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ARS Home » Northeast Area » Boston, Massachusetts » Jean Mayer Human Nutrition Research Center On Aging » Research » Publications at this Location » Publication #348233

Research Project: Nutrients, Aging, and Musculoskeletal Function

Location: Jean Mayer Human Nutrition Research Center On Aging

Title: Gamma-carboxylation and fragmentation of osteocalcin in human serum defined by mass spectrometry

Author
item Rehder, Douglas - Arizona State University
item Gundberg, Caren - Yale University
item Booth, Sarah - Jean Mayer Human Nutrition Research Center On Aging At Tufts University
item Borges, Chad - Arizona State University

Submitted to: Molecular and Cellular Proteomics
Publication Type: Peer Reviewed Journal
Publication Acceptance Date: 3/28/2015
Publication Date: 4/8/2015
Citation: Rehder, D.S., Gundberg, C.M., Booth, S.L., Borges, C.R. 2015. Gamma-carboxylation and fragmentation of osteocalcin in human serum defined by mass spectrometry. Molecular and Cellular Proteomics. 14(6):1546-1555. https://doi.org/10.1074/mcp.M114.047621.
DOI: https://doi.org/10.1074/mcp.M114.047621

Interpretive Summary: Serum osteocalcin concentrations are a highly specific measure of bone turnover, but the various circulating osteocalcin forms have not been well defined. Osteocalcin is a vitamin K-dependent protein that requires vitamin K for biochemical changes, called carboxylation, of specific amino acids that confer function to the protein. Vitamin K-dependent variants of osteocalcin are also found in circulation, but there have been no methods that can define how many of the three potential amino acid residues are fully carboxylated by vitamin K or provide their relative abundances in circulation. Recent reports that uncarboxylated and partially carboxylated osteocalcin forms have hormonal function underscore the need for precise evaluation of osteocalcin at all three potential vitamin K-dependent carboxylation sites. Two mass spectrometry immunoassay instruments were used to assess the relative abundance of the intact osteocalcin protein and its fragments and the degree of vitamin K-dependent carboxylation, respectively, in plasma from 130 patients enrolled in vitamin K supplementation trials. Human osteocalcin was found to circulate in over a dozen forms, with each form displaying anywhere from zero to three amino acid residues. The relative abundance of fragments was consistent and unaffected by vitamin K supplementation. In contrast, when compared to placebo, vitamin K supplementation dramatically increased the abundance of osteocalcin, with all three amino acid residues being carboxylated. These findings unequivocally document that increased vitamin K intake reduces the uncarboxylated form of osteocalcin. Several reports of a positive effect of vitamin K intake on insulin sensitivity in humans have demonstrated that uncarboxylation of osteocalcin, unlike in mice, is not associated with insulin resistance. Similar determinations will be useful to understand the functional significance of the vitamin K-dependent carboxylation of osteocalcin in human health and disease.

Technical Abstract: Serum osteocalcin (Oc) concentration is a highly specific measure of bone turnover, but its circulating proteoform(s) have not been well defined. Based on immunological methods, the major forms are thought to be the intact polypeptide and a large N-terminal-mid molecule fragment for which there is no consensus on the precise sequence. Vitamin K-dependent gamma-carboxylated variants of Oc are also found in circulation but there have been no methods that can define how many of the 3 potential gamma-carboxyglutamic acid (Gla) residues are gamma-carboxylated or provide their relative abundances. Recent reports that uncarboxylated and partially gamma-carboxylated Oc forms have hormonal function underscore the need for precise evaluation of Oc at all 3 potential gamma-carboxylation sites. Herein, mass spectrometric immunoassay (MSIA) was used to provide qualitative and semi-quantitative (relative percent abundance) information on Oc molecular variants as they exist in individual plasma and serum samples. Following verification that observable Oc proteoforms were accurately assigned and not simply ex vivo artifacts, MALDI-MSIA and ESI-MSIA were used to assess the relative abundance of Oc truncation and gamma-carboxylation, respectively, in plasma from 130 patients enrolled in vitamin K supplementation trials. Human Oc was found to circulate in over a dozen truncated forms with each of these displaying anywhere from 0-3 Gla residues. The relative abundance of truncated forms was consistent and unaffected by vitamin K supplementation. In contrast, when compared to placebo, vitamin K supplementation dramatically increased the fractional abundance of Oc with 3 Gla residues, corresponding to a decrease in the fractional abundance of Oc with 0 Gla residues. These findings unequivocally document that increased vitamin K intake reduces the uncarboxylated form of Oc. Several reports of a positive effect of vitamin K intake on insulin sensitivity in humans have demonstrated that un/undercarboxylation of Oc, unlike in mice, is not associated with insulin resistance. Similar determinations will be useful to understand the functional significance of Oc gamma-carboxylation in human health and disease.