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.

丝氨酸蛋白酶抑制剂 (SPI) 因其对参与免疫反应的特定蛋白酶的抑制作用而被大量报道，但与钙质贝壳相关的 SPI 数据却很少。此前，我们课题组曾报道过无核珍珠粉的蛋白质组分析，酸溶部分中含有两个 Kunitz 结构域的候选基质蛋白引起了我们的关注。本研究从三角帆蚌中获得HcKuSPI全长cDNA序列。 HcKuSPI在地幔中特异表达，杂交信号主要集中在地幔边缘的背侧上皮细胞，在地幔皮质上信号较弱，表明HcKuSPI参与了壳的形成。 嗜水气单胞菌和金黄色葡萄球菌攻击后，地幔中的 HcKuSPI 表达上调外层液体（EPF）。谷胱甘肽S转移酶(GST)-HcKuSPI重组蛋白对蛋白酶、胰蛋白酶和胰凝乳蛋白酶表现出很强的抑制活性。此外，在壳再生过程中的表膜生长和晶体沉积过程中，实验组中的HcKuSPI表达显着高于对照组，而新生棱柱和珍珠片的有机壳框架在HcKuSPI RNA干扰（RNAi）后被完全破坏。因此，外套膜分泌的HcKuSPI可以有效中和细菌感染期间EPF中过量的蛋白酶和细菌蛋白酶，并可以通过抑制蛋白酶的蛋白水解活性来防止基质蛋白胞外降解，从而保证壳生物矿化的顺利进行。此外，GST-HcKuSPI 对于晶体形态调控也至关重要。这些结果对于我们理解 SPI 在贝壳生物矿化过程中的潜在作用具有重要意义。