Molluskan hemocyanins activate in vitro the classical pathway of the human complement system, through the presence of natural serum antibodies recognizing these proteins in unsensitized donors.
-
1
Universidad de Chile, Programa Disciplinario de Immunología, Instituto de Ciencias Biomédicas, Facultad de Medicina, Chile
-
2
Universidad de Chile, Programa Disciplinario de Immunología, Instituto de Ciencias Biomédicas, Facultad de Medicina, Chile
-
3
Universidad de Chile, Programa Disciplinario de Immunología, Instituto de Ciencias Biomédicas, Facultad de Medicina, Chile
-
4
Universidad de Chile, Programa Disciplinario de Immunología, Instituto de Ciencias Biomédicas, Facultad de Medicina, Chile
-
5
Fundacion Ciencia y Tecnología para el Desarrollo (FUCITED), Chile
Hemocyanins are enormous metalloglycoproteins present in the hemolymph of mollusks, with molecular weights ranging approximately between 4 and 13.5 MDa, being their main function the transport of respiratory oxygen. These proteins are heavily glycosylated, with some studies describing carbohydrate contents up until 9% w/w, with mannose being a common and most abundant oligosaccharide. Hemocyanins elicit a potent activation of the immune system in mammals. Amongst these proteins, the keyhole limpet (Megathura crenulata) hemocyanin, KLH, has been by far the most studied, being used in experimentation as a model antigen, as carrier for haptens, as adjuvant for vaccines and as a non-specific immunostimulant in antitumor therapy of some cancers in humans. However, the relationship between the structural features of hemocyanins and the immunologic mechanisms by which they exert their potent adjuvant/immunostimulatory effects is unclear. It has been reported that in unsensitized individuals, there are natural antibodies able to bind KLH, and one study showed complement deposition on its surface, albeit without studying the specific activated pathway. On the other hand, the biodiversity of hemocyanins has prompted interest in developing new candidates with better biochemical and immunological properties because the supply depends on natural resources. Thus, the hemocyanins of Concholepas concholepas and Fisurella latimarginata (CCH and FLH respectively) have been proposed as alternatives to KLH due to improved immunostimulatory and antitumor properties. In this work, we want to study the presence, in unsensitized individuals, of antibodies that recognize these three hemocyanins and their ability to activate the human complement system in vitro, specifically through the classical pathway.
We assessed the presence of natural antibodies against CCH, FLH and KLH in the sera of 5 unsensitized individuals by dot-blot and ELISA. For these assays, nitrocellulose membranes or microplates, respectively, were sensitized with the different solutions of hemocyanins (either untreated or chemically deglycosylated by periodate treatment, named Ox-CCH, Ox-FLH and Ox-KLH), blocked with casein and incubated with diluted heat-inactivated serum from unsensitized individuals. As a positive control, we used the serum of a patient immunized with KLH, and either PBS or casein, as a negative control. Then, for dot blot assays, the membranes were developed with an AP-conjugated anti-human IgG antibody. For ELISA, microplates were developed with an HRP-coupled goat anti-human IgG antibody. For complement activation detection, we used a similar ELISA, but after sensitization, plates were incubated sequentially with donor heat-inactivated serum (as a source of antibodies), purified human C1, purified human C4, goat anti-human C4 antibody and developed with an HRP-coupled rabbit anti-goat antibody. As a positive control, wells were sensitized with human IgM.
Through dot-blot assays, we found that every hemocyanin was detected by IgG antibodies present in at least one serum donor. In contrast, using ELISA, IgG antibodies reacting with all three hemocyanins in sera from all donors were found, with expected inter-donor variability. However, contrary to what was expected, for three selected donors (based on reactivity against the different hemocyanins), chemical deglycosylation did not always reduce antibody binding. Specifically, the binding to Ox-KLH diminished for all three donors, albeit in low levels. For Ox-CCH, however, a decreased binding was observed only in one donor, which previously displayed the higher reactivity against CCH amongst all donors. Finally, in the case of FLH, the binding was remarkably increased in all three donors.
