Glycosylation of hemocyanin in Litopenaeus vannamei is an antibacterial response feature

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.