Glycosylation of proteinase 3 (PR3) is not required for its reactivity with antineutrophil cytoplasmic antibodies (ANCA) in Wegener's granulomatosis

Abstract

Objective: The glycosylation status of autoantigens appears to be crucial for the pathogenesis of some autoimmune diseases, since carbohydrates play a crucial role in the distinction of self from non-self. Proteinase 3 (PR3), the main target antigen for anti-neutrophil cytoplasmic antibodies (ANCA) in patients with Wegener's granulomatosis (WG), contains two Asn-linked glycosylation sites. The present study explores the influence of the glycosylation status of PR3 on the PR3 recognition by ANCA in a well characterized population of patients with WG.

Methods: Forty-four patients with WG (459 serum samples) who participated in a multicenter randomized trial, were tested by capture ELISA for ANCA against PR3 and deglycosylated recombinant variants of PR3.

Results: The patients were followed for a median of 27 months, and the median number of serum samples per patient was 10. At baseline, the correlation between the levels of ANCA against PR3 and against all the deglycosylated recombinant variants of PR3 were greater than 0.94 (?<0.001 for all the comparisons). Longitudinal analyses comparing the levels of ANCA against PR3 versus all the deglycosylated recombinant variants of PR3, using linear mixed models, showed no significant statistical differences (rho >or=0.90 in all cases).

Conclusion: The glycosylation status of PR3 has no impact on its recognition by ANCA in WG.

Fig. 1

Proteinase 3 and related cDNA constructs. The transfection of 293 cells with rPR3 results in the expression of a protein in the serum free media that carries both the amino- and carboxy-terminal propeptides (19, 20), precluding the folding of PR3 into its mature conformation. Therefore, all the cDNA constructs used in this study do not code for the N-terminal propeptide, resulting in the expression of PR3 variants with a mature conformation (20). Additionally, all the constructs carry the mutation S195A, which results in lack of enzymatic activity without affecting their recognition by PR3-ANCA (20). The addition of the c-myc tag to the carboxy-terminus of PR3 also does not affect binding of PR3-ANCA (19). The constructs Δ-rPR3-N113Q-S195A-c-myc, and Δ-rPR3-N159Q-S195A-c-myc carry the mutations N113Q and N159Q coding for glutamine instead of the asparagine in position 113 and 159, respectively. As a result, their corresponding expressed proteins, tagged-PR3-G1 and -G2, do not carry a glycosylation residue in position 113 and 159, respectively. The construct Δ-rPR3-N113Q/N159Q-S195A-c-myc carries both mutations, expressing a protein, tagged-PR3-G0, without any Asn-linked sugar moieties. Protein sequences are numbered based on the crystal structure correspondence with chymotrypsinogen A (5, 17).

Fig. 2

Expression of c-myc tagged deglycosylated variants of PR3 in 293 cells. All the c-myc tagged deglycosylated variants of PR3 carry indeed the c-myc tag extension, in contrast to purified native PR3. The upper panel shows purified human PR3 and all the c-myc tagged deglycosylated variants of PR3 when probed with MCPR3-2, a mouse monoclonal antibody against human PR3 (anti-PR3). In the lower panel, all c-myc tagged deglycosylated variants of PR3 could be detected when probed with the mouse monoclonal antibody against the c-myc tag polypeptide (anti-c-myc), but not purified human PR3. Proteins were precipitated in 55% trichloroacetic acid and separated by SDS-PAGE (12% gels) under non-reducing conditions. The right panel show the saturation curves of the serum-free culture media supernatants of 293 cells transfected with the c-myc tagged deglycosylated variants of PR3, using plates coated with mouse monoclonal antibody anti-c-myc (Sigma P2241) as the capturing antibody, and using the rabbit polyclonal antibody against human PR3 as detection antibody. PR3: Purified human PR3. t-PR3: tagged-PR3 from Δ-rPR3-S195A-c-myc transfected 293 cells. t-PR3-G0: tagged-PR3-G0 from Δ-rPR3-N113Q/N159Q-S195A-c-myc transfected 293 cells. t-PR3-G1: tagged-PR3-G1 from Δ-rPR3-N113Q-S195A-c-myc transfected 293 cells. t-PR3-G2: tagged-PR3-G2 from Δ-rPR3-N159Q-S195A-c-myc transfected 293 cells.

Fig. 3

Examples of patients tested over time for ANCA reactivity with tagged-PR3 and tagged deglycosylated variants of PR3 Each plot shows the levels of tagged-PR3 and tagged-PR3-G0, -G1, and -G2 over time in four different patients. In all the cases, no differences in the pattern of change could be seen among these four determinations. t-PR3: tagged-PR3. t-PR3-G0: tagged-PR3-G0. t-PR3-G1: tagged-PR3-G1. t-PR3-G2: tagged-PR3-G2.