Data from: Branchial CO2 and ammonia excretion in crustaceans: Involvement of an apical Rhesus-like glycoprotein

Aim To determine whether the crustacean Rh1 protein functions as a dual
CO2/ammonia transporter and investigate its role in branchial ammonia
excretion and acid-base regulation. Methods Sequence analysis of decapod
Rh1 proteins was used to determine the conservation of amino acid residues
putatively involved in ammonia transport and CO2 binding in human and
bacterial Rh proteins. Using the Carcinus maenas Rh1 protein (CmRh1) as a
representative of decapod Rh1 proteins, we test the ammonia and CO2
transport capabilities of CmRh1 through heterologous expression in yeast
and Xenopus oocytes coupled with site-directed mutagenesis. Quantitative
PCR was used to assess distribution of CmRh1 mRNA in various tissues.
Western blotting was used to assess CmRh1 protein expression changes in
response to high environmental ammonia and CO2. Further,
immunohistochemistry was used to assess sub-cellular localization of CmRh1
and a membrane bound carbonic anhydrase (CmCAg). Results Sequence analysis
of decapod Rh proteins revealed high conservation of several amino acid
residues putatively involved in conducting ammonia transport and CO2
binding. Expression of CmRh1 in Xenopus oocytes enhanced both ammonia and
CO2 transport which was nullified in CmRh1 D180N mutant oocytes. Transport
of the ammonia analogue methylamine by CmRh1 is dependent on both ionized
and un-ionized ammonia/methylamine species. CmRh1 was co-localized with
CmCAg to the apical membrane of the crustacean gill and only experienced
decreased protein expression in the anterior gills when exposed to high
environmental ammonia. Conclusion CmRh1 is the first identified apical
transporter-mediated route for ammonia and CO2 excretion in the crustacean
gill. Our findings shed further light on the potential universality of
dual ammonia and CO2 transport capacity of Rhesus glycoproteins in both
vertebrates and invertebrates.

研究目的：明确甲壳动物Rh1蛋白是否兼具二氧化碳（CO₂）与氨双重转运功能，并探究其在鳃部氨排泄及酸碱稳态调控中的作用。
研究方法：对十足目Rh1蛋白开展序列分析，以明确人类与细菌Rh蛋白中推定参与氨转运及CO₂结合的氨基酸残基的保守性。以欧洲绿蟹（Carcinus maenas）Rh1蛋白（CmRh1）作为十足目Rh1蛋白的代表模型，通过在酵母与非洲爪蟾卵母细胞（Xenopus oocytes）中进行异源表达并结合定点诱变技术，检测CmRh1的氨与CO₂转运能力。采用定量聚合酶链反应（Quantitative PCR）检测CmRh1 mRNA在各组织中的表达分布情况；采用蛋白质免疫印迹（Western blotting）检测CmRh1蛋白在高环境氨与CO₂胁迫下的表达变化；进一步采用免疫组织化学技术分析CmRh1与膜结合型碳酸酐酶（CmCAg）的亚细胞定位。
研究结果：十足目Rh蛋白的序列分析结果显示，多个推定参与氨转运及CO₂结合的氨基酸残基具有高度保守性。在非洲爪蟾卵母细胞中表达CmRh1可显著增强其氨与CO₂转运能力，而该转运能力在CmRh1 D180N突变体卵母细胞中完全丧失。CmRh1对氨类似物甲胺的转运依赖于离子化与非离子化形式的氨/甲胺。CmRh1与CmCAg共定位于甲壳动物鳃的顶膜，且仅在暴露于高环境氨时，其在前鳃中的蛋白表达水平出现下调。
研究结论：CmRh1是首个在甲壳动物鳃中被发现的、由顶膜转运蛋白介导的氨与CO₂排泄通路。本研究结果进一步阐明了Rh糖蛋白（Rhesus glycoproteins）在脊椎动物与无脊椎动物中兼具氨与CO₂转运能力的潜在普遍性。