Molecular cloning, mRNA expression and characterization of membrane-bound hemoglobin in oriental river prawn Macrobrachium nipponense

Most hemoglobins are respiratory proteins and are ubiquitous in animals, bacteria, fungi, protists, and plants. In this study, we describe a membrane-bound hemoglobin in the oriental river prawn Macrobrachium nipponense (MnHb), which also expresses hemocyanin. MnHb cDNA was cloned using the rapid amplification of cDNA ends (RACE) approach, which afforded a 1201 bp gene encoding a 193 amino acid polypeptide. Bioinformatic evaluation suggested MnHb is membrane anchored by N-myristoylation, and immunofluorescence confirmed its location in the membrane of chief cells in the gill. The effect of hypoxia on MnHb expression was investigated, and reverse transcription PCR (RT-PCR) and Western blotting showed that MnHb was expressed almost exclusively in the gill. Quantitative RT-PCR revealed a significant increase in expression after 6 h of hypoxia, and levels peaked at 24 h due to oxidative stress. Exposure of cultured prawns to the stress inducer H2O2 significantly up-regulated the expression of MnHb in a dose-dependent manner. MnHb may have a role in protecting cell membrane lipids from damage by reactive oxygen species.