Dataset Information

Parallel Reaction Monitoring to Assess High-Affinity N-Glycoforms of the Human Fcγ Receptor IIIa (CD16a)

ABSTRACT: Fc γ-receptor IIIa (FcγRIIIa, or CD16a) is a receptor for the Fc region of IgG antibodies and plays an essential role in the regulation of antibody effector functions. While the activation of CD16a plays a role in a healthy immune response, dysregulated CD16a activity may contribute to tissue damage and chronic inflammation by over-activating immune cells or, conversely, by compromising the removal of immune complexes. Functionally distinct glycosylated forms of CD16a exist, with CD16a glycoforms modified with high mannose N-linked glycans at site N162 demonstrating a higher binding affinity for IgG compared to other forms of CD16a. Here, we developed and tested a nano-liquid chromatography tandem mass spectrometry (nLC-MS/MS) parallel reaction monitoring (PRM) method for the site-specific study of CD16a glycosylation. The PRM method was tested by spiking recombinant CD16a (F158 or V158) into a complex matrix consisting of THP-1 cell lysate digest, then the CD16a glycopeptide PRM method was applied to measure statistically significant changes in site N162 N-glycan compositions corresponding to high affinity glycoforms of CD16a in a model of monocyte differentiation. Analyses were extended to the analysis of CD16a derived from human primary monocytes and peripheral blood mononuclear cells. CD16a site N162 N-glycosylation demonstrated remarkable similarity across the surveyed cell types, including in THP-1 cells – a monocyte cell line isolated from the peripheral blood of an acute monocytic leukemia patient, as well as primary human monocytes, and PBMCs. Critically, CD16a glycopeptide PRM facilitated measurement of changes in CD16a glycosylation at site N162, with high mannose N-glycan compositions, corresponding to high affinity glycoforms of CD16a, increasing in a model of monocyte differentiation into macrophages. Here, we have demonstrated the feasibility of utilizing a glycopeptide nLC-MS/MS-PRM targeted approach to study CD16a glycosylation in material from primary cells in a glycosylation site-specific manner. This strategy is well-suited for adaptation to clinical studies focused on understanding the role of CD16a glycosylation in physiology and disease, with a focus on immune disorders.

INSTRUMENT(S):

ORGANISM(S): Homo Sapiens (human)

TISSUE(S): Blood Cell, Monocyte, Cell Culture, Blood

DISEASE(S): Acute Leukemia,Disease Free

SUBMITTER: Carlos Pavan

LAB HEAD: Kevin Brown Chandler

PROVIDER: PXD071784 | Pride | 2026-04-27

REPOSITORIES: Pride

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Dataset's files

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			Action	DRS
	2024-02-14-FcgR-3F-PRM-0p078pico-A.raw	Raw
	2024-02-14-FcgR-3F-PRM-0p078pico-B.raw	Raw
	2024-02-14-FcgR-3F-PRM-0p078pico-C.raw	Raw
	2024-02-14-FcgR-3F-PRM-0p078pico-D.raw	Raw
	2024-02-14-FcgR-3F-PRM-0p156pico-A.raw	Raw

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Publications

Assessing High-Affinity <i>N</i>-Glycoforms of the Human Fcγ Receptor IIIa (CD16a) via Parallel Reaction Monitoring Mass Spectrometry.

Pavan Carlos H CH Desai Jay J Medina Navarro Valentina V Chandler Kevin Brown KB

Journal of proteome research 20260423

The Fc γ-receptor IIIa (FcγRIIIa), or CD16a, is a receptor for the Fc region of IgG that plays an essential role in the regulation of antibody effector functions. Dysregulated CD16a activity contributes to tissue damage and chronic inflammation or, conversely, compromises the removal of immune complexes. CD16a glycoforms with high-mannose <i>N</i>-linked glycans at site N162 demonstrate higher affinity for IgG compared to other forms. Here, we developed a targeted nanoliquid chromatography tande ...[more]

