Pizarro-Bauerle Javier, Maldonado Ismael, Sosoniuk-Roche Eduardo, Vallejos Gerardo, López Mercedes N, Salazar-Onfray Flavio, Aguilar-Guzmán Lorena, Valck Carolina, Ferreira Arturo, Becker María Inés
Immunology of Microbial Aggression Laboratory, Immunology Program, Faculty of Medicine, ICBM, Universidad de Chile , Santiago , Chile.
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.
Front Immunol. 2017 Feb 24;8:188. doi: 10.3389/fimmu.2017.00188. eCollection 2017.
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 T1 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 gastropod [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: hemocyanin (FLH) and hemocyanin (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 an complement 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.
软体动物血蓝蛋白是巨大的氧载体糖蛋白，当接种到哺乳动物体内时，表现出显著的免疫刺激特性，如产生高水平抗体、强烈的细胞反应，以及在某些类型的癌症中产生非特异性抗肿瘤免疫反应，特别是对浅表性膀胱癌。这些蛋白质能够使免疫反应偏向T1表型。然而，尽管它们目前在临床上的应用都取得了有益的结果，但尚无明确的机制来解释这些特性。考虑到人类以及其他脊椎动物物种中存在针对腹足纲动物血蓝蛋白（钥孔戚血蓝蛋白，KLH）的天然抗体的报道，我们在此首次报告，在未免疫的健康供者血清中，除了KLH之外，还存在能够识别另外两种具有免疫调节能力的腹足纲动物血蓝蛋白的抗体：血蓝蛋白（FLH）和血蓝蛋白（CCH）。通过酶联免疫吸附测定筛选，我们发现了与这些血蓝蛋白反应的IgM和IgG抗体。当研究这些抗体与去糖基化血蓝蛋白结合的能力时，未检测到相互作用的降低。此外，就FLH而言，去糖基化增加了抗体结合。我们通过补体沉积试验评估了这些抗体是否激活了人类补体系统的经典途径。结果表明，所有三种血蓝蛋白及其去糖基化对应物均引发了这种激活，由C1与免疫球蛋白结合介导。因此，这项工作有助于理解补体系统如何通过与这些蛋白质反应的天然补体激活抗体参与血蓝蛋白的免疫刺激特性。尽管不能完全排除碳水化合物的作用，但在我们的实验条件下，糖基化状态的影响有限。最后，我们的数据为进一步研究开辟了可能性，从而设计出改进的基于血蓝蛋白的诊断和免疫治疗研究工具。
