Identification and characterization of glycosylation sites on <i>Litopenaeus vannamei</i> hemocyanin

Zhang, Zehui; Li, Ruiwei; Aweya, Jude Juventus; Wang, Fan; Zhong, Min; Zhang, Yueling

doi:10.1002/1873-3468.13367

Cited by 19 publications

(9 citation statements)

References 41 publications

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“…Thus, similar or increased binding of enzymatically or chemically deglycosylated FLH and KLH to MGL might contribute to explain their remnant immunomodulatory effects. Indeed, recent studies using the arthropodan Litopenaeus vannamei hemocyanin, a galactose-rich protein with beneficial immunostimulant and antimicrobial effects, have confirmed that its beneficial effects are promoted by its galactose-rich O- glycosylations (57). It is important to note that in this study we assessed hemocyanin binding to MR, MGL, and TLR4; however, as these proteins are multiligand molecules they might bind to several other immune receptors, such as the glycan-recognizing DC-SIGN or TLR2.…”

Section: Discussionmentioning

confidence: 99%

N-Glycosylation of mollusk hemocyanins contributes to their structural stability and immunomodulatory properties in mammals

Salazar

Jiménez

Villar

et al. 2019

Journal of Biological Chemistry

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Hemocyanins are widely used as carriers, adjuvants, and nonspecific immunostimulants in cancer because they promote Th1 immunity in mammals. Hemocyanins also interact with glycan-recognizing innate immune receptors on antigen-presenting cells, such as the C-type lectin immune receptors mannose receptor (MR), macrophage galactose lectin (MGL), and the Toll-like receptors (TLRs), stimulating proinflammatory cytokine secretion. However, the role of N-linked oligosaccharides on the structural and immunological properties of hemocyanin is unclear. Mollusk hemocyanins, such as Concholepas concholepas (CCH), Fissurella latimarginata (FLH), and Megathura crenulata (KLH), are oligomeric glycoproteins with complex dodecameric quaternary structures and heterogeneous glycosylation patterns, primarily consisting of mannose-rich N-glycans. Here, we report that enzyme-catalyzed N-deglycosylation of CCH, FLH, and KLH disrupts their quaternary structure and impairs their immunogenic effects. Biochemical analyses revealed that the deglycosylation does not change hemocyanin secondary structure but alters their refolding mechanism and dodecameric structure. Immunochemical analyses indicated decreased binding of N-deglycosylated hemocyanins to the MR and MGL receptors and TLR4 and reduced endocytosis concomitant with an impaired production of tumor necrosis factor α, and interleukins 6 and 12 (IL-6 and IL-12p40, respectively) in macrophages. Evaluating the function of N-deglycosylated hemocyanins in the humoral immune response and their nonspecific antitumor effects in the B16F10 melanoma model, we found that compared with native hemocyanins N-deglycosylated hemocyanins elicited reduced antibody titers, as well as partially diminished antitumor effects and altered carrier activities. In conclusion, the glycan content of hemocyanins is, among other structural characteristics, critically required for their immunological activities and should be considered in biomedical applications.

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Section: Discussionmentioning

confidence: 99%

N-Glycosylation of mollusk hemocyanins contributes to their structural stability and immunomodulatory properties in mammals

Salazar

Jiménez

Villar

et al. 2019

Journal of Biological Chemistry

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“…In molluscan HMCs, the oligosaccharide structures of the structural subunits RvH2 from Rapana venosa hemocyanin (RvH), bc-Helix lucorum hemocyanin (bc-HlH), and Megatura crenulata keyhole limpet (KLH) reveal a complex N-glycan pattern combining typical structural features of different higher organisms; these glycosylation plays a crucial physiological role in the structural stability, immunostimulatory, and therapeutic effect of HMC (60,61). Our previous research also found that L. vannamei HMC was deglycosylated using O-glycosidase; its agglutinative activity reduced about four-to eightfold (8,38). The results from this study indicated that binding of different pathogens with HMC fractions showed diverse optimum immunological activities, and the differences in their immunological activities may be related to their glycosylation diversity.…”

Section: Discussionmentioning

confidence: 93%

“…We previously reported that HMC had an Ig-like conserved domain and could react with goat anti-human IgG, IgM, and IgA (5,19,24). Furthermore, we also found that the diversity of human IgG constituents and glycosylation levels may have functional significance (5,36,38). Therefore, to determine whether polymorphism of HMC glycosylation is associated with its functional diversity, glycan content and glycosylation level were investigated.…”

Section: Discussionmentioning

confidence: 99%

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Protein Diversity and Immune Specificity of Hemocyanin From Shrimp Litopenaeus vannamei

et al. 2021

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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.

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“…Recently, Hex-type of glycans (for glucose, mannose, and galactose) was reported in L. vannamei in addition to the mucin-type O-glycans [68]. Among the nine O-glycosylation sites in the small subunit of hemocyanin isolated from L. vannamei, those at positions Thr537, Ser539, and Thr542 in the C terminus are of importance for its immunological function [68]. The authors reported a four-fold reduction of bacterial agglutination and a 0.2-fold reduction in the antibacterial activity after replacement of all three positions with alanine.…”

Section: Glycosylation Of E Verrucosa Hemocyanin Subunit 5 and Possimentioning

confidence: 99%

Molecular Cloning, Structure and Phylogenetic Analysis of a Hemocyanin Subunit from the Black Sea Crustacean Eriphia verrucosa (Crustacea, Malacostraca)

Todorovska

Ivanov

Radkova

et al. 2021

Genes

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Hemocyanins are copper-binding proteins that play a crucial role in the physiological processes in crustaceans. In this study, the cDNA encoding hemocyanin subunit 5 from the Black sea crab Eriphia verrucosa (EvHc5) was cloned using EST analysis, RT-PCR and rapid amplification of the cDNA ends (RACE) approach. The full-length cDNA of EvHc5 was 2254 bp, consisting of a 5′ and 3′ untranslated regions and an open reading frame of 2022 bp, encoding a protein consisting of 674 amino acid residues. The protein has an N-terminal signal peptide of 14 amino acids as is expected for proteins synthesized in hepatopancreas tubule cells and secreted into the hemolymph. The 3D model showed the presence of three functional domains and six conserved histidine residues that participate in the formation of the copper active site in Domain 2. The EvHc5 is O-glycosylated and the glycan is exposed on the surface of the subunit similar to Panulirus interruptus. The phylogenetic analysis has shown its close grouping with γ-type of hemocyanins of other crustacean species belonging to order Decapoda, infraorder Brachyura.

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Identification and characterization of glycosylation sites on Litopenaeus vannamei hemocyanin

Cited by 19 publications

References 41 publications

N-Glycosylation of mollusk hemocyanins contributes to their structural stability and immunomodulatory properties in mammals

N-Glycosylation of mollusk hemocyanins contributes to their structural stability and immunomodulatory properties in mammals

Protein Diversity and Immune Specificity of Hemocyanin From Shrimp Litopenaeus vannamei

Molecular Cloning, Structure and Phylogenetic Analysis of a Hemocyanin Subunit from the Black Sea Crustacean Eriphia verrucosa (Crustacea, Malacostraca)

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