Identification of the role of Rh protein in ammonia excretion of swimming crab Portunus trituberculatus

Abstract

In Portunus trituberculatus, a full-length cDNA of Rhesus-like glycoprotein (Rh protein), the whole 478 amino acids, has been identified in gills, which plays an essential role in ammonia (NH3 /NH4+) excretion. Phylogenetic analysis of the Rh-like proteins from crabs was clustered, showing high conservation of the ammonium transporter domain and transmembrane segments essential to the function of Rh protein. Rh protein of P. trituberculatus (PtRh) was detected in all tested tissues, and showed the highest expression in gills. To further characterize the role of PtRh in ammonia metabolism and excretion, a double-stranded RNA-mediated RNA interference of PtRh was employed. The knockdown of PtRh up-regulated mRNA expression of ammonia excretion related genes aquaporin (AQP), K+-channel, vesicle associated membrane protein (VAMP), increased activities of Na+ /K+ -ATPase (NKA) and V-type H+-ATPase (V-ATPase), whereas the Na+/H+-exchanger (NHE) expression reduced firstly and then elevated. dsRNA-mediated reductions in PtRh significantly reduced ammonia excretion rate and increased ammonia and glutamine (Gln) levels in hemolymph, together with increase of glutamate dehydrogenase (GDH) and glutamine synthetase (GS) activites, indicating a central role for PtRh in ammonia excretion and detoxification mechanisms. Taken together, we conclude that the Rh protein is a primary contributor to ammonia excretion of P. trituberculatus, which may be the basis of their ability to inhabit benthic water with high ammonia levels.