Glucosamine: fructose-6-phosphate amidotransferase in the white shrimp Litopenaeus vannamei: characterization and regulation under alkaline and cadmium stress.

Heavy metal residues and chemical contaminators considered as relevant sources of aquatic environmental pollutants have a generally immunosuppressive effect on aquatic organisms, depressing metabolic activities and immune response. Glutamine: fructose-6-phosphate aminotransferase (GFAT, EC2.6.1.16) is the first, and rate-limiting, enzyme in the hexosamine biosynthetic pathway, and is involved in the regulation of chitin biosynthesis and glycosylation of proteins. We have isolated and characterized GFAT from the white shrimp Litopenaeus vannamei. Amino acid sequence similarity of the Lv-GFAT (L.vannamei-GFAT) was highest to GFATs isolated from insects and mammals (83 % similarity to that of Haemaphysalis longicornis). The open-reading frame of the Lv-GFAT codes for a protein of 41.6 kDa with a calculated isoelectric point of 5.03. RT-PCR assays showed that endogenous Lv-GFAT mRNA is most strongly expressed in the intestine. Further analysis of Lv-GFAT gene expression in hepatopancreas by quantitative real-time PCR demonstrated that Lv-GFAT transcript levels increased when the shrimp were exposed to alkaline pH (9.3) and cadmium stress, but the time when its mRNA expression level peaked differed under these stresses. We also first expressed the recombinant protein of GFAT from shrimps in Escherichia coli. Western blot analyses confirmed that the Lv-GFAT protein was strongly expressed in the hepatopancreas after exposure to the LC-Cd stress. These results suggest that Lv-GFAT expression is stimulated by alkaline pH and cadmium stress and that it may play important roles in resistance of shrimp to environmental stresses.