The glyceraldehyde-3-phosphate dehydrogenase of the shrimp Litopenaeus vannamei: Molecular cloning, characterization and expression during hypoxia.

Some marine crustaceans like the white shrimp Litopenaeus vannamei are tolerant to environmental hypoxia. Under oxygen deprivation, shrimp tissues obtain energy by enhancing anaerobic glycolysis. In mammals, hypoxia increases the expression of the glycolytic enzyme glyceraldehyde-3-phosphate dehydrogenase (GAPDH), which has been shown a "moonlighting" role in cells. However, the effect of hypoxia on the GAPDH expression has not been studied in crustaceans. In the present work, we obtained a 2744 bp gene sequence with a 999 bp ORF split by a single intron. The deduced protein is 332 amino acids and corresponds to the L. vannamei GAPDH (LvGAPDH), which is highly similar in sequence and structure to other animal GAPDHs. During hypoxia, LvGAPDH expression is significantly induced in gills but not in hepatopancreas, suggesting that it may play a role in the molecular and cellular response of shrimp to hypoxia.

Camacho-Jiménez L ，Peregrino-Uriarte AB ，Martínez-Quintana JA ，Yepiz-Plascencia G ... -  《-》

Regulation of glyceraldehyde-3-phosphate dehydrogenase by hypoxia inducible factor 1 in the white shrimp Litopenaeus vannamei during hypoxia and reoxygenation.

Hypoxia is a frequent source of stress in the estuarine habitat of the white shrimp Litopenaeus vannamei. During hypoxia, L. vannamei gill cells rely more heavily on anaerobic glycolysis to obtain ATP. This is mediated by transcriptional up-regulation of glycolytic enzymes including glyceraldehyde-3-phosphate dehydrogenase (GAPDH). The hypoxia inducible factor 1 (HIF-1) is an important transcriptional activator of several glycolytic enzymes during hypoxia in diverse animals, including crustaceans. In this work, we cloned and sequenced a fragment corresponding to the 5' flank of the GAPDH gene and identified a putative HIF-1 binding site, as well as sites for other transcription factors involved in the hypoxia signaling pathway. To investigate the role of HIF-1 in GAPDH regulation, we simultaneously injected double-stranded RNA (dsRNA) into shrimp to silence HIF-1α and HIF-1β under normoxia, hypoxia, and hypoxia followed by reoxygenation, and then measured gill HIF-1α, HIF-1β expression, GAPDH expression and activity, and glucose and lactate concentrations at 0, 3, 24 and 48 h. During normoxia, HIF-1 silencing induced up-regulation of GAPDH transcripts and activity, suggesting that expression is down-regulated via HIF-1 under these conditions. In contrast, HIF-1 silencing during hypoxia abolished the increases in GAPDH expression and activity, glucose and lactate concentrations. Finally, HIF-1 silencing during hypoxia-reoxygenation prevented the increase in GAPDH expression, however, those changes were not reflected in GAPDH activity and lactate accumulation. Altogether, these results indicate that GAPDH and glycolysis are transcriptionally regulated by HIF-1 in gills of white shrimp.

Camacho-Jiménez L ，Leyva-Carrillo L ，Peregrino-Uriarte AB ，Duarte-Gutiérrez JL ，Tresguerres M ，Yepiz-Plascencia G ... -  《-》

The bifunctional 6-phosphofructokinase-2/fructose-2,6-bisphosphatase from the shrimp Litopenaeus vannamei: Molecular characterization and down-regulation of expression in response to severe hypoxia.

