RESUMO
In marine animals, glycine betaine is one of the main osmolytes accumulated under osmotic stress conditions; nevertheless, in penaeids, shrimps little is known about the pathways involved in glycine betaine biosynthesis. In animal cells, glycine betaine is synthesized by the enzyme betaine aldehyde dehydrogenase (BADH). We herein investigated the salinity effect on the synthesis and concentration of glycine betaine on white shrimp Litopenaeus vannamei. Shrimps were subjected to 10, 20, 35, 40, 50, and 60â¯ppt salinity conditions for seven days. BADH activity increased in hepatopancreas and gills of shrimps subjected to salinities above 35â¯ppt salinity. In muscle, the BADH activity decreased at 35â¯ppt salinity. In hepatopancreas from shrimps subjected to 50 and 60â¯ppt salinities, BADH activity increased 1.1 and 1.7-fold. At 60â¯ppt salinity, BADH activity increased 1.5-fold respect to 35â¯ppt in gills. Glycine betaine concentration increased in hepatopancreas, gills, muscle, and hemolymph in shrimps subjected to salinities above 35â¯ppt. Glycine betaine concentration also increased at 20â¯ppt salinity, while at 10â¯ppt, not detected significant differences. The catch of glycine betaine from hemolymph by the cell likely is carried out to avoid protein denaturalization. Ammonia concentration in the aquarium's water only increased at salinities of 20â¯ppt and 10â¯ppt (1.1-fold relative to 35â¯ppt). Our data demonstrated that in L. vannamei, salinity regulates BADH activity and glycine betaine content in a tissue-specific manner.
Assuntos
Betaína-Aldeído Desidrogenase/metabolismo , Betaína/metabolismo , Osmorregulação , Pressão Osmótica , Penaeidae/metabolismo , Salinidade , Animais , Hemolinfa/metabolismo , Hepatopâncreas/metabolismo , Penaeidae/efeitos dos fármacosRESUMO
The enzyme betaine aldehyde dehydrogenase (BADH) catalyzes the irreversible oxidation of betaine aldehyde to glycine betaine (GB), a very efficient osmolyte accumulated during osmotic stress. In this study, we determined the nucleotide sequence of the cDNA for the BADH from the white shrimp Litopenaeus vannamei (LvBADH). The cDNA was 1882 bp long, with a complete open reading frame of 1524 bp, encoding 507 amino acids with a predicted molecular mass of 54.15 kDa and a pI of 5.4. The predicted LvBADH amino acid sequence shares a high degree of identity with marine invertebrate BADHs. Catalytic residues (C-298, E-264 and N-167) and the decapeptide VTLELGGKSP involved in nucleotide binding and highly conserved in BADHs were identified in the amino acid sequence. Phylogenetic analyses classified LvBADH in a clade that includes ALDH9 sequences from marine invertebrates. Molecular modeling of LvBADH revealed that the protein has amino acid residues and sequence motifs essential for the function of the ALDH9 family of enzymes. LvBADH modeling showed three potential monovalent cation binding sites, one site is located in an intra-subunit cavity; other in an inter-subunit cavity and a third in a central-cavity of the protein. The results show that LvBADH shares a high degree of identity with BADH sequences from marine invertebrates and enzymes that belong to the ALDH9 family. Our findings suggest that the LvBADH has molecular mechanisms of regulation similar to those of other BADHs belonging to the ALDH9 family, and that BADH might be playing a role in the osmoregulation capacity of L. vannamei.
Assuntos
Betaína-Aldeído Desidrogenase/metabolismo , Betaína/metabolismo , Modelos Moleculares , Penaeidae/enzimologia , Motivos de Aminoácidos , Sequência de Aminoácidos , Animais , Betaína-Aldeído Desidrogenase/classificação , Betaína-Aldeído Desidrogenase/genética , Sítios de Ligação , Biocatálise , Clonagem Molecular , Humanos , Dados de Sequência Molecular , Filogenia , Estrutura Terciária de Proteína , Proteínas Recombinantes/biossíntese , Proteínas Recombinantes/química , Proteínas Recombinantes/isolamento & purificação , Alinhamento de SequênciaRESUMO
Crustaceans overcome osmotic disturbances by regulating their intracellular concentration of ions and osmolytes. Glycine betaine (GB), an osmolyte accumulated in response to hyperosmotic stress, is synthesized by betaine aldehyde dehydrogenase (BADH EC 1.2.1.8) through the oxidation of betaine aldehyde. A partial BADH cDNA sequence from the white shrimp Litopenaeus vannamei was obtained and its organ-specific expression during osmotic stress (low and high salinity) was evaluated. The partial BADH cDNA sequence (LvBADH) is 1103bp long and encodes an open reading frame for 217 protein residues. The amino acid sequence of LvBADH is related to that of other BADHs, TMABA-DH and ALDH9 from invertebrate and vertebrate homologues, and includes the essential domains of their function and regulation. LvBADH activity and mRNA expression were detected in the gills, hepatopancreas and muscle with the highest levels in the hepatopancreas. LvBADH mRNA expression increased 2-3-fold in the hepatopancreas and gills after 7days of osmotic variation (25 and 40ppt). In contrast, LvBADH mRNA expression in muscle decreased 4-fold and 15-fold after 7days at low and high salinity, respectively. The results indicate that LvBADH is ubiquitously expressed, but its levels are organ-specific and regulated by osmotic stress, and that LvBADH is involved in the cellular response of crustaceans to variations in environmental salinity.