γ-Glutamate and β–Hydroxyaspartate in Proteins
Vitamin K-dependent coagulation plasma proteins possess from 9–12 residues of γ-carboxyglutamic acid (Gla) distributed over a ca. 45 amino acid peptide sequence, i.e., the Gla domain, which encompasses the NH2-terminal region. In addition, epidermal growth factor (EGF) homology units present in many of these same proteins contain β-hydroxyaspartate (Hya) residues, which is a modification decoupled from γ-carboxylation. The function of Gla residues in these proteins, viz., prothrombin, coagulation factors VII, IX, and X, along with anticoagulant protein C and protein S, is to coordinate Ca2+. This results in a large conformational alteration in the proteins or peptides, which allows adsorption to membrane phospholipids (PL), an event that is critical is to their proper functions in the blood coagulation system. Less certain is the role of Hya in EGF domains, but it has been proposed that modification at this residue may negatively regulate fucosylation of these regions. In several proteins, these modules also interact with Ca2+, but it has been shown that although the particular aspartate containing the β-OH group is critical to that interaction, β-hydroxylation of that Asp residue is not.
Because of their widespread distribution, quantitative detection protocols for both Gla and Hya are of importance. It is the purpose of this communication to detail a reliable method for these analyses that is employed in our laboratories.
Key Wordsγ-Carboxyglutamic acid (Gla) β-hydroxyaspartate (Hya) vitamin K-dependent proteins
Rabiet, M-J., Jorgensen, M. J., Furie B., and Furie, B. C. (1987) Effect of propeptide mutations on post-translational processing of factor IX. Evidence that β-hydroxylation and g-carboxylation are independent events. J. Biol. Chem.
, 14895–14898.PubMedGoogle Scholar
Nelsestuen, G. L. (1976) Role of γ-carboxyglutamic acid. An unusual protein transition required for the calcium-dependent binding of prothrombin to phospholipid. J. Biol. Chem.
, 5648–5656.PubMedGoogle Scholar
Nelsestuen, G. L., Broderius, M. and Martin, G. (1976) Role of γ-carboxyglutamic acid. Cation specificity of prothrombin and factor X-phospholipid binding. J. Biol. Chem.
, 6886–6993.PubMedGoogle Scholar
Harris, R. J., Ling, V. T., and Spellman, M. W. (1992) O-Linked fucose is present in the first epidermal growth factor domain of factor XII but not protein C. J. Biol. Chem.
, 5102–5107.PubMedGoogle Scholar
Haack, J. A., Rivier, J., Parks, T. N., Mena, E. E., Cruz, L. J., and Olivera, B. M. (1990) Conantokin-T. A γ-carboxyglutamate-containing peptide with N-methyl-D-aspartate antagonist activity. J. Biol. Chem.
, 6025–6029.PubMedGoogle Scholar
Skolnick, P., Boje, K., Miller, R., Pennington, M., and Maccecchini, M.-L. (1992) Noncompetitive inhibition of N-methyl-D-aspartate by conantokin-G: evidence for an allosteric interaction at polyamine sites. J. Neurochem.
, 1516–1521.CrossRefPubMedGoogle Scholar
Blandl, T., Prorok, M., and Castellino, F. J. (1998) NMDA-receptor antagonist requirements in conantokin-G. FEBS Lett.
, 257–262.CrossRefPubMedGoogle Scholar
McIntosh, J., Olivera, B. M., Cruz, L., and Gray, W. (1984) γ-Carboxyglutamate in a neuroac-tive toxin. J. Biol. Chem.
, 14343–14346.PubMedGoogle Scholar
Price, P. A., Poser, J. W., and Raman, N. (1976) Primary structure of the gamma
-carboxy-glutamic acid-containing protein from bovine bone. Proc. Natl. Acad. Sci. USA
, 3374–3375.CrossRefPubMedGoogle Scholar
Hauschka, P. V., Frenkel, J., DeMuth, R., and Gundberg, C. M. (1983) Presence of osteocalcin and related higher molecular weight 4-carboxyglutamic acid-containing proteins in developing bone. J. Biol. Chem.
, 176–182.PubMedGoogle Scholar
Price, P. A., Urist, M. R., and Otawara, Y. (1983) Matrix Gla protein, a new gamma
-carboxy-glutamic acid-containing protein which is associated with the organic matrix of bone. Biochem. Biophys. Res. Comm.
, 765–771.CrossRefPubMedGoogle Scholar
Price, P. A., Williamson, M. K. (1985) Primary structure of bovine matrix Gla protein, a new vitamin K-dependent bone protein. J Biol Chem
, 14971–14975.PubMedGoogle Scholar
Soute, B. A., Muller-Esterl, W., de Boer-van den Berg, M. A., Ulrich, M., and Vermeer, C. (1985) Discovery of a gamma
-carboxyglutamic acid-containing protein in human spermatozoa. FEBS Lett.
