title: Molluskan Hemocyanins Activate the Classical Pathway of the Human Complement System through Natural Antibodies.
abstract: Molluskan hemocyanins are enormous oxygen-carrier glycoproteins that show remarkable immunostimulatory properties when inoculated in mammals, such as the generation of high levels of antibodies, a strong cellular reaction, and generation of non-specific antitumor immune responses in some types of cancer, particularly for superficial bladder cancer. These proteins have the ability to bias the immune response toward a Th1 phenotype. However, despite all their current uses with beneficial clinical outcomes, a clear mechanism explaining these properties is not available. Taking into account reports of natural antibodies against the hemocyanin of the gastropodMegathura crenulata[keyhole limpet hemocyanin (KLH)] in humans as well as other vertebrate species, we report here for the first time, the presence, in sera from unimmunized healthy donors, of antibodies recognizing, in addition to KLH, two other hemocyanins from gastropods with documented immunomodulatory capacities:Fisurella latimarginatahemocyanin (FLH) andConcholepas concholepashemocyanin (CCH). Through an ELISA screening, we found IgM and IgG antibodies reactive with these hemocyanins. When the capacity of these antibodies to bind deglycosylated hemocyanins was studied, no decreased interaction was detected. Moreover, in the case of FLH, deglycosylation increased antibody binding. We evaluated through anin vitrocomplement deposition assay whether these antibodies activated the classical pathway of the human complement system. The results showed that all three hemocyanins and their deglycosylated counterparts elicited this activation, mediated by C1 binding to immunoglobulins. Thus, this work contributes to the understanding on how the complement system could participate in the immunostimulatory properties of hemocyanins, through natural, complement-activating antibodies reacting with these proteins. Although a role for carbohydrates cannot be completely ruled out, in our experimental setting, glycosylation status had a limited effect. Finally, our data open possibilities for further studies leading to the design of improved hemocyanin-based research tools for diagnosis and immunotherapy.
authors.affiliation: Immunology of Microbial Aggression Laboratory, Immunology Program, Faculty of Medicine, ICBM, Universidad de Chile , Santiago , Chile.
authors.forename: Javier
authors.initials: J
authors.lastname: Pizarro-Bauerle
authors.affiliation: Immunology of Microbial Aggression Laboratory, Immunology Program, Faculty of Medicine, ICBM, Universidad de Chile , Santiago , Chile.
authors.forename: Ismael
authors.initials: I
authors.lastname: Maldonado
authors.affiliation: Immunology of Microbial Aggression Laboratory, Immunology Program, Faculty of Medicine, ICBM, Universidad de Chile , Santiago , Chile.
authors.forename: Eduardo
authors.initials: E
authors.lastname: Sosoniuk-Roche
authors.affiliation: Immunology of Microbial Aggression Laboratory, Immunology Program, Faculty of Medicine, ICBM, Universidad de Chile , Santiago , Chile.
authors.forename: Gerardo
authors.initials: G
authors.lastname: Vallejos
authors.affiliation: Faculty of Medicine, Millennium Institute on Immunology and Immunotherapy, ICBM, Universidad de Chile, Santiago, Chile; Immunology Program, Faculty of Medicine, ICBM, Universidad de Chile, Santiago, Chile.
authors.forename: Mercedes N
authors.initials: MN
authors.lastname: López
authors.affiliation: Faculty of Medicine, Millennium Institute on Immunology and Immunotherapy, ICBM, Universidad de Chile, Santiago, Chile; Immunology Program, Faculty of Medicine, ICBM, Universidad de Chile, Santiago, Chile.
authors.forename: Flavio
authors.initials: F
authors.lastname: Salazar-Onfray
authors.affiliation: Faculty of Veterinary Medicine and Livestock Sciences, University of Chile , Santiago , Chile.
authors.forename: Lorena
authors.initials: L
authors.lastname: Aguilar-Guzmán
authors.affiliation: Immunology of Microbial Aggression Laboratory, Immunology Program, Faculty of Medicine, ICBM, Universidad de Chile , Santiago , Chile.
authors.forename: Carolina
authors.initials: C
authors.lastname: Valck
authors.affiliation: Immunology of Microbial Aggression Laboratory, Immunology Program, Faculty of Medicine, ICBM, Universidad de Chile , Santiago , Chile.
authors.forename: Arturo
authors.initials: A
authors.lastname: Ferreira
authors.affiliation: Biosonda Corporation, Santiago, Chile; Fundación Ciencia y Tecnología para el Desarrollo (FUCITED), Santiago, Chile.
authors.forename: María Inés
authors.initials: MI
authors.lastname: Becker
keywords: adjuvants; carbohydrates; classical pathway; complement system; molluskan hemocyanins; natural antibodies