Gamma-carboxylation and fragmentation of osteocalcin in human serum defined by mass spectrometry - PubMed (original) (raw)

Randomized Controlled Trial

. 2015 Jun;14(6):1546-55.

doi: 10.1074/mcp.M114.047621. Epub 2015 Apr 8.

Affiliations

Randomized Controlled Trial

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

Douglas S Rehder et al. Mol Cell Proteomics. 2015 Jun.

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 (γ)-carboxylated variants of Oc are also found in circulation but there have been no methods that can define how many of the three potential γ-carboxyglutamic acid (Gla) residues are γ-carboxylated or provide their relative abundances. Recent reports that uncarboxylated and partially γ-carboxylated Oc forms have hormonal function underscore the need for precise evaluation of Oc at all three potential γ-carboxylation sites. Herein, mass spectrometric immunoassay (MSIA) was used to provide qualitative and semiquantitative (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 γ-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 with placebo, vitamin K supplementation dramatically increased the fractional abundance of Oc with three Gla residues, corresponding to a decrease in the fractional abundance of Oc with zero 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 shown that un- or undercarboxylation of Oc, unlike in mice, is not associated with insulin resistance. Analyses similar to those described here will be useful to understand the functional significance of Oc γ-carboxylation in human health and disease.

© 2015 by The American Society for Biochemistry and Molecular Biology, Inc.

PubMed Disclaimer

Figures

Fig. 1.

Fig. 1.

Oc from a single human blood plasma sample analyzed by MALDI-MSIA. The intact, full length protein is the most abundant form, but several previously undocumented N- and C-terminally truncated forms are also evident (Table I). Evidence described in the text indicates that these did not appear to form artifactually ex vivo. Because of prompt fragmentation induced by the laser (–23), γ-carboxyl post-translational modifications could not be observed by MALDI-MS. The spectrum shown is a single representative of spectra from 130 specimens analyzed (

supplemental Fig. S5

). Lower mass shoulder peaks as observed on peaks A, D, and F correspond to in-source neutral loss of water or ammonia. Higher mass shoulder peaks correspond to sodium adducts [M + Na]+.

Fig. 2.

Fig. 2.

Carboxylated (COOH) and uncarboxylated (unCOOH) bovine Oc fortified into human serum and plasma at 120 ng/ml and incubated at 25 °C for up to 4 h followed by analysis by MALDI-MSIA. Cleavage of bovine Oc was minimal; the most striking change was a slight increase in L[2–49]V after 4 h incubation in serum or plasma as shown in the inset. The adjacent small peak at m/z 5620 is Y[1–48]P and did not increase in relative abundance over time. a) Pure COOH Oc standard, b) COOH Oc fortified into plasma then immediately extracted, c) COOH Oc fortified into plasma and incubated for 4 h, d) COOH Oc fortified into serum then immediately extracted, e) COOH Oc fortified into serum and incubated for 4 h, f) Pure unCOOH Oc standard, g) unCOOH Oc fortified into plasma then immediately extracted, h) unCOOH Oc fortified into plasma and incubated for 4 h, i) unCOOH Oc fortified into serum then immediately extracted, and j) unCOOH Oc fortified into serum and incubated for 4 h.

Fig. 3.

Fig. 3.

ESI-MSIA mass spectrum of Oc extracted from human blood plasma donated by a 24-yr old female. This sample was purchased from a commercial biobank for the purposes of initial assay development and was not part of Studies A-C described in the text. γ-carboxyl post-translational modifications were readily detected and mass mapped – revealing the precise number of γ-carboxyl groups present on each protein molecule. Nine differentially truncated forms of Oc were detected by ESI-MSIA (Table I). Magnesium and chromium adducts (which have previously been documented (39, 40) are likely derived from the metal electrospray needle. All mass spectral peak area integrals were calculated and used to determine the relative percent abundance of Oc with 0, 1, 2, and 3 γ-carboxyl groups. Peaks appear as solid black because protein ions are monoisotopically resolved (inset). * Indicates nonspecific detection of a multiply charged form of apolipoprotein A–I. Qualitatively, the lack of spacing between the isotopes indicates that the peak does not arise from Oc.

Fig. 4.

Fig. 4.

Detailed molecular comparison provided by ESI-MSIA of circulating Oc γ-carboxylation in volunteers receiving a vitamin K supplement or placebo (Study B). The relative percent abundance of circulating Oc molecules bearing 0, 1, 2, and 3 Gla residues is shown for each group. Samples were collected after 24 months of treatment. Error bars represent 95% confidence intervals. * Indicates statistical significance as determined by a t test (p < 0.001).

Fig. 5.

Fig. 5.

Circulating Oc γ-carboxylation in volunteers from Study C after vitamin K depletion (d13) and vitamin K supplementation (d41). The relative percent abundance of circulating Oc molecules bearing 0, 1, 2, and 3 Gla residues is shown for each group. Specimens analyzed were collected 7 days after initiation of each treatment. Error bars represent 95% confidence intervals. * Indicates statistical significance as determined by a paired t test (* for p < 0.001; † for p < 0.01).

Similar articles

Cited by

References

    1. Hauschka P. V., Lian J. B., Cole D. E., Gundberg C. M. (1989) Osteocalcin and matrix Gla protein: vitamin K-dependent proteins in bone. Physiol. Rev. 69, 990–1047 - PubMed
    1. Eastell R., Hannon R. A. (2008) Biomarkers of bone health and osteoporosis risk. Proc. Nutr. Soc. 67, 157–162 - PubMed
    1. Garnero P., Grimaux M., Demiaux B., Preaudat C., Seguin P., Delmas P. D. (1992) Measurement of serum osteocalcin with a human-specific two-site immunoradiometric assay. J. Bone Miner Res. 7, 1389–1398 - PubMed
    1. Booth S. L., Martini L., Peterson J. W., Saltzman E., Dallal G. E., Wood R. J. (2003) Dietary phylloquinone depletion and repletion in older women. J. Nutr. 133, 2565–2569 - PubMed
    1. Booth S. L., Al Rajabi A. (2008) Determinants of vitamin K status in humans. Vitam. Horm. 78, 1–22 - PubMed

Publication types

MeSH terms

Substances

Grants and funding

LinkOut - more resources