Molecular Cloning and Expression Analysis of Lactate Dehydrogenase from the Oriental River Prawn Macrobrachium nipponense in Response to Hypoxia

Abstract

Metabolic adaption to hypoxic stress in crustaceans implies a shift from aerobic to anaerobic metabolism. Lactate dehydrogenase (LDH) is a key enzyme in glycolysis in prawns. However, very little is known about the role of LDH in hypoxia inducible factor (HIF) pathways of prawns. In this study, full-length cDNA of LDH (MnLDH) was obtained from the oriental river prawn Macrobrachium nipponense, and was characterized. The full-length cDNA is 2267-bp with an open reading frame of 999 bp coding for a protein of 333 amino acids with conserved domains important for function and regulation. Phylogenetic analysis showed that MnLDH is close to LDHs from other invertebrates. Quantitative real-time PCR revealed that MnLDH is expressed in various tissues with the highest expression level in muscle. MnLDH mRNA transcript and protein abundance in muscle, but not in hepatopancreas, were induced by hypoxia. Silencing of hypoxia-inducible factor 1 (HIF-1) α or HIF-1β subunits blocked the hypoxia-dependent increase of LDH expression and enzyme activity in muscle. A series of MnLDH promoter sequences, especially the full-length promoter, generated an increase in luciferase expression relative to promoterless vector; furthermore, the expression of luciferase was induced by hypoxia. These results demonstrate that MnLDH is probably involved a HIF-1-dependent pathway during hypoxia in the highly active metabolism of muscle.

Effects of Low Temperature on Antioxidant and Heat Shock Protein Expression Profiles and Transcriptomic Responses in Crayfish (Cherax destructor)

“…CSPs are a critical factor required for cellular adaptation to low temperatures and overcoming the deleterious effects of cold stress [ 94 , 95 ]. Currently, increasing the expression level of CSP at low temperatures has been found in crustaceans such as M. nipponense , C. quadricarinatus , and Alvinocaris longirostris [ 96 , 97 , 98 ]. Similar results were also found in our experiments.…”

Glucose transporter GLUT1 expression is important for oriental river prawn (Macrobrachium nipponense) hemocyte adaptation to hypoxic conditions

“…Our previous studies in the oriental river prawn confirmed that the glycolytic pathway is regulated by HIF-1α via transcriptional activation of glycolytic enzyme genes in prawn muscle tissue in response to hypoxia, including LDH and HK ( 55 , 56 ). Furthermore, the present study confirmed that the GLUT1 promoter contains an HIF-1α-binding HRE using ChIP.…”

“…Similarly, we constructed plasmids pGL3-GLUT2-1, pGL3-GLUT2-2, and pGL3-GLUT2-3. The protocols detailed in a previous study were used for the luciferase assay and transfection experiments under normoxic and hypoxic conditions ( 56 ). Briefly, the pGL3-reporter plasmids containing one copy of each potential HRE site were transfected into hemocytes using the X-trem-eGENE HP DNA transfection reagent (Roche).…”

Identifying Relationships between Glutathione S-Transferase-2 Single Nucleotide Polymorphisms and Hypoxia Tolerance and Growth Traits in Macrobrachium nipponense

“…Currently, SNP studies in aquatic animals have predominantly focused on growth traits; previous studies successfully identified growth-related SNP loci in several shrimp species, including M. japonicus [ 33 ], M. rosenbergii [ 34 ], and Litopenaeus vannamei [ 35 ]. Previous studies also reported that lactate dehydrogenase [ 36 ], glutathione peroxidase [ 37 ], p53 [ 38 ], GST [ 9 ], and caspase-3 [ 39 ] were involved in hypoxia tolerance in M. nipponense . GST-2 is an important detoxification enzyme, with key roles in responses to hypoxia [ 40 ].…”