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

Shengming Sun,Wenyi Zhang,Xugan Wu,Jian Zhu,Hongtuo Fu,Xianping Ge,Hui Qiao,Shubo Jin

doi:10.3390/ijms19071990

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.

Highlights

The higher lactate levels of hemolymph indicate that anaerobic metabolism makes a significant contribution to energy production in crustaceans during hypoxia [1]
Lactate dehydrogenase (LDH, EC 1.1.1.27) as an enzyme is found in almost all organisms, LDH catalyzes the conversion of lactate to pyruvic acid and back, as it converts NAD+ to NADH and back [2]
Amino acid sequence comparison showed that highly conserved residues were found in all LDHs, for example for substrate binding, and dimer/tetramer interface formation

Summary

Introduction

The higher lactate levels of hemolymph indicate that anaerobic metabolism makes a significant contribution to energy production in crustaceans during hypoxia [1]. Different LDH subunits (isoforms) can be included in LDH tetramers, such as LDH-A (mainly present in anaerobic muscle tissue), LDH-B (mainly present in aerobic heart tissue), and LDH-C (present only in mature testis), which show different tissue expression, kinetic, physicochemical and immunochemical properties [3]. Most invertebrates preferentially oxidize L-lactic acid, several species of mollusks, a few arthropods and polychaetes were found to have exclusively D-LDH enzymatic activity [4,5], and all LDHs from crustaceans have tetrameric structure [6,7,8] with around 140 kDa, 36 kDa per subunit [9]. Various invertebrates, including northern krill Meganyctiphanes norvegica [10,11], the Antarctic krill Euphausia superba [12] and the snow crab Chionoecetes opilio [13] contain multiple forms of LDH

Methods

Results

Conclusion