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

Abstract

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.

A heat-shocked melanoma cell lysate vaccine enhances tumor infiltration by prototypic effector T cells inhibiting tumor growth

“…9 10 In contrast, TRIMEL seems to generate the appropriate combination of DAMPs capable of inducing an effective DC maturation. 8 9 CCH has potent immunostimulatory effects, whose adjuvant properties could be partially explained by its carbohydrate-based high complexity and stability, 24 which activate APCs via the interaction with the C-type lectin and Toll-like receptors (TLRs) such as mannose receptor and TLR4, respectively, 25 strengthening innate and adaptive immune responses in mammals, making them useful in cancer immunotherapy. 26 30 Notably, treatment of B16F10 tumor-bearing mice with TRIMEL/CCH or combined cell lysates in the absence of CCH did not prevent tumor growth ( figure 2B), indicating that every vaccine component is crucial for induction of protective responses in our model.…”

“… 23 Meanwhile, CCH is a high molecular weight glycoprotein related to oxygen transport in mollusks. It has been described that the highly complex CCH molecule shows a structural stability that contribute to their potent immunostimulatory effects, 24 strengthening innate and adaptive immunity in mammals and making it useful in cancer immunotherapy. 25 26 Although both hemocyanins show comparable immunogenic characteristics, 27 CCH is made up of intermixed subunits organized as heterododecamers that do not require divalent ions, given greater comparative stability.…”

Hemocyanins from Helix and Rapana Snails Exhibit in Vitro Antitumor Effects in Human Colorectal Adenocarcinoma

“…Due to these structural properties, hemocyanins stimulate the mammalian immune system nonspecifically by interacting with macrophages, polymorphonuclears, CD4+, and CD8+ cells and induce potent humoral and cellular immune response [ 11 , 13 ]. Moreover, significant direct antitumor effects of hemocyanins have been established in various in vitro and in vivo tumor models [ 4 , 14 , 15 , 16 ].…”

De Novo Structural Determination of the Oligosaccharide Structure of Hemocyanins from Molluscs

“…Molluscan hemocyanins are oligomeric glycoproteins with complex dodecameric quaternary structures and heterogeneous glycosylation patterns, primarily consisting of mannose-rich N -glycans, which contribute to their structural stability and immunomodulatory properties in mammals [ 46 ]. The enzyme-catalyzed N -deglycosylation of Concholepas concholepas [ 47 ], Fissurella latimarginata [ 2 ], and KLH disrupts their quaternary structure and impairs their immunogenic effects.…”