Glycosylation of hemocyanin in Litopenaeus vannamei is an antibacterial response feature

Abstract

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.