Then, when sera from these three donors was assessed for its capacity to activate the classical pathway of the human complement system, we found that all three hemocyanins and their deglycosylated counterparts elicited this activation. However, and again contrary to what was expected, we only observed an effect for deglycosylation on FLH, with a remarkable increase in the ability of Ox-FLH to activate the classical pathway, correlating to its ability to bind more antibodies than its untreated counterpart.
Based on these results, we propose that these natural antibodies, present in the serum of unsensitized donors, are accountable, at least in part, for the immunogenic properties of molluskan hemocyanins as a group, through activation of the classical pathway of the complement system: However, it is important to note that deglycosylation did not always reduce antibody binding, but increased it in some cases. This can be explained by the possibility that not all antibodies are directed against carbohydrate epitopes but also to protein epitopes (which raises the question of how they originated in unsensitized individuals). Alternatively or concomitantly, the deglycosylation process, either through generation of Schiff bases or through exposure of underlying carbohydrate motifs, could generate neo-epitopes on hemocyanins, leading to an increased antibody binding. We can conclude that, although carbohydrates may not be the main reason behind the great immunogenicity of molluskan hemocyanins in mammals, they are still important, in at least some cases, and should be considered when studying these molecules. Finally, we are also currently studying an eventual role of the lectin pathway in the immunogenicity of hemocyanins, given their high mannose content. We may thus obtain a more complete picture of the interaction of these proteins with the human complement system, contributing to the understanding of their remarkable immunostimulant properties.
Acknowledgements
Beca de Magister Complementario, CONICYT, to JPB; Proyecto FONDECYT Regular 1130099 and Proyecto Anillo ACT-112 to AF.
Keywords:
Molluskan hemocyanins,
complement system,
natural antibodies,
classical pathway,
Carbohydrates,
Hemocyanins,
adjuvants
Conference:
IMMUNOCOLOMBIA2015 - 11th Congress of the Latin American Association of Immunology - 10o. Congreso de la Asociación Colombiana de Alergia, Asma e Inmunología, Medellin, Colombia, 13 Oct - 16 Oct, 2015.
Presentation Type:
Poster Presentation
Topic:
Innate Immunity
Citation:
Pizarro-Bauerle
J,
Maldonado
I,
López
MN,
Salazar-Onfray
F,
Valck
C,
Becker
M and
Ferreira
A
(2015). Molluskan hemocyanins activate in vitro the classical pathway of the human complement system, through the presence of natural serum antibodies recognizing these proteins in unsensitized donors..
Front. Immunol.
Conference Abstract:
IMMUNOCOLOMBIA2015 - 11th Congress of the Latin American Association of Immunology - 10o. Congreso de la Asociación Colombiana de Alergia, Asma e Inmunología.
doi: 10.3389/conf.fimmu.2015.05.00021
Copyright:
The abstracts in this collection have not been subject to any Frontiers peer review or checks, and are not endorsed by Frontiers.
They are made available through the Frontiers publishing platform as a service to conference organizers and presenters.
The copyright in the individual abstracts is owned by the author of each abstract or his/her employer unless otherwise stated.
Each abstract, as well as the collection of abstracts, are published under a Creative Commons CC-BY 4.0 (attribution) licence (https://creativecommons.org/licenses/by/4.0/) and may thus be reproduced, translated, adapted and be the subject of derivative works provided the authors and Frontiers are attributed.
For Frontiers’ terms and conditions please see https://www.frontiersin.org/legal/terms-and-conditions.
Received:
15 Apr 2015;
Published Online:
14 Sep 2015.
*
Correspondence:
Dr. María Inés Becker, Fundacion Ciencia y Tecnología para el Desarrollo (FUCITED), Santiago, RM, 7750269, Chile, mariaines.becker@fucited.cl
Dr. Arturo Ferreira, Universidad de Chile, Programa Disciplinario de Immunología, Instituto de Ciencias Biomédicas, Facultad de Medicina, Santiago, RM, 8380453, Chile, aferreira_uchile@yahoo.com