IDENTIFICATION OF HEMOCYANIN GLYCOSYLATED SITES FROM SHRIMP Litopenaeus vennamei AND STUDY ON ITS CORRECTION WITH ANTIMICROBIAL FUNCTION

Hemocyanin (HMC) was documented to act as a important immune factors in invertebrates. In this study, we purified HMCs from Litopenaeus vannamei with gel-filtration chromatography and affinity chromatography (named as S-HMC and A-HMC). Only deglycosylated HMC by O-glycosidase leaded to decreased in agglutinative activities. Mass spectrometry analysis showed that L. vannamei HMC had O-glycan modifications, which has not been previously reported. 46 and 12 glycosylation sites were identified in S-HMC and A-HMC. Specifically, 23 and 20 glycosylation sites were identified in HMC subunit with higher and lower molecule weight, respectively.

Furthermore, together with our previous publications, the glycosylation sites of hemocyanin subunit C-terminal Thr537, Ser539 and Thr542 were replaced to Ala and expressed in yeast Pichia pastoris (named as wt-HMC and mut-HMC). Later results indicated that agglutination activities of mut-HMC were less than wt-HMC. We interestingly found that, compared with wt-HMC, the antimicrobial activities of mut-HMC were reducted significantly. Taken together, Thr537,Ser539 and Thr542 appeared to be closely associated with hemocyanin's antimicrobial activity, these results will be contributed to investigate hemocyanin function diversity mechanism of sugar molecules.

Key word：Litopenaeus vannamei; hemocyanin; glycosylation site; antibacterial function