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A Novel Kunitz-Type Serine Protease Inhibitor (HcKuSPI) is Involved in Antibacterial Defense in Innate Immunity and Participates in Shell Formation of Hyriopsis cumingii

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Abstract

Serine protease inhibitors (SPIs) are abundantly reported for its inhibition against specific proteases involved in the immune responses, but SPI data related to calcareous shells are scarce. Previously, our research group has reported the proteome analysis of non-nucleated pearl powder, and a candidate matrix protein containing two Kunitz domains in the acid soluble fraction caught our attention. In the present study, the full-length cDNA sequence of HcKuSPI was obtained from Hyriopsis cumingii. HcKuSPI was specifically expressed in the mantle, with hybridization signals mainly concentrated to dorsal epithelial cells at the mantle edge and weak signals at the mantle pallium, suggesting HcKuSPI was involved in shell formation. HcKuSPI expression in the mantle was upregulated after Aeromonas hydrophila and Staphylococcus aureus challenge to extrapallial fluids (EPFs). A glutathione S transferase (GST)-HcKuSPI recombinant protein showed strong inhibitory activity against the proteases, trypsin and chymotrypsin. Moreover, HcKuSPI expression in an experimental group was significantly higher when compared with a control group during pellicle growth and crystal deposition in shell regeneration processes, while the organic shell framework of newborn prisms and nacre tablets was completely destroyed after HcKuSPI RNA interference (RNAi). Therefore, HcKuSPI secreted by the mantle may effectively neutralize excess proteases and bacterial proteases in the EPF during bacterial infection and could prevent matrix protein extracellular degradation by suppressing protease proteolytic activity, thereby ensuring a smooth shell biomineralization. In addition, GST-HcKuSPI was also crucial for crystal morphology regulation. These results have important implications for our understanding of the potential roles of SPIs during shell biomineralization.

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All relevant data used to support the findings of this study are included within the article.

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Funding

This work was supported by The National Natural Science Foundation of China (32202921) and the Sci-tech Special Commissioners Project of Zhejiang Province-Pearl Quality Improvement and Industrialization (2020–2024).

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  1. School of Life and Environmental Sciences, Shaoxing University, Shaoxing, 312000, People’s Republic of China

    Can Jin, Kang Cheng, Rui Jiang, Yihang Zhang & Wen Luo

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  1. Can Jin
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  2. Kang Cheng
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  3. Rui Jiang
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Can Jin: Investigation, project administration, writing-original draft and funding acquisition. Kang Cheng and Rui Jiang: methodology, validation, experiment. Yihang Zhang: writing-review and editing. Wen Luo: supervision, funding acquisition. All authors contributed to the article and approved the submitted version.

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Correspondence to Wen Luo.

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Jin, C., Cheng, K., Jiang, R. et al. A Novel Kunitz-Type Serine Protease Inhibitor (HcKuSPI) is Involved in Antibacterial Defense in Innate Immunity and Participates in Shell Formation of Hyriopsis cumingii. Mar Biotechnol 26, 37–49 (2024). https://doi.org/10.1007/s10126-023-10275-8

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