Abstract
Carbonic anhydrase 8 (CA8), an isozyme of α-carbonic anhydrases, lacks the ability to catalyze the reversible hydration of CO2 to bicarbonate and proton. Previous studies have shown that single point mutations of CA8, CA8-S100P, and CA8-G162R, are associated with novel syndromes including congenital ataxia and mild cognitive impairment. Our previous results demonstrated that overexpression of wild type (WT) CA8 promoted cell proliferation, neurite outgrowth, anti-apoptosis, invasion and migration abilities in neuronal cells. In this study, we examined the expressions and functions of CA8-S100P and CA8-G162R in neuroblastoma cells lines, compared with those of WT CA8. Our results show that the protein expressions of mutant CA8-S100P and CA8-G162R were significantly decreased in Neuro-2a and SK-N-SH cells. Interestingly, CA8-S100P demonstrated a significant increase in cell proliferation in both Neuro-2a and SK-N-SH cells. However, both CA8 mutations showed significantly decreased effects on cell protection and migration in SK-N-SH cells. Surprisingly, a significant increase of invasive ability was observed in SK-N-SH cells with overexpression of CA8-S100P as compared with those with overexpression of WT CA8 under retinoic acid (RA) treatment. In addition, we found that Neuro-2a cells with overexpression of CA8-S100P and CA8-G162R showed significantly increased neurite outgrowth. Taken together, our data suggest that the expressions of CA8-S100P and CA8-G162R in neuronal cells alter cell morphology, proliferation, mobility and viability; indicating that the homozygous point mutations of CA8 lead to not only the loss of WT CA8 function, but also the gain of novel functions leading to neuromuscular dysfunction.
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Abbreviations
- α-CAs:
-
α-Carbonic anhydrases
- CA8:
-
Carbonic anhydrase related protein 8
- CHX:
-
Cycloheximide
- DAPI:
-
4,6-Diamidino-2-phenylindole
- DEPC:
-
Diethylpyrocarbonate
- DMEM:
-
Dulbecco’s Modified Eagle’s Medium
- DRB:
-
5,6-Dichloro-1-β-d-ribofuranosylbenzimidazole
- DTT:
-
Dithiothreitol
- ER:
-
Endoplasmic reticulum
- FBS:
-
Fetal bovine serum
- IVT:
-
In vitro Transcription/translation
- IP3 :
-
1,4,5-Trisphosphate
- IP3RI:
-
1,4,5-Trisphosphate receptor type I
- MALS:
-
Macroautophagy-lysosome system
- PA:
-
Paraformaldehyde
- PI:
-
Propidium iodide
- RA:
-
Retinoic acid
- RYRs:
-
Ryanodine receptors
- STS:
-
Staurosporine
- UPS:
-
Ubiquitin–proteasome system
- wdl :
-
Waddle
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Acknowledgements
We thank the National RNAi Core Facility at Academia Sinica in Taiwan for providing the RNAi reagents and related services.
Funding
This work was supported by grants from the Ministry of Science and Technology of the Republic of China (MOST105-2320-B-029-001-MY3; MOST104-2320-B-029-001; MOST103-2311-B-029-002; MOST102-2311-B-029-002; NSC101-2311-B-029-001).
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MH and THC conceived and designed the study; THC, THH performed the experiments; THC, BYH and PSL analyzed data; BYH and THC provided statistical analysis; THC, BYH, and MH wrote and revised the manuscript. All authors read and approved the final manuscript.
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10571_2020_907_MOESM1_ESM.pptx
Figure S1. Protein structure simulation of the wild type CA8. From RCSB protein data bank, CA8 structure prediction was obtained (PDB ID: 2W2J). Molecular graphics images were produced using Jmol software. (A) Serine at amino acid position 100 is at the C-terminal region of a β-strand (as indicated by arrow) (B) Glycine at amino acid position 162 is in the middle of a loop which connects two β-strands (as indicated by arrow) (PPTX 372 kb)
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Chi, TH., Hsieh, B.Y., Liang, PS. et al. Expression and Functional Study of Single Mutations of Carbonic Anhydrase 8 in Neuronal Cells. Cell Mol Neurobiol 41, 1355–1371 (2021). https://doi.org/10.1007/s10571-020-00907-1
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DOI: https://doi.org/10.1007/s10571-020-00907-1