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.
Highlights
Hemocyanins are widely used as carriers, adjuvants, and nonspecific immunostimulants in cancer because they promote Th1 immunity in mammals
We have shown that hemocyanins promote the secretion of proinflammatory cytokines by antigen-presenting cells (APCs), such as TNF␣, IL-6, and IL-12p40, with varying intensity and temporality for each hemocyanin [35]
To verify the removal of N-glycans, as a first approach we analyzed the samples by dot blots with periodic acid-Schiff (PAS) staining, which revealed the presence of sugars in a nonspecific manner (Fig. 1A), and later with concanavalin A (ConA), which revealed the presence of mannoserich N-glycans (Fig. 1B)
Summary
Hemocyanins are widely used as carriers, adjuvants, and nonspecific immunostimulants in cancer because they promote Th1 immunity in mammals. 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. Mollusk hemocyanins are large oxygen-carrier glycoproteins present in the hemolymph of some mollusks These glycoproteins are widely used in biomedicine and biotechnology because they induce Th1 immune responses when inoculated in mammals. Mollusk hemocyanins are high-molecular-weight oligomeric glycoproteins (4 – 8 MDa) characterized by a complex quaternary didecameric structure with repeated epitopes [3]. These glycoproteins are composed of 10 subunits, which form hollow cylindrical structures known as decamers. Biophysical techniques suggest that hemocyanins are stable in a long range of pH values, which is probably a consequence of the interactions between subunits and the high degree of oligomerization that stabilize the quaternary structure [7, 8, 15, 16]
Sign-in/Register to view highlights & summary Talk to us
Join us for a 30 min session where you can share your feedback and ask us any queries you have
Schedule a callDisclaimer: All third-party content on this website/platform is and will remain the property of their respective owners and is provided on "as is" basis without any warranties, express or implied. Use of third-party content does not indicate any affiliation, sponsorship with or endorsement by them. Any references to third-party content is to identify the corresponding services and shall be considered fair use under The CopyrightLaw.
