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-carboxyglutamate 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 belonging to the genus Conus, suggesting wider prevalence of gamma-carboxylation. We demonstrate that an open reading frame from a Drosophila melanogaster cDNA clone encodes a protein with vitamin K-dependent gamma-carboxylase 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 are 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 fixed embryos indicated ubiquitous presence of carboxylase mRNA throughout embryogenesis. Northern blot analysis revealed increased mRNA levels in 12-24-h embryos. The continued presence of carboxylase mRNA suggests that it plays an important role during embryogenesis. Although the model substrate FLEEL is carboxylated by the enzyme, a substrate containing the propeptide of a Conus carboxylase substrate, conantokin G, is poorly carboxylated. Its occurrence 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 post-translational modification in biological systems.

The Drosophila schnurri gene acts in the Dpp/TGF beta signaling pathway and encodes a transcription factor homologous to the human MBP family.

Decapentaplegic (dpp), a TGF beta-related ligand, plays a key role in Drosophila development. Although dpp receptors have been isolated, the downstream components of the signaling pathway remain to be identified. We have cloned the schnurri (shn) gene and show that it encodes a putative zinc finger transcription factor homologous to the human major histocompatibility complex-binding proteins 1 and 2. Mutations in shn affect multiple events that require dpp signaling as well as the transcription of dpp-responsive genes. Genetic interactions and the strikingly similar phenotypes of mutations in shn and the dpp receptors encoded by thick veins and punt suggest that shn plays a downstream role in dpp signaling.

Trypanosoma cruzi: identification of a surface antigen restricted to the flagellar region of the infective form of the parasite.

A hybridoma cell line was derived from spleen cells of B6D2 mice infected with the Peru strain of Trypanosoma cruzi. The monoclonal antibody produced by this hybridoma, designated mAb20H1, reacts exclusively with molecular components of trypomastigotes, the infective form of the parasite. The results of indirect immunofluorescence and of immunoelectron microscopy with gold-tagged antibodies indicate that the 20H1 antigen is restricted to the surface of the part of the flagellum in contact with the cell body and to the surface of the cell body in the immediate vicinity of this organelle. Western blot analysis showed that the 20H1 antigen consists of four to five different molecules with sizes between 34 and 41 kDa, and that these molecules are glycoproteins with affinity for concanavalin A. In other strains of T. cruzi, mAb20H1 reacts with glycoproteins with apparent sizes that range between 37 and 43 kDa in the CL, Esmeraldo and Y strains, and between 41 and 45 kDa in the Silvio strain.