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.

Hemocyanin from Shrimp Litopenaeus vannamei Has Antiproliferative Effect against HeLa Cell In Vitro.

Hemocyanin (HMC) has been shown to participate in multiple roles of immune defence. In this study, we investigated the antiproliferative effect and underpinning mechanism of HMC from Litopenaeus vannamei in vitro. Sulforhodamine B (SRB) assay indicated that HMC could dramatically inhibit the growth of HeLa cells, but not 293T cells under the same conditions. Moreover, typical morphological features of apoptosis in HeLa cells including the formation of apoptotic body-like vesicles, chromatin condensation and margination were observed by using 4, 6-diamidino-2- phenylindole dihydrochloride (DAPI) staining and fluorescence analysis. An apoptotic DNA ladder from 180 to 300 bp was also detected. Furthermore, 10 variation proteins associated with apoptosis pathway, viz. G3PDH isoforms 1/2 (G3PDH1/2), aldosereductase, ectodemal dysplasia receptor associated death receptor domain isoform CRA_a (EDARADD), heat shock 60kD protein 1 variant 1 (HSP60), heat shock 70kDa protein 5 precursor (HSP70), heat shock protein 90kDa beta member 1 precursor (HSP90), 14-3-3 protein ζ/δ, Ran and ubiquitin activating enzyme E1(UBE1), were identified from HMC-treated HeLa cells by the proteomic and quantitative real-time RT-PCR strategies. Importantly, the reactive oxygen species (ROS), mitochondrial membrane potential (Δψm) and caspase-9/3 activities were changed significantly in HMC-treated HeLa cells. Together, the data suggests that L. vannamei HMC mediates antiproliferative properties through the apoptosis mechanism involving the mitochondria triggered pathway.