On a potential global role for vitamin K-dependent gamma-carboxylation in animal systems - Evidence for a gamma-glutamyl carboxylase in Drosophila

The vitamin K-dependent gamma -carboxylation of glutamate to gamma -carboxy glutamate was originally well characterized in the mammalian blood clotting cascade. gamma -Carboxyglutamate has also been found in a number of other mammalian proteins and in neuropeptides from the venoms of marine snails be longing to the genus Conus, suggesting wider prevalence of gamma -carboxyla tion, We demonstrate that an open reading frame from a Drosophila melanogas ter cDNA clone encodes a protein with vitamin K-dependent gamma -carboxylas e activity. The open reading frame, 670 amino acids in length, is truncated at the C-terminal end compared with mammalian gamma -carboxylase, which is 758 amino acids. The mammalian gene has 14 introns; in Drosophila there ar e two much shorter introns but in positions precisely homologous to two of the mammalian introns. In addition, a deletion of 6 nucleotides is observed when cDNA and genomic sequences are compared. In situ hybridization to fix ed embryos indicated ubiquitous presence of carboxylase mRNA throughout emb ryogenesis. Northern blot analysis revealed increased mRNA levels in 12-24- h embryos. The continued presence of carboxylase mRNA suggests that it play s an important role during embryogenesis. Although the model substrate FLEE L is carboxylated by the enzyme, a substrate containing the propeptide of a Conus carboxylase substrate, conantokin G, is poorly carboxylated. Its occ urrence in vertebrates, molluscan systems (i.e. Conus), and Drosophila and the apparently strong homology between the three systems suggest that this is a highly conserved and widely distributed posttranslational modification in biological systems.