Affinity Purification and Comparative Biosensor Analysis of Citrulline-Peptide-Specific Antibodies in Rheumatoid Arthritis

Szarka, Eszter; Aradi, Petra; Huber, Krisztina; Pozsgay, Judit; Végh, Lili; Magyar, Anna; Gyulai, Gergö; Nagy, György; Rojkovich, Bernadette; Kiss, Éva; Hudecz, Ferenc; Sármay, Gabriella

doi:10.3390/ijms19010326

Cited by 13 publications

(9 citation statements)

References 48 publications

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“…However, our findings related to glycine are consistent with the findings of others. The presence of glycine next to citrulline has been observed in the citrullinome of 30 human tissues , in motifs targeted by the citrullinating peptidylarginine deiminases 2 and 4 , and in autoantibody binding in RA . The preference for citrullination of or IgG binding to peptides with citrulline next to serine has not been previously described.…”

Section: Discussionmentioning

confidence: 97%

Disordered Antigens and Epitope Overlap Between Anti–Citrullinated Protein Antibodies and Rheumatoid Factor in Rheumatoid Arthritis

Zheng

Mergaert

Fahmy

et al. 2019

Arthritis & Rheumatology

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Objective. Anti-citrullinated protein antibodies (ACPAs) and rheumatoid factor (RF) are commonly present in rheumatoid arthritis (RA) without a clear rationale for their coexistence. Moreover, autoantibodies develop against proteins with different posttranslational modifications and native proteins without obvious unifying characteristics of the antigens. We undertook this study to broadly evaluate autoantibody binding in seronegative and seropositive RA to identify novel features of reactivity.Methods. An array was created using a total of 172,828 native peptides, citrulline-containing peptides, and homocitrulline-containing peptides derived primarily from proteins citrullinated in the rheumatoid joint. IgG and IgM binding to peptides were compared between cyclic citrullinated peptide (CCP)-positive RF+, CCP+RF−, CCP−RF+, and CCP−RF− serum from RA patients (n = 48) and controls (n = 12). IgG-bound and endogenously citrullinated peptides were analyzed for amino acid patterns and predictors of intrinsic disorder, i.e., unstable 3-dimensional structure. Binding to IgG-derived peptides was specifically evaluated. Enzyme-linked immunosorbent assay confirmed key results.Results. Broadly, CCP+RF+ patients had high citrulline-specific IgG binding to array peptides and CCP+RF− and CCP−RF+ patients had modest citrulline-specific IgG binding (median Z scores 3.02, 1.42, and 0.75, respectively; P < 0.0001). All RA groups had low homocitrulline-specific binding. CCP+RF+ patients had moderate IgG binding to native peptides (median Z score 2.38; P < 0.0001). The highest IgG binding was to citrulline-containing peptides, irrespective of protein identity, especially if citrulline was adjacent to glycine or serine, motifs also seen in endogenous citrullination in the rheumatoid joint. Highly bound peptides had multiple features predictive of disorder. IgG from CCP+RF+ patients targeted citrulline-containing IgG-derived peptides.Conclusion. Disordered antigens, which are frequently citrullinated, and common epitopes for ACPAs and RF are potentially unifying features for RA autoantibodies.

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Section: Discussionmentioning

confidence: 97%

Disordered Antigens and Epitope Overlap Between Anti–Citrullinated Protein Antibodies and Rheumatoid Factor in Rheumatoid Arthritis

Zheng

Mergaert

Fahmy

et al. 2019

Arthritis & Rheumatology

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“…We collected sera from anti-cyclic citrullinated peptide (CCP)-positive antibodies in diagnosed RA patients and from healthy controls with ethical permission and purified the IgG fractions by protein G affinity chromatography. Sera of RA patients were tested for ACPA-IgG by ELISA using different citrulline-containing peptides corresponding to citrullinated epitopes in filaggrin, collagen, and a multi-epitope citrulline peptide characterized by us previously (see Table in Section 8 ) [ 32 ]. The ACPA reactivity and demographical and clinical data of the patients are summarized in Table 1 .…”

Section: Resultsmentioning

confidence: 99%

“…The ACPA content of the acquired serum samples was detected with an indirect ELISA as previously described [ 32 , 46 , 47 ]. Shortly, ELISA plates were pre-coated with 5 μg/mL neutravidine overnight at 4 °C to enhance peptide binding, and then were coated with selected citrulline or arginine-containing biotinylated peptides (see Table 2 for amino acid sequences) at 1 μg/mL concentration for an hour at 37 °C.…”

