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Zebrafish Galectin 3 binding protein (Lgals3bp) is the target antigen of the microglial 4C4 monoclonal antibody

View ORCID ProfileMireia Rovira, View ORCID ProfileAlice Montanari, Latifa Hammou, View ORCID ProfileLaura Chomette, View ORCID ProfileMagali Miserocchi, View ORCID ProfileJennifer Pozo, View ORCID ProfileVirginie Imbault, View ORCID ProfileXavier Bisteau, View ORCID ProfileValérie Wittamer
doi: https://doi.org/10.1101/2022.04.05.486973
Mireia Rovira
1Institut de Recherche Interdisciplinaire en Biologie Humaine et Moléculaire (IRIBHM), Université Libre de Bruxelles (ULB), Brussels 1070, Belgium
2ULB Institute of Neuroscience (UNI), Université Libre de Bruxelles (ULB), Brussels 1070, Belgium
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Alice Montanari
1Institut de Recherche Interdisciplinaire en Biologie Humaine et Moléculaire (IRIBHM), Université Libre de Bruxelles (ULB), Brussels 1070, Belgium
2ULB Institute of Neuroscience (UNI), Université Libre de Bruxelles (ULB), Brussels 1070, Belgium
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Latifa Hammou
1Institut de Recherche Interdisciplinaire en Biologie Humaine et Moléculaire (IRIBHM), Université Libre de Bruxelles (ULB), Brussels 1070, Belgium
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Laura Chomette
1Institut de Recherche Interdisciplinaire en Biologie Humaine et Moléculaire (IRIBHM), Université Libre de Bruxelles (ULB), Brussels 1070, Belgium
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Magali Miserocchi
1Institut de Recherche Interdisciplinaire en Biologie Humaine et Moléculaire (IRIBHM), Université Libre de Bruxelles (ULB), Brussels 1070, Belgium
2ULB Institute of Neuroscience (UNI), Université Libre de Bruxelles (ULB), Brussels 1070, Belgium
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Jennifer Pozo
1Institut de Recherche Interdisciplinaire en Biologie Humaine et Moléculaire (IRIBHM), Université Libre de Bruxelles (ULB), Brussels 1070, Belgium
2ULB Institute of Neuroscience (UNI), Université Libre de Bruxelles (ULB), Brussels 1070, Belgium
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Virginie Imbault
1Institut de Recherche Interdisciplinaire en Biologie Humaine et Moléculaire (IRIBHM), Université Libre de Bruxelles (ULB), Brussels 1070, Belgium
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Xavier Bisteau
1Institut de Recherche Interdisciplinaire en Biologie Humaine et Moléculaire (IRIBHM), Université Libre de Bruxelles (ULB), Brussels 1070, Belgium
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Valérie Wittamer
1Institut de Recherche Interdisciplinaire en Biologie Humaine et Moléculaire (IRIBHM), Université Libre de Bruxelles (ULB), Brussels 1070, Belgium
2ULB Institute of Neuroscience (UNI), Université Libre de Bruxelles (ULB), Brussels 1070, Belgium
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  • For correspondence: valerie.wittamer@ulb.be
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ABSTRACT

Background Two decades ago, the fish-specific monoclonal antibody 4C4 was found to be highly reactive to zebrafish microglia, the macrophages of the central nervous system. This has resulted in 4C4 being widely used, in combination with available fluorescent transgenic reporters to identify and isolate microglia. However, the target protein of 4C4 remains unidentified, which represents a major caveat. In addition, whether the 4C4 expression pattern is strictly restricted to microglial cells in zebrafish has never been investigated.

Results Having demonstrated that 4C4 is able to capture its native antigen from adult brain lysates, we used immunoprecipitation/mass-spectrometry, coupled to recombinant expression analyses, to identify its target. The cognate antigen was found to be a paralog of Galectin 3 binding protein (Lgals3bpb), known as MAC2-binding protein in mammals. Notably, 4C4 did not recognize other paralogs, demonstrating specificity. Moreover, our data show that Lgals3bpb expression, while ubiquitous in microglia, also identifies leukocytes in the periphery, including populations of gut and liver macrophages.

Conclusions The 4C4 monoclonal antibody recognizes Lgals3bpb, a predicted highly glycosylated protein whose function in the microglial lineage is currently unknown. Identification of Lgals3bpb as a new pan-microglia marker will be fundamental in forthcoming studies using the zebrafish model.

Competing Interest Statement

The authors have declared no competing interest.

Copyright 
The copyright holder for this preprint is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under a CC-BY-NC-ND 4.0 International license.
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Posted April 07, 2022.
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Zebrafish Galectin 3 binding protein (Lgals3bp) is the target antigen of the microglial 4C4 monoclonal antibody
Mireia Rovira, Alice Montanari, Latifa Hammou, Laura Chomette, Magali Miserocchi, Jennifer Pozo, Virginie Imbault, Xavier Bisteau, Valérie Wittamer
bioRxiv 2022.04.05.486973; doi: https://doi.org/10.1101/2022.04.05.486973
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Zebrafish Galectin 3 binding protein (Lgals3bp) is the target antigen of the microglial 4C4 monoclonal antibody
Mireia Rovira, Alice Montanari, Latifa Hammou, Laura Chomette, Magali Miserocchi, Jennifer Pozo, Virginie Imbault, Xavier Bisteau, Valérie Wittamer
bioRxiv 2022.04.05.486973; doi: https://doi.org/10.1101/2022.04.05.486973

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