Department of Biology and Guangdong Provincial Key laboratory of Marine Biotechnology, Shantou University, Shantou 515063, China.
Department of Biology and Guangdong Provincial Key laboratory of Marine Biotechnology, Shantou University, Shantou 515063, China.
Immunol Lett. 2017 Dec;192:42-47. doi: 10.1016/j.imlet.2017.10.008. Epub 2017 Oct 16.
Hemocyanin is an important multifunctional non-specific immune molecule. In this study, we purified lectin binding and non-lectin binding hemocyanin from Litopenaeus vannamei using Concanavalin A (ConA) lectin affinity chromatography (designated HMC-C and HMC-NC, respectively). Analysis of the carbohydrate content showed that HMC-C had about 20 times as much carbohydrate as HMC-NC. 54 and 42 peaks were observed in HMC-C and HMC-NC by HPLC, which reduced to 49 and 6 peaks, respectively, when digested with trypsin and repurified with ConA lectin column. Further, the agglutinative activity of HMC-C against two pathogenic bacteria, Vibrio alginolyticus and Vibrio fluvialis, was about 8-fold and 4-fold, respectively, to that of HMC-NC. While the antibacterial activity of HMC-NC was about 30% lower compared with HMC-C. Similarly, when HMC was deglycosylated using O-glycosidase, its agglutinative activity reduced about 4-8 fold. Most importantly, when shrimps were challenged with V. alginolyticus or V. fluvialis, the glycan content of hemocyanin increased dramatically and remained high at the earlier time points (24-72h) post infection, only decreasing after 96 hpi. Taken together, these results suggest that hemocyanin glycosylation plays an important role in its antibacterial properties.
血蓝蛋白是一种重要的多功能非特异性免疫分子。在本研究中，我们使用刀豆球蛋白 A（ConA）亲和层析法从凡纳滨对虾中纯化了具有凝集素结合和非凝集素结合功能的血蓝蛋白（分别命名为 HMC-C 和 HMC-NC）。糖含量分析表明，HMC-C 的糖含量约为 HMC-NC 的 20 倍。HMC-C 和 HMC-NC 通过 HPLC 分别观察到 54 和 42 个峰，经胰蛋白酶消化并再次用 ConA 亲和层析柱纯化后分别减少到 49 和 6 个峰。此外，HMC-C 对两种致病性细菌——溶藻弧菌和鳗弧菌的凝集活性分别比 HMC-NC 高约 8 倍和 4 倍。而 HMC-NC 的抗菌活性比 HMC-C 低约 30%。同样，当使用 O-糖苷酶对 HMC 进行去糖基化处理时，其凝集活性降低了约 4-8 倍。最重要的是，当虾受到溶藻弧菌或鳗弧菌的攻击时，血蓝蛋白的糖含量会急剧增加，并在感染后早期（24-72 小时）保持高水平，仅在 96 小时后才下降。综上所述，这些结果表明血蓝蛋白的糖基化在其抗菌特性中起着重要作用。
