FcγR Binding and ADCC Activity of Human IgG Allotypes

Taeye, Steven W. de; Bentlage, Arthur E. H.; Mebius, Mirjam M.; Meesters, Joyce; Lissenberg-Thunnissen, Suzanne N.; Falck, David; Sénard, Thomas; Salehi, Niloufar; Wuhrer, Manfred; Schuurman, Janine; Labrijn, Aran F.; Rispens, Theo; Vidarsson, Gestur

doi:10.3389/fimmu.2020.00740

Cited by 134 publications

(116 citation statements)

References 74 publications

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“…Between donors, however, the IGHG3 * 14 allotype seems to exhibit a higher degree of galactosylation and sialylation in comparison with IGHG3 * 11/12 allotypes. This observation, was also previously described for mAbs by de Taeye et al (11). So far, we only observed 12 of the 27 existing allotypes, with few combinations of them.…”

Section: Allotype-specific Characterization Of Glycosylationsupporting

confidence: 88%

MS-Based Allotype-Specific Analysis of Polyclonal IgG-Fc N-Glycosylation

et al. 2020

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Current approaches to study glycosylation of polyclonal human immunoglobulins G (IgG) usually imply protein digestion or glycan release. While these approaches allow in-depth characterization, they also result in a loss of valuable information regarding certain subclasses, allotypes and co-occuring post-translational modifications (PTMs). Unfortunately, the high variability of polyclonal IgGs makes their intact mass spectrometry (MS) analysis extremely challenging. We propose here a middle-up strategy for the analysis of the intact fragment crystallizable (Fc) region of human plasma IgGs, with the aim of acquiring integrated information of the N-glycosylation and other PTMs of subclasses and allotypes. Human plasma IgG was isolated using Fcspecific beads followed by an on-bead C H 2 domain digestion with the enzyme IdeS. The obtained mixture of Fc subunits was analyzed by capillary electrophoresis (CE) and hydrophilic interaction liquid chromatography (HILIC) hyphenated with MS. CE-MS provided separation of different IgG-subclasses and allotypes, while HILIC-MS allowed resolution of the different glycoforms and their oxidized variants. The orthogonality of these techniques was key to reliably assign Fc allotypes. Five individual donors were analyzed using this approach. Heterozygosis was observed in all the analyzed donors resulting in a total of 12 allotypes identified. The assignments were further confirmed using recombinant monoclonal IgG allotypes as standards. While the glycosylation patterns were similar within allotypes of the same subclass, clear differences were observed between IgG subclasses and donors, highlighting the relevance of the proposed approach. In a single analysis, glycosylation levels specific for each allotype, relative abundances of subclasses and information on co-occurring modifications are obtained. This middle-up method represents an important step toward a comprehensive analysis of immunoglobulin G-Fc variants.

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Section: Allotype-specific Characterization Of Glycosylationsupporting

confidence: 88%

MS-Based Allotype-Specific Analysis of Polyclonal IgG-Fc N-Glycosylation

et al. 2020

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“…Of note, IgG allotypes were found to correlate with serum IgG levels ( 19 , 43 ) but also with the switching between IgG isotypes suggesting that IgG allotypes may aﬀect the humoral antibody response. Moreover, it was recently demonstrated that allotypic variants within the IgG3 subclass substantially aﬀect FcgRIIIA binding and Antibody-Dependent Cellular Cytotoxicity ( 44 ). To our knowledge, no work has previously shown associations between Gm phenotypes comprising G3m24 or G3m24 alone with a higher risk of malaria infections, while G3m24 is prevalent among sub-Saharan African populations ( 45 , 46 ).…”

Section: Discussionmentioning

confidence: 99%

Susceptibility to Plasmodium falciparum Malaria: Influence of Combined Polymorphisms of IgG3 Gm Allotypes and Fc Gamma Receptors IIA, IIIA, and IIIB

