Alternative splicing generates two lactate dehydrogenase subunits differentially expressed during hypoxia via HIF-1 in the shrimp Litopenaeus vannamei

Abstract

Metabolic adjustment to low oxygen exposure (hypoxia) in the white shrimp Litopenaeus vannamei implies a shift to anaerobic metabolism. Lactate dehydrogenase (LDH) is a key enzyme of the anaerobic metabolism described in most organisms. The structure and expression of the LDH gene, as well as the LDH isoenzymes in marine crustacean are not well defined. In the present study we characterized a gene that codes for two LDH subunits, measured their expression and detected the isoenzymes in tissues from white shrimp. We also silenced the transcriptional activator hypoxia inducible factor 1 (HIF-1) to elucidate the regulation of LDH in tissues from white shrimp in response to hypoxia. The complete LDH gene coding sequence is 7571 bp (LvanLDH) and encodes two different LDH subunits (LDHvan-1 and LDHvan-2) generated by alternative splicing and composed of 332 amino acids with conserved domains important for the function and regulation. Phylogenetic analysis shows that LvanLDH -1 and -2 are closer to the invertebrate counterparts. The LDHvan-1 transcript increased 2.5-fold after hypoxia in gills but not in hepatopancreas, while the LDHvan-2 transcript decreased 14-fold in muscle but not in gills and hepatopancreas. Three bands with LDH activity of ∼60–90 kDa were detected in hepatopancreas, while one band of ∼140 kDa was detected in gills and muscle. The silencing of HIF-1 blocked the increase of LDH mRNA and activity produced by hypoxia in gills. These results demonstrate a single gene for LDH (LvanLDH) that by alternative splicing generates two different LDH subunits (LDHvan-1 and LDHvan-2) that are expressed in a tissue-specific manner during hypoxia via the HIF-1 pathway.