Anjugam Mahalingam, Vaseeharan Baskaralingam, Iswarya Arokiadhas, Amala Muthu, Govindarajan Marimuthu, Alharbi Naiyf S, Kadaikunnan Shine, Khaled Jamal M, Benelli Giovanni
Biomaterials and Biotechnology in Animal Health Lab, Department of Animal Health and Management, Alagappa University, Karaikudi 630004, Tamil Nadu, India.
Biomaterials and Biotechnology in Animal Health Lab, Department of Animal Health and Management, Alagappa University, Karaikudi 630004, Tamil Nadu, India.
Mol Immunol. 2017 Dec;92:1-11. doi: 10.1016/j.molimm.2017.09.013. Epub 2017 Oct 10.
The present study reports the purification of novel immune molecule β-1, 3 glucan binding protein from the heamolymph of the Indian white shrimp, Fenneropenaeus indicus (Fiβ-GBP). The purified Fiβ-GBP had 95kDa molecular weight in SDS-PAGE analysis. MALDI-TOF/TOF analysis revealed that the purified Fiβ-GBP showed similarity to various crustacean proteins; 48 and 46% similarity was observed for β-1, 3 glucan binding protein of Chinese white shrimp Fenneropenaeus chinensis and banana shrimp Fenneropenaeus merguiensis, with MOWSE score of 3.11e+12 and 2.05e+8, respectively. The phenoloxidase activity (PO) of Fiβ-GBP was evaluated and, in the presence of laminarin, PO activity increased significantly. Substrate specificity assay demonstrated that Fiβ-GBP had the specific binding site for soluble or insoluble β-glucan (laminarin), since the PO activity increased in the presence of laminarin when compared to other sugars. Enzymatic activities revealed that the optimum temperature and pH for Fiβ-GBP activating PO were 40°C and pH 7-8. Moreover, even at 100°C Fiβ-GBP enhanced PO activity highlighting that Fiβ-GBP was thermostable and thermophilic in nature. Among various divalent metallic ions, Fiβ-GBP significantly promoted the PO activity in presence of Mg and Ca. The breakdown of para nitroanilide from N-Benzoyl-l-Arginine 4-Nitroanilide hydrochloride showed that serine protease activity was induced by Fiβ-GBP and also increased concentration of Fiβ-GBP evoked the activity. Furthermore, hemolytic activity tests revealed that PO reaction product induced RBC membrane damage and cell shrinkage. Lastly, Baclight bacterial viability assays showed maximum killing effect of PO reaction product on both Gram positive and Gram negative bacteria.
本研究报道了从印度白对虾凡纳滨对虾血淋巴中纯化新型免疫分子β-1,3-葡聚糖结合蛋白（Fiβ-GBP）。在SDS-PAGE分析中，纯化后的Fiβ-GBP分子量为95kDa。基质辅助激光解吸电离飞行时间串联质谱（MALDI-TOF/TOF）分析表明，纯化后的Fiβ-GBP与多种甲壳类蛋白质具有相似性；中国对虾凡纳滨对虾和墨吉对虾的β-1,3-葡聚糖结合蛋白与之的相似性分别为48%和46%，MOWSE分数分别为3.11e+12和2.05e+8。对Fiβ-GBP的酚氧化酶活性（PO）进行了评估，在海带多糖存在的情况下，PO活性显著增加。底物特异性分析表明，Fiβ-GBP对可溶性或不溶性β-葡聚糖（海带多糖）具有特异性结合位点，因为与其他糖类相比，在海带多糖存在时PO活性增加。酶活性显示，Fiβ-GBP激活PO的最佳温度和pH分别为40°C和pH 7-8。此外，即使在100°C时，Fiβ-GBP仍能增强PO活性，这突出表明Fiβ-GBP本质上是耐热和嗜热的。在各种二价金属离子中，Fiβ-GBP在Mg和Ca存在时显著促进PO活性。对盐酸N-苯甲酰-L-精氨酸4-硝基苯胺中对硝基苯胺的分解表明，Fiβ-GBP诱导了丝氨酸蛋白酶活性，并且Fiβ-GBP浓度的增加也引发了该活性。此外，溶血活性测试表明，PO反应产物会导致红细胞膜损伤和细胞收缩。最后，Baclight细菌活力测定表明，PO反应产物对革兰氏阳性菌和革兰氏阴性菌均具有最大杀伤作用。
