Abstract
Hemocyanin is an important non-specific innate immune defense molecule with phenoloxidase, antiviral, antibacterial, hemolytic, and antitumor activities. To better understand the mechanism of functional diversity, proteomics approach was applied to characterize hemocyanin (HMC) expression profiles from Litopenaeus vannamei. At first, hemocyanin was purified by Sephadex G-100 and DEAE-cellulose (DE-52) columns from shrimp serum, and 34 protein spots were identified as HMC on the 2-DE gels. Furthermore, we found that 9 HMC spots about 75 or 77 kDa were regulated by Streptococcus agalactiae and Vibrio parahaemolyticus infection at 6, 12, and 24 h. In addition, 6 different pathogen-binding HMC fractions, viz., HMC-Mix, HMC-Vp, HMC-Va, HMC-Vf, HMC-Ec, and HMC-Sa, showed different agglutinative and antibacterial activities. Moreover, lectin-blotting analysis showed significant differences in glycosylation level among HMC isomers and bacteria-binding HMC fractions. Particularly, the agglutinative activities of the HMC fractions were almost completely abolished when HMC was deglycosylated by O-glycosidase, which suggest that O-linked sugar chains of HMC played important roles in the innate immune recognition. Our findings demonstrated for the first time that L. vannamei HMC had molecular diversity in protein level, which is closely associated with its ability to recognize diverse pathogens, whereas glycan modification probably contributed to HMC’s diversity and multiple immune activities.
Highlights
Hemocyanin (HMC) is a large copper-containing respiratory protein found in the hemolymph of mollusks and arthropods
25 spots, ranging from 25 to 66 kDa, showed homology with HMC, suggesting that these may be HMC fragments or modified proteins. These results indicate that L. vannamei HMC might possess high molecular diversity at the protein level
The HMC-Va carbohydrate content was 6.38%, which was about 2.6- and 5.4-fold to HMCVf and HMC fractions bound directly to mixed bacteria (HMC-Mix) (Figure 5D). These results suggest that glycosylation diversity might exist in HMC and HMC fractions bound to different bacteria
Summary
Hemocyanin (HMC) is a large copper-containing respiratory protein found in the hemolymph of mollusks and arthropods. The immune significance of HMC was dismissed until the late 1990s when Decker and Rimke demonstrated unequivocally its phenoloxidase activity [1]. It has been reported that HMC could be functionally converted into phenoloxidase. Our previous findings indicated that HMC from shrimp Litopenaeus vannamei reacted with human Ig as an antigen [5, 19, 24] and acted as an immune-enhancing protein [25, 26]. These results show HMC’s roles in many immune activities and its importance in invertebrate innate immune system. Little is known about the molecular mechanism of HMC’s functional diversity in L. vannamei
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