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.