The involvement of a novel calmodulin-like protein isoform from oyster Crassostrea gigas in transcription factor regulation provides new insight into acclimation to ocean acidification

• Published in: Aquatic toxicology 2023-04, Vol.257, p.106423-106423, Article 106423
• Main Authors: Li, Changmei, Wang, Yilin, Wei, Manman, Wang, Xiudan
• Format: Article
• Language: English
• Subjects: Acclimatization; Alternative splicing; Animals; Calmodulin - genetics; Calmodulin - metabolism; Calmodulin-like protein; Carbon Dioxide - metabolism; Carbon Dioxide - toxicity; Crassostrea - metabolism; Hemocytes - metabolism; Hydrogen-Ion Concentration; Ocean Acidification; Pacific oyster; Protein Isoforms - genetics; Protein Isoforms - metabolism; Seawater; Transcription factor; Transcription Factors - metabolism; Water Pollutants, Chemical - toxicity
• ISSN: 0166-445X; 1879-1514
• DOI: 10.1016/j.aquatox.2023.106423

•A novel CgCaLP-2 gene was identified in Crassostrea gigas and it underwent alternative splicing.•Two protein isoforms were detected and the 23 kD form mainly responded to long-term CO2 exposure.•CgCaLP-2 redistributed to nuclear membrane after long-term CO2 exposure.•The expression of a transcription factor CgSIX4 was associated with CgCaLP-2 under CO2 exposure and in vivo knock down further verified their relevance. Marine organisms need to adapt to improve organismal fitness under ocean acidification (OA). Recent studies have shown that marine calcifiers can achieve acclimation by stimulating calcium binding/signaling pathways. Here, a CaM-like gene (CgCaLP-2) from oyster Crassostrea gigas which typically responded to long-term CO2 exposure (two months) rather than short-term exposure (one week) was characterized. The cloned cDNA was 678 bp and was shorter than the retrieved sequence from NCBI (1125 bp). The two sequences, designated as CgCaLP-2-v1 and CgCaLP-2-v2, were demonstrated to be different splice variants by the genome sequence analysis. Western blotting analysis revealed two bands of 23 kD and 43 kD in mantle and hemocytes, corresponding to predicted molecular weight of CgCaLP-2-v1 and CgCaLP-2-v2, respectively. The isoform CgCaLP-2-v1 (the 23 kD band) was highly stimulated in response to long-term CO2 exposure (42-day and 56-day treatment) in hemocytes and mantle tissue. The fluorescence signal of CgCaLP-2 in mantle and hemocytes became more intensive after long-term CO2 exposure. Besides, in hemocytes, CgCaLP-2 presented a higher localization on the nuclear membrane after long-term CO2 exposure (56 d). The target gene network of CgCaLP-2 was predicted, and a transcription factor (TF) gene annotated as Homeobox protein SIX4 (CgSIX4) showed a similar expressive trend to CgCaLP-2 during CO2 exposure. Suppression of CgCaLP-2 via RNA interference significantly reduced the mRNA expression of CgSIX4. The results suggested that CgCaLP-2 might mediate the Ca2+-CaLP-TF signal transduction pathway under long-term CO2 exposure. This study serves as an example to reveal that alternative splicing is an important mechanism for generation multiple protein isoforms and thus shape the plastic responses under CO2 exposure, providing new insight into the potential acclimation ability of marine calcifiers to future OA.