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

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.

Supplementary data:

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
Research affiliation: Kiel University > Kiel Marine Science
OceanRep > The Future Ocean - Cluster of Excellence
Refereed: Yes
DOI etc.: 10.1016/j.jprot.2016.02.031
ISSN: 1874-3919
Projects: Future Ocean
Date Deposited: 25 Feb 2017 07:03
Last Modified: 01 Feb 2018 12:11
URI: http://eprints.uni-kiel.de/id/eprint/36146

Actions (login required)

View Item View Item