Institute of Marine Sciences and Guangdong Provincial Key Laboratory of Marine Biotechnology, Shantou University, Shantou, China.
College of Fisheries, Henan Normal University, Xinxiang, China.
Front Immunol. 2021 Dec 7;12:772091. doi: 10.3389/fimmu.2021.772091. eCollection 2021.
Hemocyanin is an important non-specific innate immune defense molecule with phenoloxidase, antiviral, antibacterial, hemolytic, and antitumor activities. To better understand the mechanism of functional diversity, proteomics approach was applied to characterize hemocyanin (HMC) expression profiles from . At first, hemocyanin was purified by Sephadex G-100 and DEAE-cellulose (DE-52) columns from shrimp serum, and 34 protein spots were identified as HMC on the 2-DE gels. Furthermore, we found that 9 HMC spots about 75 or 77 kDa were regulated by and infection at 6, 12, and 24 h. In addition, 6 different pathogen-binding HMC fractions, viz., HMC-Mix, HMC-Vp, HMC-Va, HMC-Vf, HMC-Ec, and HMC-Sa, showed different agglutinative and antibacterial activities. Moreover, lectin-blotting analysis showed significant differences in glycosylation level among HMC isomers and bacteria-binding HMC fractions. Particularly, the agglutinative activities of the HMC fractions were almost completely abolished when HMC was deglycosylated by O-glycosidase, which suggest that O-linked sugar chains of HMC played important roles in the innate immune recognition. Our findings demonstrated for the first time that HMC had molecular diversity in protein level, which is closely associated with its ability to recognize diverse pathogens, whereas glycan modification probably contributed to HMC's diversity and multiple immune activities.
血蓝蛋白是一种重要的非特异性先天免疫防御分子，具有酚氧化酶、抗病毒、抗菌、溶血和抗肿瘤活性。为了更好地理解功能多样性的机制，采用蛋白质组学方法来描绘 虾血清中血蓝蛋白（HMC）的表达谱。首先，通过 Sephadex G-100 和 DEAE-纤维素（DE-52）柱从虾血清中纯化血蓝蛋白，在 2-DE 凝胶上鉴定出 34 个蛋白斑点为 HMC。此外，我们发现 9 个约 75 或 77 kDa 的 HMC 斑点在 6、12 和 24 h 时受到 和 感染的调节。此外，6 种不同的病原体结合 HMC 部分，即 HMC-Mix、HMC-Vp、HMC-Va、HMC-Vf、HMC-Ec 和 HMC-Sa，显示出不同的凝集和抗菌活性。此外，凝集素印迹分析显示 HMC 异构体和细菌结合 HMC 部分之间的糖基化水平存在显著差异。特别是，当 HMC 被 O-糖基酶去糖基化时，HMC 部分的凝集活性几乎完全被抑制，这表明 HMC 的 O-连接糖链在先天免疫识别中起着重要作用。我们的研究结果首次表明， HMC 在蛋白质水平上具有分子多样性，这与其识别多种病原体的能力密切相关，而糖基化修饰可能有助于 HMC 的多样性和多种免疫活性。