PMID: 42026724

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Project description:Influenza A virus (IAV) mutates rapidly, preventing a single seasonal vaccine from targeting more than one strain, and year-to-year vaccine effectiveness is low. One challenge in designing effective vaccines is that genetic mutations frequently cause amino acid variations in IAV envelope protein hemagglutinin (HA) that create new N-glycosylation sequons; resulting N-glycans cause antigenic shielding, allowing viral escape from adaptive immune responses. Vaccine candidate strain selection currently involves correlating antigenicity with HA protein sequence among circulating strains, but quantitative comparison of site-specific glycosylation information is likely to improve the ability to design vaccines with broader effectiveness against evolved strains. However, there is poor understanding of the influence of glycosylation on immunodominance, antigenicity, and immunogenicity of HA, and there are no well-tested methods for comparing glycosylation similarity among virus samples. Here, we present a method for statistically rigorous quantification of similarity between two related virus strains that considers the presence and abundance of glycopeptide glycoforms. We demonstrate the strength of our approach by determining that there was a quantifiable difference in glycosylation at the protein level between wild-type IAV HA from A/Switzerland/9715293/2013 (SWZ13) and a mutant strain of SWZ13, even though no N-glycosylation sequons were changed. We determined site-specifically that WT and mutant HA have varying similarity at the glycosylation sites of the head domain, reflecting competing pressures to evade host immune response while retaining viral fitness. To our knowledge, our results are the first to quantify changes in glycosylation state that occur in related proteins of considerable glycan heterogeneity. Our results provide a method for understanding how changes in glycosylation state are correlated with variations in protein sequence, which is necessary for improving IAV vaccine strain selection. Understanding glycosylation will be especially important as we find new expression vectors for vaccine production, as glycosylation state depends greatly on the host species.

Project description:As biopharmaceuticals, recombinant proteins have become indispensable tools in medicine. An increasing demand, not only in quantity but also in diversity, drives the constant development and improvement of production platforms. The N-glycosylation pattern on biopharmaceuticals plays important roles in activity, serum half-life and immunogenicity. Therefore, production platforms with tailored protein N-glycosylation are of great interest. Plant-based systems have already demonstrated their potential to produce pharmaceutically relevant recombinant proteins, although their N-glycan patterns differ from those in humans. Plants have shown great plasticity towards the manipulation of their glycosylation machinery, and some have already been glyco-engineered in order to avoid the attachment of plant-typical, putatively immunogenic sugar residues. This resulted in complex-type N-glycans with a core structure identical to the human one. Compared to humans, plants lack the ability to elongate these N-glycans with β1,4-linked galactoses and terminal sialic acids. However, these modifications, which require the activity of several mammalian enzymes, have already been achieved for Nicotiana benthamiana and the moss Physcomitrella. Here, we present the first step towards sialylation of recombinant glycoproteins in Physcomitrella, human β1,4-linked terminal N-glycan galactosylation, which was achieved by the introduction of a chimeric β1,4-galactosyltransferase (FTGT). This chimeric enzyme consists of the moss α1,4-fucosyltransferase transmembrane domain, fused to the catalytic domain of the human β1,4-galactosyltransferase. Stable FTGT expression led to the desired β1,4-galactosylation. However, additional pentoses of unknown identity were also observed. The nature of these pentoses was subsequently determined by Western blot and enzymatic digestion followed by mass spectrometric analysis and resulted in their identification as α-linked arabinoses. Since a pentosylation of β1,4-galactosylated N-glycans was reported earlier, e.g. on recombinant human erythropoietin produced in glyco-engineered Nicotiana tabacum, this phenomenon is of a more general importance for plant-based production platforms. Arabinoses, which are absent in humans, may prevent the full humanization of plant-derived products. Therefore, the identification of these pentoses as arabinoses is important as it creates the basis for their abolishment to ensure the production of safe biopharmaceuticals in plant-based systems.

Dataset Information

Parallel Reaction Monitoring to Assess High-Affinity N-Glycoforms of the Human Fcγ Receptor IIIa (CD16a)

Dataset's files

Publications

Assessing High-Affinity <i>N</i>-Glycoforms of the Human Fcγ Receptor IIIa (CD16a) via Parallel Reaction Monitoring Mass Spectrometry.

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Dataset Information

Parallel Reaction Monitoring to Assess High-Affinity N-Glycoforms of the Human Fcγ Receptor IIIa (CD16a)

Dataset's files

Publications

Assessing High-Affinity &lt;i&gt;N&lt;/i&gt;-Glycoforms of the Human Fcγ Receptor IIIa (CD16a) via Parallel Reaction Monitoring Mass Spectrometry.

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OmicsDI is part of the ELIXIR infrastructure

Tweets

Assessing High-Affinity <i>N</i>-Glycoforms of the Human Fcγ Receptor IIIa (CD16a) via Parallel Reaction Monitoring Mass Spectrometry.