Junprung Wisarut, Supungul Premruethai, Sangklai Nutthapon, Tassanakajon Anchalee
Center of Excellence for Molecular Biology and Genomics of Shrimp, Department of Biochemistry, Faculty of Science, Chulalongkorn University, Bangkok, Thailand; and.
Aquatic Molecular Genetics and Biotechnology Research Team, National Center for Genetic Engineering and Biotechnology, National Science and Technology Development Agency, Pathumthani, Thailand.
J Immunol. 2022 Aug 1;209(3):582-592. doi: 10.4049/jimmunol.2100804.
Recent studies have initiated a paradigm shift in understanding heat shock protein 70 (HSP70) functions in the shrimp immune system. However, the mechanism by which Litopenaeus vannamei (Lv)HSP70 modulates the innate immune response remains unclear. This study shows that LvHSP70 binds to the pattern recognition receptor LPS and β-1,3-glucan-binding protein (LvLGBP), and subsequently leads to the activation of the prophenoloxidase system. Injection of shrimp with rLvHSP70 significantly (p < 0.05) upregulated the gene and protein expression of the key pattern recognition receptor LvLGBP. A coimmunoprecipitation and ELISA-based binding assay strongly confirmed the binding of LvHSP70 to LvLGBP at polysaccharide recognition motifs (PLS motifs) with a Kd of 4.44 μM and its competitive binding with LPS (IC50) is 8.036 μM. Conversely, LPS efficiently competed with LvHSP70 for binding to LvLGBP in a concentration-dependent manner with an IC50 of 7.662 μM, indicating that both are ligands of LvLGBP and likely bind at the same site. Binding of LvHSP70 to LvLGBP highly activated phenoloxidase activity in shrimp hemocyte lysate supernatants. Gene silencing of LvLGBP impaired the activation of phenoloxidase activity in shrimp by rLvHSP70, indicating that LvHSP70-LvLGBP interaction was essential for stimulating the immune cascade. Taken together, these results demonstrated that LvHSP70 is a ligand of LvLGBP similar to LPS and acts as a damage-associated molecular pattern to modulate the shrimp immune system via the prophenoloxidase system, eventually leading to the production of melanin and toxic reactive intermediates against invading pathogens.
最近的研究引发了对热休克蛋白70（HSP70）在对虾免疫系统中功能理解的范式转变。然而，凡纳滨对虾（Lv）HSP70调节先天免疫反应的机制仍不清楚。本研究表明，LvHSP70与模式识别受体脂多糖（LPS）和β-1,3-葡聚糖结合蛋白（LvLGBP）结合，随后导致酚氧化酶原系统的激活。用重组LvHSP70注射对虾显著（p < 0.05）上调了关键模式识别受体LvLGBP的基因和蛋白表达。基于免疫共沉淀和酶联免疫吸附测定的结合试验有力地证实了LvHSP70与LvLGBP在多糖识别基序（PLS基序）处结合，解离常数（Kd）为4.44 μM，其与LPS的竞争性结合半抑制浓度（IC50）为8.036 μM。相反，LPS以浓度依赖方式与LvHSP70有效竞争结合LvLGBP，IC50为7.662 μM，表明二者都是LvLGBP的配体且可能结合于同一位点。LvHSP70与LvLGBP的结合高度激活了对虾血细胞裂解液上清中的酚氧化酶活性。LvLGBP基因沉默削弱了重组LvHSP70对虾酚氧化酶活性的激活，表明LvHSP70-LvLGBP相互作用对于刺激免疫级联反应至关重要。综上所述，这些结果表明，LvHSP70是与LPS类似的LvLGBP配体，作为损伤相关分子模式通过酚氧化酶原系统调节对虾免疫系统，最终导致黑色素和针对入侵病原体的毒性反应性中间体的产生。