Section: Methodsmentioning

confidence: 99%

“…ACPA were affinity purified from IgG fractions of RA patients on citrulline-containing peptides (provided by Anna Magyar and Katalin Uray at ELTE Peptide Chemistry Research Group) immobilized on NHS (n-hydroxi-succinimide) columns (Hi Trap ® , GE Healthcare, Uppsala, Sweden) as previously described [ 32 ]. The concentration of the eluted fractions was determined and adjusted to the 0.5–1 mg/mL range.…”

Section: Methodsmentioning

confidence: 99%

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The Role of IgG Fc Region N-Glycosylation in the Pathomechanism of Rheumatoid Arthritis

Gyebrovszki

Ács

Szabó

et al. 2022

IJMS

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Anti-citrullinated protein antibodies (ACPAs) are involved in the pathogenesis of rheumatoid arthritis. N-glycosylation pattern of ACPA-IgG and healthy IgG Fc differs. The aim of this study is to determine the relative sialylation and galactosylation level of ACPAs and control IgG to assess their capability of inducing TNFα production, and furthermore, to analyze the correlations between the composition of Fc glycans and inflammatory markers in RA. We isolated IgG from sera of healthy volunteers and RA patients, and purified ACPAs on a citrulline-peptide column. Immunocomplexes (IC) were formed by adding an F(ab)2 fragment of anti-human IgG. U937 cells were used to monitor the binding of IC to FcγR and to trigger TNFα release determined by ELISA. To analyze glycan profiles, control IgG and ACPA-IgG were digested with trypsin and the glycosylation patterns of glycopeptides were analyzed by determining site-specific N-glycosylation using nano-UHPLC-MS/MS. We found that both sialylation and galactosylation levels of ACPA-IgG negatively correlate with inflammation-related parameters such as CRP, ESR, and RF. Functional assays show that dimerized ACPA-IgG significantly enhances TNFα release in an FcγRI-dependent manner, whereas healthy IgG does not. TNFα production inversely correlates with the relative intensities of the G0 glycoform, which lacks galactose and terminal sialic acid moieties.

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“…There have been several attempts to characterize the affinity of antibodies associated with autoimmunity [ 35 , 36 , 37 , 38 ], infectious disease [ 39 , 40 ], vaccination [ 41 ] and allergy [ 14 , 42 , 43 , 44 , 45 ]. The strength of binding of serum antibodies is determined by the distribution of clones with different affinities specific for the tested epitope.…”

Section: Discussionmentioning

confidence: 99%

Absolute Quantitation of Serum Antibody Reactivity Using the Richards Growth Model for Antigen Microspot Titration

Papp¹,

Kovács

Orosz

et al. 2022

Sensors

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In spite of its pivotal role in the characterization of humoral immunity, there is no accepted method for the absolute quantitation of antigen-specific serum antibodies. We devised a novel method to quantify polyclonal antibody reactivity, which exploits protein microspot assays and employs a novel analytical approach. Microarrays with a density series of disease-specific antigens were treated with different serum dilutions and developed for IgG and IgA binding. By fitting the binding data of both dilution series to a product of two generalized logistic functions, we obtained estimates of antibody reactivity of two immunoglobulin classes simultaneously. These estimates are the antigen concentrations required for reaching the inflection point of thermodynamic activity coefficient of antibodies and the limiting activity coefficient of antigen. By providing universal chemical units, this approach may improve the standardization of serological testing, the quality control of antibodies and the quantitative mapping of the antibody–antigen interaction space.

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Affinity Purification and Comparative Biosensor Analysis of Citrulline-Peptide-Specific Antibodies in Rheumatoid Arthritis

Cited by 13 publications

References 48 publications

Disordered Antigens and Epitope Overlap Between Anti–Citrullinated Protein Antibodies and Rheumatoid Factor in Rheumatoid Arthritis

Disordered Antigens and Epitope Overlap Between Anti–Citrullinated Protein Antibodies and Rheumatoid Factor in Rheumatoid Arthritis

The Role of IgG Fc Region N-Glycosylation in the Pathomechanism of Rheumatoid Arthritis

Absolute Quantitation of Serum Antibody Reactivity Using the Richards Growth Model for Antigen Microspot Titration

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