Data from: Branchial CO2 and ammonia excretion in crustaceans: Involvement of an apical Rhesus-like glycoprotein
Data files
Jan 21, 2025 version files 15.22 KB
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Figure_2A.csv
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Figure_2C.csv
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Figure_2D.csv
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Figure_4A.csv
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Figure_4B.csv
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Figure_4C.csv
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Figure_5.csv
525 B
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README.md
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Abstract
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.
README: Branchial CO2 and ammonia excretion in crustaceans: Involvement of an apical Rhesus-like glycoprotein
This data set provides data collected from 4 experiments.
Experiment 1:Gene expression of CmRh1 across different tissues and Protein expression in gills during exposure to high environmental ammonia and hypercapnia
Experiment 2:Time dependent radiolabelled methyl amine uptake in Xenopus oocytes expressing CmRh1, CmRh1 D180N variant and water-injected sham
Experiment 3: Effect of environmental ammonia, methyl amine concentration, and pH on radiolabelled methyl amine uptake in Xenopus oocytes expressing CmRh1, CmRh1 D180N variant and water-injected sham
Experiment 4: CO2 excretion rate in Xenopus oocytes expressing CmRh1, CmRh1 D180N variant and water injected sham
Description of the data and file structure
Figure 2A data is organized by gene assayed, sample, tissue type, treatment, starting quantity, and then a normalized gene expression comparing CmRh1 (gene of interest) to normalizing house keeping gene (arginine kinase or ribosomal protein S3). N/A indicates that there was no detectable expression for that particular sample.
Figure 2C+D data is organized by sample, tissue type, treatment, and relative protein expression of CmRh1. N/A indicates a missing protein sample for the particular individual.
Figure 4A data is organized by what was injected into the oocyte (water=sham, CmRh1 cRNA, or CmRh1 D180N variant cRNA), the incubation time, and the dependent variable which was rate of methyl amine uptake. N/A indicates an oocyte had burst prior to scintillation counting result in no reliable data for that replicate.
Figure 4B+C data is organized by what was injected into the oocyte (water=sham, CmRh1 cRNA, or CmRh1 D180N variant cRNA), the ovary used (1 or 2), the concentration of unlabelled methyl amine, pH,concentration of ammonium chloride, and the dependent variable which was rate of methyl amine uptake, and the final column reflects methyl amine uptake rate with subtraction of the average sham rate for each group. N/A indicates an oocyte had burst prior to scintillation counting result in no reliable data for that replicate or sham subtraction rate is not applicable for the particular sample.
Figure 5 data is organized by what was injected into the oocyte (water=sham, CmRh1 cRNA, or CmRh1 D180N variant cRNA) and the CO2 excretion rate measured. N/A indicates that the oocyte had burst resulting in no reliable measurement for that replicate.