Biochemical characterization of Drosophilaγ‐glutamyl carboxylase and its role in fly development

Abstract

To investigate structure-function relationships in gamma-glutamyl carboxylases, the enzyme from Drosophila melanogaster was characterized. Four cysteine residues were shown to be important determinants for enzymatic activity. Native Drosophila substrates have not yet been identified, but propeptides of human prothrombin and factor IX are recognized by the Drosophila enzyme. The presence of the propeptide region increased apparent affinity by approximately 200-fold, and mutation of a hydrophobic residue of factor IX propeptide (F-16A) decreased carboxylation by 90%, as in the human enzyme. Substrate recognition appears to be highly conserved between the human and Drosophila gamma-glutamyl carboxylases. Inactivation of Drosophila gamma-glutamyl carboxylase by non-sense mutations or insertional mutagenesis by P-element insertion have no apparent effects on growth and fertility under laboratory conditions.

Fatal hemorrhage in mice lacking γ-glutamyl carboxylase

“…However, a recent analysis of flies deficient in ␥-carboxylase demonstrated no obvious phenotype. 6 Taken together with evidence for the widespread expression of ␥-carboxylase in the mammalian embryo, 3 these observations suggest broad functional significance of ␥-carboxylation, potentially affecting multiple biologic processes both in the developing embryo and the adult animal. We were thus surprised to observe grossly normal development of the completely ␥-carboxylase-deficient mouse embryo through embryonic day 9.5, with approximately half of the embryos developing normally to term.…”

“…1,24,38 Four Gla-domain containing proteins have been identified in C. intestinalis, including a receptor tyrosine kinase and a possible homolog of a Notch receptor family ligand. 5 The function and specific substrates of ␥-carboxylase in Drosophila are unknown, 6 although some studies suggest a possible role for insect carboxylase in the regulation of circadian rhythm. However, a recent analysis of flies deficient in ␥-carboxylase demonstrated no obvious phenotype.…”

“…The enzyme functions posttranslationally to convert selected glutamic acid (Glu) residues within a specific subset of proteins to Gla. 1,2 ␥-carboxylase functional activity can be detected in most or all mammalian tissues, 3 and the carboxylase gene has been demonstrated to be conserved not only in other vertebrates such as zebrafish, 4 but also in chordates such as Ciona intestinalis 5 and diverse invertebrates including Drosophila 6 and Conus, the cone snail. 7,8 The most thoroughly characterized ␥-carboxylase substrates are the vitamin K-dependent coagulation factors prothrombin, factor VII, factor IX, factor X, protein C, protein S, and protein Z.…”

A small intron of Drosophila gamma carboxylase, not predictable in silico for mammalian splicing machinery, is spliced improperly in human cells

“…Genes encoding γ-carboxylase (γC) have been identified in vertebrates and two invertebrate species, mollusks and D. melanogaster (Goodstadt and Ponting, 2004;Robertson, 2004;Bandyopadhyay et al, 2006). The potential of the Drosophila γC (DγC) for carboxylation of some human vitamin-K-dependent (VKD) proteins was demonstrated previously in both in vitro (Bandyopadhyay et al, 2006) and in vivo (Vatandoost et al, 2012) studies.…”

“…The potential of the Drosophila γC (DγC) for carboxylation of some human vitamin-K-dependent (VKD) proteins was demonstrated previously in both in vitro (Bandyopadhyay et al, 2006) and in vivo (Vatandoost et al, 2012) studies. In order to investigate the potential of the DγC to γ-carboxylate a human substrate in a mammalian host after cloning of the DγC gene, 2144 base pairs (bp), and its corresponding cDNA (2019 bp) in separate expression vectors, we studied the expression of DγC in a mammalian cell line (in preparation).…”