Molecular characterization of three Rhesus glycoproteins from the gills of the African lungfish, Protopterus annectens, and effects of aestivation on their mRNA expression levels and protein abundance

African lungfishes are ammonotelic in water. They can aestivate for long periods on land during drought. During aestivation, the gills are covered with dried mucus and ammonia excretion ceases. In fishes, ammonia excretion through the gills involves Rhesus glycoproteins (RhGP/Rhgp). This study aimed to obtain the complete cDNA coding sequences of rhgp from the gills of Protopterus annectens, and to determine their branchial mRNA and protein expression levels during the induction, maintenance and arousal phases of aestivation. Three isoforms of rhgp (rhag, rhbg and rhcg) were obtained in the gills of P. annectens. Their complete cDNA coding sequences ranged between 1311 and 1398 bp, coding for 436 to 465 amino acids with estimated molecular masses between 46.8 and 50.9 kDa. Dendrogramic analyses indicated that Rhag was grouped closer to fishes, while Rhbg and Rhcg were grouped closer to tetrapods. During the induction phase, the protein abundance of Rhag, but not its transcript level, was down-regulated in the gills, suggesting that there could be a decrease in the release of ammonia from the erythrocytes to the plasma. Furthermore, the branchial transcript levels of rhbg and rhcg decreased significantly, in preparation for the subsequent shutdown of gill functions. During the maintenance phase, the branchial expression levels of rhag/Rhag, rhbg/Rhbg and rhcg/Rhcg decreased significantly, indicating that their transcription and translation were down-regulated. This could be part of an overall mechanism to shut down branchial functions and save metabolic energy used for transcription and translation. It could also be regarded as an adaptive response to stop ammonia excretion. During the arousal phase, it is essential for the lungfish to regain the ability to excrete ammonia. Indeed, the protein abundance of Rhag, Rhbg and Rhcg recovered to the corresponding control levels after 1 day or 3 days of recovery from 6 months of aestivation.非洲肺鱼在水环境中为排氨动物。在干旱时期，它们可在陆地上进行长期夏眠。夏眠期间，鳃会被干燥的黏液覆盖，氨排泄过程随之停止。在鱼类中，经鳃进行的氨排泄依赖于Rh蛋白（Rhesus glycoproteins，RhGP/Rhgp）。本研究旨在获取安氏原鳍肺鱼（Protopterus annectens）鳃组织中rhgp基因的完整cDNA编码序列，并测定其在夏眠的诱导期、维持期与觉醒期的鳃部mRNA及蛋白表达水平。研究人员从安氏原鳍肺鱼的鳃组织中获得了3种rhgp同工型（rhag、rhbg与rhcg）。其完整cDNA编码序列长度介于1311至1398 bp之间，编码的氨基酸残基数为436至465，预估分子质量介于46.8至50.9 kDa之间。系统树分析结果显示，Rhag与鱼类的亲缘关系更近，而Rhbg与Rhcg则更接近四足动物。在夏眠诱导期，Rhag的蛋白丰度（而非其转录水平）出现下调，这提示氨从红细胞向血浆的释放过程可能有所减弱。此外，rhbg与rhcg的鳃部转录水平显著下降，为后续鳃功能的关闭做好准备。在夏眠维持期，rhag/Rhag、rhbg/Rhbg及rhcg/Rhcg的鳃部表达水平均显著降低，表明其转录与翻译过程均被下调。这一现象可能是整体关闭鳃功能、节省转录与翻译所需代谢能的机制之一，也可被视为停止氨排泄的适应性响应。在夏眠觉醒期，肺鱼需要重新获得氨排泄能力。实验结果证实，经6个月夏眠后，在恢复饲养1天或3天时，Rhag、Rhbg与Rhcg的蛋白丰度即可恢复至相应对照组水平。