TY - JOUR
T1 - FcγRIIA-specific DARPins as novel tools in blood cell analysis and platelet aggregation
AU - Riechert, Vanessa
AU - Hein, Sascha
AU - Visser, Mayken
AU - Zimmermann, Mathias
AU - Wesche, Jan
AU - Adams, Philipp A.
AU - Theuerkauf, Samuel A.
AU - Jamali, Arezoo
AU - Wangorsch, Andrea
AU - Reuter, Andreas
AU - Pasternak, Alexander O.
AU - Hartmann, Jessica
AU - Greinacher, Andreas
AU - Herrera-Carrillo, Elena
AU - Berkhout, Ben
AU - Cichutek, Klaus
AU - Buchholz, Christian J.
N1 - Funding Information: This work was supported by grant 1R01AI145045-01 from the National Institutes of Health (NIH) to B. B., E. H.-C., and C. J. B. The content is solely the responsibility of the authors and does not necessarily represent the official views of the National Institutes of Health. Publisher Copyright: © 2023 The Authors
PY - 2023/6/1
Y1 - 2023/6/1
N2 - Fc receptors are involved in a variety of physiologically and disease-relevant responses. Among them, FcγRIIA (CD32a) is known for its activating functions in pathogen recognition and platelet biology, and, as potential marker of T lymphocytes latently infected with HIV-1. The latter has not been without controversy due to technical challenges complicated by T-B cell conjugates and trogocytosis as well as a lack of antibodies distinguishing between the closely related isoforms of FcγRII. To generate high-affinity binders specific for FcγRIIA, libraries of designed ankyrin repeat proteins (DARPins) were screened for binding to its extracellular domains by ribosomal display. Counterselection against FcγRIIB eliminated binders cross-reacting with both isoforms. The identified DARPins bound FcγRIIA with no detectable binding for FcγRIIB. Their affinities for FcγRIIA were in the low nanomolar range and could be enhanced by cleavage of the His-tag and dimerization. Interestingly, complex formation between DARPin and FcγRIIA followed a two-state reaction model, and discrimination from FcγRIIB was based on a single amino acid residue. In flow cytometry, DARPin F11 detected FcγRIIA+ cells even when they made up less than 1% of the cell population. Image stream analysis of primary human blood cells confirmed that F11 caused dim but reliable cell surface staining of a small subpopulation of T lymphocytes. When incubated with platelets, F11 inhibited their aggregation equally efficient as antibodies unable to discriminate between both FcγRII isoforms. The selected DARPins are unique novel tools for platelet aggregation studies as well as the role of FcγRIIA for the latent HIV-1 reservoir.
AB - Fc receptors are involved in a variety of physiologically and disease-relevant responses. Among them, FcγRIIA (CD32a) is known for its activating functions in pathogen recognition and platelet biology, and, as potential marker of T lymphocytes latently infected with HIV-1. The latter has not been without controversy due to technical challenges complicated by T-B cell conjugates and trogocytosis as well as a lack of antibodies distinguishing between the closely related isoforms of FcγRII. To generate high-affinity binders specific for FcγRIIA, libraries of designed ankyrin repeat proteins (DARPins) were screened for binding to its extracellular domains by ribosomal display. Counterselection against FcγRIIB eliminated binders cross-reacting with both isoforms. The identified DARPins bound FcγRIIA with no detectable binding for FcγRIIB. Their affinities for FcγRIIA were in the low nanomolar range and could be enhanced by cleavage of the His-tag and dimerization. Interestingly, complex formation between DARPin and FcγRIIA followed a two-state reaction model, and discrimination from FcγRIIB was based on a single amino acid residue. In flow cytometry, DARPin F11 detected FcγRIIA+ cells even when they made up less than 1% of the cell population. Image stream analysis of primary human blood cells confirmed that F11 caused dim but reliable cell surface staining of a small subpopulation of T lymphocytes. When incubated with platelets, F11 inhibited their aggregation equally efficient as antibodies unable to discriminate between both FcγRII isoforms. The selected DARPins are unique novel tools for platelet aggregation studies as well as the role of FcγRIIA for the latent HIV-1 reservoir.
KW - DARPin
KW - Fc receptors
KW - HIV
KW - binding site
KW - surface plasmon resonance
KW - thrombocytes
UR - http://www.scopus.com/inward/record.url?scp=85159558651&partnerID=8YFLogxK
U2 - https://doi.org/10.1016/j.jbc.2023.104743
DO - https://doi.org/10.1016/j.jbc.2023.104743
M3 - Article
C2 - 37100283
SN - 0021-9258
VL - 299
JO - Journal of Biological Chemistry
JF - Journal of Biological Chemistry
IS - 6
M1 - 104743
ER -