et al. 2020

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The binding of immunoglobulin (Ig) to Fc gamma receptors (FcgR) at the immune cell surface is an important step to initiate immunological defense against malaria. However, polymorphisms in receptors and/or constant regions of the IgG heavy chains may modulate this binding. Here, we investigated whether polymorphisms located in FcgR and constant regions of the heavy chain of IgG are associated with susceptibility to P. falciparum malaria. For this purpose, a clinical and parasitological follow-up on malaria was conducted among 656 infants in southern Benin. G3m allotypes (from total IgG3) were determined by a serological method of hemagglutination inhibition. FcgRIIA 131R/H and FcgRIIIA 176F/V genotypes were determined using the TaqMan method and FcgRIIIB NA1/NA2 genotypes were assessed by polymerase chain reaction using allele-specific primers. Association analyses between the number of malaria infections during the follow-up and polymorphisms in IgG G3m allotypes and FcgR were studied independently by zero inflated binomial negative regression. The influence of combinations of G3m allotypes and FcgRIIA/FcgRIIIA/FcgRIIIB polymorphisms on the number of P. falciparum infections, and their potential interaction with environmental exposure to malaria was assessed by using the generalized multifactor dimensionality reduction (GMDR) method. Results showed that individual carriage of G3m24 single allotype and of G3m5,6,10,11,13,14,24 phenotype was independently associated with a high risk of malaria infection. A risk effect for G3m6 was observed only under high environmental exposure. FcgRIIIA 176VV single genotype and combined carriage of FcgRIIA 131RH/FcgRIIIA 176VV/FcgRIIIB NA1NA2, FcgRIIA 131HH/FcgRIIIA 176FF/FcgRIIIB NA1NA1, FcgRIIA 131HH/FcgRIIIA 176VV/FcgRIIIB NA2NA2 and FcgRIIA 131HH/FcgRIIIA 176VV/FcgRIIIB NA1NA2 genotypes were related to a high number of malaria infections. The risk was accentuated for FcgRIIIA 176VV when considering the influence of environmental exposure to malaria. Finally, the GMDR analysis including environmental exposure showed strengthened associations with a malaria risk when FcgRIIA/FcgRIIIA/FcgRIIIB genotypes were combined to G3m5,6,11,24 and G3m5,6,10,11,13,15,24 phenotypes or G3m10 and G3m13 single allotypes. Our results highlight the relevance of studying IgG heavy chain and FcgR polymorphisms, independently as well as in combination, in relation to the individual susceptibility to P. falciparum infection. The intensity of individual exposure to mosquito bites was demonstrated to impact the relationships found.

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“… 8 , 9 Subclass, allotype, and glycosylation, especially fucosylation of IgG impacts FcγR binding. 10 − 12 FcγR-mediated IgG effector functions are partially regulated by varying FcγR expression on different immune cells. 13 For example, FcγRIIIb or CD16b is exclusively expressed as a monomeric protein mainly on granulocytes, while the homologous FcγRIIIa or CD16a is expressed on NK cells, monocytes, macrophages, and dendritic cells.…”

Section: Introductionmentioning

confidence: 99%

Site-Specific Glycosylation Mapping of Fc Gamma Receptor IIIb from Neutrophils of Individual Healthy Donors

et al. 2020

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Fc gamma receptors (FcγRs) translate antigen recognition by immunoglobulin G (IgG) into various immune responses. A better understanding of this key element of immunity promises novel insights into mechanisms of (auto-/allo-)immune diseases and more rationally designed antibody-based drugs. Glycosylation on both IgG and FcγR impacts their interaction dramatically. Regarding FcγR glycosylation profiling, major analytical challenges are associated with the presence of multiple glycosylation sites in close proximity and large structural heterogeneity. To address these challenges, we developed a straightforward and comprehensive analytical methodology to map FcγRIIIb glycosylation in primary human cells. After neutrophil isolation and immunoprecipitation, glycopeptides containing a single site each were generated by a dual-protease in-gel digestion. The complex mixture was resolved by liquid chromatography–tandem mass spectrometry (LC-MS/MS) providing information on the level of individual donors. In contrast to recently published alternatives for FcγRIIIb, we assessed its site-specific glycosylation in a single LC-MS/MS run and simultaneously determined the donor allotype. Studying FcγRIIIb derived from healthy donor neutrophils, we observed profound differences as compared to the soluble variant and the homologous FcγRIIIa on natural killer cells. This method will allow assessment of differences in FcγRIII glycosylation between individuals, cell types, subcellular locations, and pathophysiological conditions.

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FcγR Binding and ADCC Activity of Human IgG Allotypes

Cited by 134 publications

References 74 publications

MS-Based Allotype-Specific Analysis of Polyclonal IgG-Fc N-Glycosylation

MS-Based Allotype-Specific Analysis of Polyclonal IgG-Fc N-Glycosylation

Susceptibility to Plasmodium falciparum Malaria: Influence of Combined Polymorphisms of IgG3 Gm Allotypes and Fc Gamma Receptors IIA, IIIA, and IIIB

Site-Specific Glycosylation Mapping of Fc Gamma Receptor IIIb from Neutrophils of Individual Healthy Donors

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