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

• Published in: PloS one Vol. 10; no. 8; p. e0135374
• Main Authors: Annis, Douglas S, Ma, Hanqing, Balas, Danika M, Kumfer, Kraig T, Sandbo, Nathan, Potts, Gregory K, Coon, Joshua J, Mosher, Deane F
• Format: Journal Article
• Language: English
• Published: United States Public Library of Science 14.08.2015; Public Library of Science (PLoS)
• Subjects: Acids; Biochemistry; Blood; Blood coagulation; Carboxylation; Cell Adhesion Molecules - genetics; Cell Adhesion Molecules - metabolism; Coagulation; Coagulation factor VII; Coagulation factors; Cornea; Cytokines; Epithelial cells; Extracellular matrix; Female; Fibroblasts; Fibrosis; Gels; Gene expression; Glutamates; Glutamic acid; Growth factors; HEK293 Cells; Humans; Immunoglobulins; Lung - metabolism; Lung - pathology; Lung cancer; Lungs; Male; Mass spectrometry; Mass spectroscopy; Matrix protein; Menaquinones; Metabolic Engineering; Monoclonal antibodies; Peptides; Protein Processing, Post-Translational; Proteins; Proteomics; Pulmonary Fibrosis - genetics; Pulmonary Fibrosis - metabolism; Pulmonary Fibrosis - pathology; Recombinant Proteins - genetics; Recombinant Proteins - metabolism; Reductase; Residues; Scientific imaging; Structure-function relationships; Vitamin K; Vitamin K - metabolism; Western blotting; United States > US; Wisconsin
• ISSN: 1932-6203; 1932-6203
• DOI: 10.1371/journal.pone.0135374

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.