title: Immunodominant role of CCHA subunit of Concholepas hemocyanin is associated with unique biochemical properties.
abstract: Hemocyanin, the oxygen transporter metallo-glycoprotein from mollusks, shows strong relationship between its notable structural features and intrinsic immunomodulatory effects. Here we investigated the individual contribution of CCHA and CCHB subunits from Concholepas hemocyanin (CCH) to in vivo humoral immune response and their pre-clinical evaluation as immunotherapeutic agent in a mice bladder cancer model, in relation to their biochemical properties. To this end, subunits were purified and well characterized. Homogeneous subunits were obtained by anionic exchange chromatography, and its purity assessed by electrophoretic and immunochemical methods. While each CCH subunit contains eight functional units showing partial cross reaction, the vibrational spectral analysis showed several spectral differences, suggesting structural differences between them. In addition, we demonstrated differences in the carbohydrate content: CCHA had a 3.6% w/w sugar with both N- and O-linked moieties. In turn, CCHB had a 2.5% w/w sugar with N-linked, while O-linked moieties were nearly absent. Considering these differences, it was not possible to predict a priori whether the immunogenic and immunotherapeutic properties of subunits might be similar. Surprisingly, both subunits by itself induced a humoral response, and showed an antitumor effect in the bladder carcinoma cell line MBT-2. However, when immunologic parameters were analyzed, CCHA showed better efficiency than CCHB. No allergic reactions or any toxic effects were observed in mice treated with CCHA, sustaining its potential therapeutic use. Our study supports that CCHA subunit accounts for the most important features involved in the immunogenicity of CCH, such as better hydrophilicity and higher content of carbohydrates.
authors.affiliation: Fundación Ciencia y Tecnología para el Desarrollo, Avenida Eduardo Castillo Velasco 2902, Santiago, Chile. mib@biosonda.cl
authors.forename: María Inés
authors.initials: MI
authors.lastname: Becker
authors.forename: Alejandra
authors.initials: A
authors.lastname: Fuentes
authors.forename: Miguel
authors.initials: M
authors.lastname: Del Campo
authors.forename: Augusto
authors.initials: A
authors.lastname: Manubens
authors.forename: Esteban
authors.initials: E
authors.lastname: Nova
authors.forename: Harold
authors.initials: H
authors.lastname: Oliva
authors.forename: Fernando
authors.initials: F
authors.lastname: Faunes
authors.forename: María Antonieta
authors.initials: MA
authors.lastname: Valenzuela
authors.forename: Marcelo
authors.initials: M
authors.lastname: Campos-Vallette
authors.forename: Alvaro
authors.initials: A
authors.lastname: Aliaga
authors.forename: Jorge
authors.initials: J
authors.lastname: Ferreira
authors.forename: Alfredo E
authors.initials: AE
authors.lastname: De Ioannes
authors.forename: Pablo
authors.initials: P
authors.lastname: De Ioannes
authors.forename: Bruno
authors.initials: B
authors.lastname: Moltedo
mesh_terms: D000818:Animals; D000917:Antibody Formation; D000970:Antineoplastic Agents / Q000737:chemistry / Q000276:immunology* / Q000627:therapeutic use; D002277:Carcinoma / Q000188:drug therapy* / Q000276:immunology; D045744:Cell Line, Tumor; D003429:Cross Reactions / Q000276:immunology; D049851:Gastropoda / Q000737:chemistry*; D006433:Hemocyanins / Q000737:chemistry / Q000276:immunology* / Q000627:therapeutic use; D007167:Immunotherapy; D053208:Kaplan-Meier Estimate; D051379:Mice; D008807:Mice, Inbred BALB C; D008809:Mice, Inbred C3H; D008810:Mice, Inbred C57BL; D021122:Protein Subunits / Q000737:chemistry / Q000276:immunology / Q000627:therapeutic use; D001749:Urinary Bladder Neoplasms / Q000188:drug therapy* / Q000276:immunology
chemical_list: D000970:Antineoplastic Agents; D021122:Protein Subunits; D006433:Hemocyanins