INHIBITORS OF 2-KETOGLUTARATE-DEPENDENT DIOXYGENASES BLOCK ASPARTYL BETA-HYDROXYLATION OF RECOMBINANT HUMAN FACTOR-IX IN SEVERAL MAMMALIAN EXPRESSION SYSTEMS

  • 25 April 1989
    • journal article
    • research article
    • Vol. 264  (12) , 6615-6618
Abstract
While a role has been ascribed to the .gamma.-carboxyglutamate (Gla) residues in vitamin K-dependent coagulation proteins and the enzyme catalyzing this postranslational modification has been identified and partially characterized, both the functional significance of a second posttranslationally synthesized amino acid found in these proteins, .beta.-hydroxyaspartate (Hya), and the aspartyl .beta.-hydroxylating enzyme remain to be determined. We now report that inhibitors of 2-ketoglutarate-dependent dioxygenases, such as dipyridyl, O-phenanthroline, and pyridine 2,4-dicarboxylate block hydroxylation of Asp64 in recombinant factor IX molecules produced in three different mammalian expression systems. This hydroxylation was not inhibited by the specific copper chelators 2,9-dimethylphenanthroline or D-penicillamine. The Gla levels in these proteins were unaffected by these compounds and demonstrate that carboxylation proceeds independently of hydroxylation. Using these Hya-deficient recombinant factor IX molecules we demonstrate that this residue does not play a significant role in factor IX binding to endothelial cells under equilibrium conditions. From additional binding studies we have concluded that the Gla domain of factor IX is a major cell binding domain of factor IX. Furthermore, in contrast to studies demonstrating a marked loss of one-stage clotting activity in recombinant factors IX following site-directed mutations of Asp64 to neutral or basic residues (Rees, D. J. G., Jones, I. M., Handford, P. A., Walter, S. J., Esnouf, M. P., Smith, K. J. and Brownlee, G. J. (1988) EMBO J. 7, 2053-2061), we have not found a decrease of one-stage clotting activity with Hya-deficient factor IX. Hya-deficient proteins produced in this manner may prove to be more appropriate to elucidate the function of Hya than those produced by site-directed mutagenesis.