A Contra Capture Protein Array Platform for Studying Post-translationally Modified (PTM) Auto-antigenomes

Abstract

Aberrant modifications of proteins occur during disease development and elicit disease-specific antibody responses. We have developed a protein array platform that enables the modification of many proteins in parallel and assesses their immunogenicity without the need to express, purify, and modify proteins individually. We used anticitrullinated protein antibodies (ACPAs) in rheumatoid arthritis (RA) as a model modification and profiled antibody responses to ∼190 citrullinated proteins in 20 RA patients. We observed unique antibody reactivity patterns in both clinical anticyclic citrullinated peptide assay positive (CCP+) and CCP- RA patients. At individual antigen levels, we detected antibodies against known citrullinated autoantigens and discovered and validated five novel antibodies against specific citrullinated antigens (osteopontin (SPP1), flap endonuclease (FEN1), insulin like growth factor binding protein 6 (IGFBP6), insulin like growth factor I (IGF1) and stanniocalcin-2 (STC2)) in RA patients. We also demonstrated the utility of our innovative array platform in the identification of immune-dominant epitope(s) for citrullinated antigens. We believe our platform will promote the study of post-translationally modified antigens at a breadth that has not been achieved before, by both identifying novel autoantigens and investigating their roles in disease development. The developed platforms can potentially be used to study many autoimmune disease-relevant modifications and their immunogenicity.

Fig. 1.

Principle of the contra capture protein array platform.

Fig. 2.

Heat map of quantitative analysis of sero-reactivity to 11 known citrullinated auto-antigens and Epstein-Barr Nuclear Antigen (EBNA) on contra captured protein arrays. Thirty serum samples (12 CCP+ RA patients, 8 CCP- RA patients, 10 healthy controls) were used for this study. The left half of the heat map shows sero-reactivity to native protein arrays and the right half shows sero-reactivity to citrullinated protein arrays. EBNA was used as a positive sero-profiling control because most adults show sero-positivity to this antigen. Blue, low reactivity; red, high reactivity.

Fig. 3.

Representative images of native and citrullinated arrays probed with serum samples. All samples showed reactivity to both citrullinated and native EBNA.

Fig. 4.

Quantitative comparison of sero-reactivity of MBP. Results obtained from the contra capture platform versus the ELISA platform against anti-citrullinated MBP for 30 serum samples. Please refer to Methods for assay details.

Fig. 5.

Sero-profiling of ACPAs in RA patients on contra capture protein arrays against ∼190 antigens and validation of novel ACPAs identified on contra capture arrays by ELISA. A, Heat map depicting overall reactivity of 20 CCP+ and CCP- serum samples to 190 genes printed on array. CCP- negative samples were assay as pools of two samples as annotated on the top of the figure. Blue, low reactivity; red, high reactivity. B, Example array images probed with CCP+ RA patient serum samples (S366 and S451). C, Blinded validation of selected novel ACPAs by ELISA using an independent set of 150 sera comprising 50 CCP+, 50 CCP- and 50 control samples. HC, healthy control. * indicates t test p < 0.05, **, p < 0.01 and ****, p < 0.0001. D, Heat map depicting sero-reactivity against citrullinated antigens assayed by ELISA in different groups of the independent sample set.

Fig. 6.

Epitope mapping for MBP. Eight C-terminal deletion fragments were constructed for each isoform of MBP. Two response patterns were observed. In pattern 1, reactivity to deletion mutants MBP 1.7, MBP 1.8, MBP 2.2 and MBP 2.3 were observed. In pattern 2, reactivity to deletion mutants MBP 1.8 and MBP 2.3 were observed, but not to MBP 1.7 and MBP 2.2. MBP 1.7 and MBP 2.2 shared the same sequence and MBP 1.8 and MBP 2.3 shared same sequence.

Fig. 7.

Detection of antibodies against Tn-glycosylated proteins on contra capture arrays. A, Tn glycosylation by GalNac-T2, T3 and T11. After protein expression, arrays were untreated (native) or treated with one of three GalNac-Ts, labeled as GalNac-T2, GalNac-T3 or GalNac-T11, respectively. Features in boxes represents positive and negative control proteins. Green boxes on GalNac-T2, -T3 and -T11 represent MUC1-TR. The yellow box on GalNac-T2 represents NRP2 and that on GalNac-T11 represents POMC. The two yellow boxes on GalNac-T3 represent APOE (right) and CD55 (left), respectively. The APOE mutant (unmodifiable) is shown in a purple box. B, VVA and anti-HaloTag staining of arrays displaying 87 different proteins Tn-glycosylated by GalNac-T3. C, Antibody profiling against the 87 proteins shown in (B) with (left column) and without (right column) Tn-glycosylation by GALNT3. S1571-IgA and S1571-IgG were the same array detecting AAb of IgA and IgG classes in sample S1571, simultaneously. Same for S1660-IgG and S1660-IgA.