, 137–141.CrossRefPubMedGoogle Scholar
Kulman, J. D., Harris, J. E., Haldeman, B. A., and Davie, E. W. (1997) Primary structure and tissue distribution of two novel proline-rich gamma
-carboxyglutamic acid proteins. Proc. Natl. Acad. Sci. USA
, 9058–9062.CrossRefPubMedGoogle Scholar
Linde, A., Bhown, M., Cothran, W. C., Hoglund, A., and Butler, W. T. (1982) Evidence for several gamma
-carboxyglutamic acid-containing proteins in dentin. Biochim. Biophys. Acta
, 235–239.CrossRefPubMedGoogle Scholar
Drakenberg, T., Fernlund, P., Roepstorff, P., and Stenflo, J. (1983) β-Hydroxyaspartic acid in vitamin K-dependent protein C. Proc. Natl. Acad. Sci. USA
, 1802–1806.CrossRefPubMedGoogle Scholar
Stenflo, J., Ohlin, A.-K., Owen, W. G., and Schneider, W. J. (1988) β-Hydroxyaspartic acid or β-hydroxyasparagine in bovine low density lipoprotein receptor and in bovine thrombomodulin. J. Biol. Chem.
, 21–24.PubMedGoogle Scholar
Thielens, N. M., Van Dorsselaer, A., Gagnon, J., and Arlaud, G. J. (1990) Chemical and functional characterization of a fragment of C1-s containing the epidermal growth factor homology region. Biochemistry
, 3570–3578.CrossRefPubMedGoogle Scholar
Prorok, M., Warder, S. E., Blandl, T., and Castellino, F. J. (1996) Calcium binding properties of synthetic g-carboxyglutamic acid containing marine cone snail “sleeper” peptides, conantokin-G and conantokin-T. Biochemistry
, 16528–16534.CrossRefPubMedGoogle Scholar
Colpitts, T. L. and Castellino, F. J. (1994) Calcium and phospholipid binding properties of synthetic gamma
-carboxyglutamic acid-containing peptides with sequence counterparts in human protein C. Biochemistry
, 3501–3508CrossRefPubMedGoogle Scholar
Zhang, L. and Castellino, F. J. (1990) A γ-carboxyglutamic acid variant (γ6
D) of human activated protein C displays greatly reduced activity as an anticoagulant. Biochemistry
, 10828–10834.CrossRefPubMedGoogle Scholar
Zhang, L. and Castellino, F. J. (1991) Role of the hexapeptide disulfide loop present in the γ-carboxyglutamic acid domain of protein C in its activation properties and in the in vitro anticoagulant activity of activated protein C. Biochemistry
, 6696–6704.CrossRefPubMedGoogle Scholar
Zhang, L., Jhingan, A., and Castellino, F. J. (1992) Role of individual gamma
-carboxyglutamic acid residues of activated human protein C in defining its in vitro
anticoagulant activity. Blood
, 942–952.PubMedGoogle Scholar
Zhang, L. and Castellino, F. J. (1992) Influence of specific γ-carboxyglutamic acid residues on the integrity of the calcium-dependent conformation of human protein C. J. Biol. Chem.
, 26078–26084.PubMedGoogle Scholar
Zhang, L. and Castellino, F. J. (1993) The contributions of individual gamma
-carboxyglutamic acid residues in the calcium-dependent binding of recombinant human protein C to acidic phospholipid vesicles. J. Biol. Chem.
, 12040–12045.PubMedGoogle Scholar
Yu, S., Zhang, L., Jhingan, A., Christiansen, W. T., and Castellino, F. J. (1994) Construction, expression, and properties of a recombinant human protein C with replacement of its growth factor-like domains by those of human coagulation factor IX. Biochemistry
, 823–831.CrossRefPubMedGoogle Scholar
Zhang, L. and Castellino, F. J. (1994) The binding energy of human coagulation protein C to acidic phospholipid vesicles contains a major contribution from leucine-5 in the gamma
-carboxy-glutamic acid domain. J. Biol. Chem.
, 3590–3595.PubMedGoogle Scholar
Cheng, C.-H., Geng, J.-P., and Castellino, F. J. (1997) The functions of the first epidermal growth factor homology region of human protein C as revealed by a charge-to-alanine scanning mutagenesis investigation. Biol. Chem.
, 1491–1500.CrossRefPubMedGoogle Scholar
Stenflo, J. (1976) A new vitamin K-dependent protein. Purification from bovine plasma and preliminary characterization. J. Biol. Chem.
, 355–363.PubMedGoogle Scholar
Kuwada, M. and Katayama, K. (1983) An improved method for determination of gamma
-carboxyglutamic acid in proteins, bone and urine. Anal. Biochem.
, 173–179.CrossRefPubMedGoogle Scholar
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