Hypoxia is a frequent stressor in marine environments with multiple adverse effects on marine species. The white shrimp Litopenaeus vannamei withstands hypoxic conditions by activating anaerobic metabolism with tissue-specific changes in glycolytic and gluconeogenic enzymes. In animal cells, glycolytic/gluconeogenic fluxes are highly controlled by the levels of fructose-2,6-bisphosphate (F-2,6-P2), a signal metabolite synthesized and degraded by the bifunctional enzyme 6-phosphofructo-2-kinase/fructose-2,6-bisphosphatase (PFK-2/FBPase-2). PFK-2/FBPase-2 has been studied in vertebrates and some invertebrates, but as far as we know, there are no reports on PFK-2/FBPase-2 from crustaceans. In the present work, we obtained cDNA nucleotide sequences corresponding to two mRNAs for PFK-2/FBPase-2 and named them PFKFBP1 (1644 bp) and PFKFBP2 (1566 bp), from the white shrimp L. vannamei. The deduced PFKFBP1 and PFKFBP2 are 547 and 521 amino acids long, respectively. Both proteins share 99.23% of identity, and only differ in 26 additional amino acids present in the kinase domain of the PFKFBP1. The kinase and phosphatase domains are highly conserved in sequence and structure between both isoforms and other proteins from diverse taxa. Total expression of PFKFBP1-2 is tissue-specific, more abundant in gills than in hepatopancreas and undetectable in muscle. Moreover, severe hypoxia (1 mg/L of DO) decreased expression of PFKFBP1-2 in gills while anaerobic glycolysis was induced, as indicated by accumulation of cellular lactate. These results suggest that negative regulation of PFKFBP1-2 at expression level is necessary to set up anaerobic glycolysis in the cells during the response to hypoxia.

Flores-Sauceda M ，Camacho-Jiménez L ，Peregrino-Uriarte AB ，Leyva-Carrillo L ，Arvizu-Flores A ，Yepiz-Plascencia G ... -  《-》

Molecular cloning and modeling of the Tp53-induced glycolysis and apoptotic regulator (TIGAR) from the Pacific white shrimp Litopenaeus vannamei and its expression in response to hypoxia.

Hypoxia is a common stressor for aquaculture species. The Pacific white shrimp Litopenaeus vannamei survives low dissolved oxygen (DO) conditions by adjusting its energy metabolism. In vertebrates, the transcription factor p53 regulates glucose metabolism under stress through diverse target genes like the Tp53-induced glycolysis and apoptotic regulator (TIGAR), a protein similar to fructose-2,6-bisphosphatase that has a pro-survival role in cells participating in the defense against oxidative damage. Until now, TIGAR has been not reported in any invertebrate species, including crustaceans. In this work, we report the molecular cloning of the white shrimp TIGAR. The cDNA sequence is 765 bp encoding a 254 amino acid protein. Bioinformatics analyses predicted that although the overall sequence identities of L. vannamei TIGAR and vertebrate proteins are not very high (33.61%-35.34%), they have a remarkable predicted structural similarity with full conservation of catalytic residues, secondary and three-dimensional structures. Gene expression analysis by RT-qPCR revealed that the mRNA abundance of TIGAR in white shrimp is tissue-specific under normal oxygen conditions, with higher expression in gills than hepatopancreas and muscle. Also, gene expression in gills and hepatopancreas is modified by environmental hypoxia, suggesting that TIGAR participates in the cellular tolerance of L. vannamei to this stressor.

Camacho-Jiménez L ，Felix-Portillo M ，Nuñez-Hernandez DM ，Yepiz-Plascencia G ... -  《-》

White shrimp Litopenaeus vannamei catalase: gene structure, expression and activity under hypoxia and reoxygenation.

Catalase (EC 1.11.1.6) is an antioxidant enzyme involved in redox equilibrium, regulating hydrogen peroxide (H(2)O(2)) concentration, a harmful reactive oxygen species (ROS) that is produced during hypoxia. Hypoxia occurs commonly in aquatic environments and in shrimp farms. We studied the catalase gene of the shrimp Litopenaeus vannamei and tested its expression and enzyme activity during hypoxia (1.5mg/L O(2); 6 and 24h) and reoxygenation (1h after hypoxia). The complete gene is 2974bp long and has four introns of 821, 223, 114 and 298bp, respectively. The first intron has tree microsatellites, with GT and (T)AT(GT) repeated sequences. L. vannamei catalase is part of an invertebrate clade including crustaceans and rotifers. Catalase expression and activity is different in gills and hepatopancreas. Expression in gills increased 3.2 and 3-fold in response to hypoxia and reoxygenation (6 and 24h hypoxia, followed by 1h reoxygenation) compared to normoxia, while no differences were detected in the expression and activity in hepatopancreas. Catalase activity in gills had a contrary response to expression in hypoxia and reoxygenation.

Trasviña-Arenas CH ，Garcia-Triana A ，Peregrino-Uriarte AB ，Yepiz-Plascencia G ... -  《-》