NDLI: Absence of Vitamin K-Dependent γ-Carboxylation in Human Periostin Extracted from Fibrotic Lung or Secreted from a Cell Line Engineered to Optimize γ-Carboxylation

Content Provider	PubMed Central
Author	Annis, Douglas S. Ma, Hanqing Balas, Danika M. Kumfer, Kraig T. Sandbo, Nathan Potts, Gregory K. Coon, Joshua J. Mosher, Deane F.
Editor	Frutos, Pablo Garcia De
Copyright Year	2015
Abstract	Periostin (PN, gene name POSTN) is an extracellular matrix protein that is up-regulated in bronchial epithelial cells and lung fibroblasts by TH-2 cytokines. Its paralog, TGF-β-induced protein (βig-h3, gene name TGFBI), is also expressed in the lung and up-regulated in bronchial myofibroblasts by TGF-β. PN and βig-h3 contain fasciclin 1 modules that harbor putative recognition sequences for γ-glutamyl carboxylase and are annotated in UniProt as undergoing vitamin K-dependent γ-carboxylation of multiple glutamic acid residues. γ-carboxylation profoundly alters activities of other proteins subject to the modification, e.g., blood coagulation factors, and would be expected to alter the structure and function of PN and βig-h3. To analyze for the presence of γ-carboxylation, proteins extracted from fibrotic lung were reacted with monoclonal antibodies specific for PN, βig-h3, or modification with γ-carboxyglutamic acid (Gla). In Western blots of 1-dimensional gels, bands stained with anti-PN or -βig-h3 did not match those stained with anti-Gla. In 2-dimensional gels, anti-PN-positive spots had pIs of 7.0 to >8, as expected for the unmodified protein, and there was no overlap between anti-PN-positive and anti-Gla-positive spots. Recombinant PN and blood coagulation factor VII were produced in HEK293 cells that had been transfected with vitamin K 2, 3-epoxide reductase C1 to optimize γ-carboxylation. Recombinant PN secreted from these cells did not react with anti-Gla antibody and had pIs similar to that found in extracts of fibrotic lung whereas secreted factor VII reacted strongly with anti-Gla antibody. Over 67% coverage of recombinant PN was achieved by mass spectrometry, including peptides with 19 of the 24 glutamates considered targets of γ-carboxylation, but analysis revealed no modification. Over 86% sequence coverage and three modified glutamic acid residues were identified in recombinant fVII. These data indicate that PN and βig-h3 are not subject to vitamin K-dependent γ-carboxylation.
Related Links	http://dx.doi.org/10.1371/journal.pone.0135374
Starting Page	135374
File Format	PDF
ISSN	19326203
e-ISSN	19326203
Journal	PLoS ONE
Issue Number	8
Volume Number	10
Language	English
Publisher	Public Library of Science
Publisher Date	2015-08-01
Access Restriction	Open
Rights Holder	Public Library of Science
Subject Keyword	Biochemistry, Genetics and Molecular Biology(all) Agricultural and Biological Sciences(all) Medicine(all) Research in Higher Education
Content Type	Text
Resource Type	Article
Subject	Multidisciplinary

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