characterization of overexpressed proteins to deduce important protein modifications. (C) 2016 Elsevier B.V. All rights reserved.

Document Type:	Article
Additional Information:	Times Cited: 0 Hung, Chien-Wen Koudelka, Tomas Anastasi, Cyril Becker, Alexander Moali, Catherine Tholey, Andreas
Keywords:	LC-MS, Disulfide bond, Posttranslational modification, Protein characterization, Protein mass spectrometry
Research affiliation:	Kiel University > Kiel Marine Science OceanRep > The Future Ocean - Cluster of Excellence
Refereed:	Yes
Open Access Journal?:	No
Publisher:	Elsevier
Projects:	Future Ocean
Date Deposited:	25 Feb 2017 07:03
Last Modified:	23 May 2019 09:43
URI:	https://oceanrep.geomar.de/id/eprint/36146

Actions (login required)

View Item