Cloning and characterisation of mitochondrial manganese superoxide dismutase (mtMnSOD) from the giant freshwater prawn Macrobrachium rosenbergii

Abstract

A cDNA encoding a mitochondrial manganese superoxide dismutase (mtMnSOD) was cloned from the hepatopancreas of giant freshwater prawn Macrobrachium rosenbergii using reverse transcription polymerase chain reaction (RT-PCR) by degenerate primers. Both 3'- and 5'-regions were isolated by rapid amplification of cDNA end (RACE) PCR method. Analysis of nucleotide sequence revealed that the mtMnSOD full-length cDNA consists of 1202bp containing an open reading frame of 654bp, which encodes a protein consisting of 218 amino acids including a signal peptide of 16 amino acid residues. The calculated molecular mass of the mature proteins (202 amino acids) is 24kDa with an estimated pI of 7.12. Two putative N-glycosylation sites, NXT and NXS were observed in the mtMnSOD. Manganese superoxide dismutase signatures from 180 to 187 (DVWEHAYY), and four conserved amino acids responsible for binding manganese were observed (H48, H96, D180 and H184). Sequence comparison showed that the mtMnSOD deduced amino acid sequence of Macrobrachium rosenbergii has similarity of 88%, 78%, 56%, 54% and 46% to that of blue crab Callinectes sapidus, crucifix crab Charybdis feriatus, brown shrimp Farfantepenaeus aztecus, European lobster Palinurus vulgaris, and grass shrimp Palaemontes pugio, respectively, and has similarity of 45%, 44%, 43%, 26% and 25% to cytMnSOD (cytosolic MnSOD) deduced amino acid sequence of blue crab C. sapidus, prawn M. rosenbergii, tiger shrimp Penaeus monodon, grass shrimp P. pugio and brown shrimp F. aztecus, respectively. Quantitative real-time RT-PCR analysis showed that levels of mtMn-SOD transcripts in hepatopancreas and haemocytes were not significantly different between the M. rosenbergii injected with Lactococcus garvieae, and that injected with saline after 3h to 24h.