Immunodominant role of CCHA subunit of Concholepas hemocyanin is associated with unique biochemical properties.
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.
Becker
María Inés
MI
Fundación Ciencia y Tecnología para el Desarrollo, Avenida Eduardo Castillo Velasco 2902, Santiago, Chile. mib@biosonda.cl
Fuentes
Alejandra
A
Del Campo
Miguel
M
Manubens
Augusto
A
Nova
Esteban
E
Oliva
Harold
H
Faunes
Fernando
F
Valenzuela
María Antonieta
MA
Campos-Vallette
Marcelo
M
Aliaga
Alvaro
A
Ferreira
Jorge
J
De Ioannes
Alfredo E
AE
De Ioannes
Pablo
P
Moltedo
Bruno
B
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
D000970:Antineoplastic Agents; D021122:Protein Subunits; D006433:Hemocyanins