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.
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.
Department of Biomolecular Chemistry, University of Wisconsin-Madison, Madison, Wisconsin, United States of America.
Douglas S
DS
Annis
Department of Biomolecular Chemistry, University of Wisconsin-Madison, Madison, Wisconsin, United States of America.
Hanqing
H
Ma
Department of Biomolecular Chemistry, University of Wisconsin-Madison, Madison, Wisconsin, United States of America.
Danika M
DM
Balas
Morgridge Institute for Research, University of Wisconsin-Madison, Madison, Wisconsin, United States of America.
Kraig T
KT
Kumfer
Department of Medicine, University of Wisconsin-Madison, Madison, Wisconsin, United States of America.
Nathan
N
Sandbo
Department of Chemistry, University of Wisconsin-Madison, Madison, Wisconsin, United States of America.
Gregory K
GK
Potts
Department of Biomolecular Chemistry, University of Wisconsin-Madison, Madison, Wisconsin, United States of America; Department of Chemistry, University of Wisconsin-Madison, Madison, Wisconsin, United States of America.
Joshua J
JJ
Coon
Department of Biomolecular Chemistry, University of Wisconsin-Madison, Madison, Wisconsin, United States of America; Department of Medicine, University of Wisconsin-Madison, Madison, Wisconsin, United States of America.
Deane F
DF
Mosher
D015815:Cell Adhesion Molecules / Q000235:genetics / Q000378:metabolism*; D005260:Female; D057809:HEK293 Cells; D006801:Humans; D008168:Lung / Q000378:metabolism* / Q000473:pathology; D008297:Male; D060847:Metabolic Engineering; D011499:Protein Processing, Post-Translational; D011658:Pulmonary Fibrosis / Q000235:genetics / Q000378:metabolism* / Q000473:pathology; D011994:Recombinant Proteins / Q000235:genetics / Q000378:metabolism; D014812:Vitamin K / Q000378:metabolism*
D015815:Cell Adhesion Molecules; C512835:POSTN protein, human; D011994:Recombinant Proteins; D014812:Vitamin K