Evidence of Alternative Modes of B Cell Activation Involving Acquired Fab Regions of N-Glycosylation in Antibody-Secreting Cells Infiltrating the Labial Salivary Glands of Patients With Sjögren's Syndrome

Abstract

Objective: To better understand the role of B cells, the potential mechanisms responsible for their aberrant activation, and the production of autoantibodies in the pathogenesis of Sjögren's syndrome (SS), this study explored patterns of selection pressure and sites of N-glycosylation acquired by somatic mutation (acN-glyc) in the IgG variable (V) regions of antibody-secreting cells (ASCs) isolated from the minor salivary glands of patients with SS and non-SS control patients with sicca symptoms.

Methods: A novel method to produce and characterize recombinant monoclonal antibodies (mAb) from single cell-sorted ASC infiltrates was applied to concurrently probe expressed genes (all heavy- and light-chain isotypes as well as any other gene of interest not related to immunoglobulin) in the labial salivary glands of patients with SS and non-SS controls. V regions were amplified by reverse transcription-polymerase chain reaction, sequenced, and analyzed for the incidence of N-glycosylation and selection pressure. For specificity testing, the amplified regions were expressed as either the native mAb or mutant mAb lacking the acN-glyc motif. Protein modeling was used to demonstrate how even an acN-glyc site outside of the complementarity-determining region could participate in, or inhibit, antigen binding.

Results: V-region sequence analyses revealed clonal expansions and evidence of secondary light-chain editing and allelic inclusion, of which neither of the latter two have previously been reported in patients with SS. Increased frequencies of acN-glyc were found in the sequences from patients with SS, and these acN-glyc regions were associated with an increased number of replacement mutations and lowered selection pressure. A clonal set of polyreactive mAb with differential framework region 1 acN-glyc motifs was also identified, and removal of the acN-glyc could nearly abolish binding to autoantigens.

Conclusion: These findings support the notion of an alternative mechanism for the selection and proliferation of some autoreactive B cells, involving V-region N-glycosylation, in patients with SS.

Figure 1

Somatic hypermutations, replacement mutations and amino acid changes in sequences with and without acN-glycs. A – C) Sequence analyses of V-regions from SS patient and DNMC control minor salivary gland ASCs with (black columns, n = 31) and without (white columns, n = 91) acN-glycs. A) Number of heavy and light chain V-region somatic hypermutations. B) Number of heavy and light chain replacement mutations. C) Number of heavy and light chain V-region amino acid changes.

Figure 2

BASELINe comparison of selection pressure differences between groups. The histograms provide a visualization of selection pressures for the CDRs (top half of plots, red lines) and the FWRs (bottom half of plots, blue lines) and an overlay (lower left quadrant) showing the differences between the compared groups, where the solid lines represent the column variable (ie. DNMC in panel A) and the dashed lines represent the row variable (ie. SS in panel A). Statistically derived relative differences (binomial statistical test used by BASELINe) are shown (upper right quadrant) for each comparison, where red color and positive numbers are representative of positive selection, and green color and negative numbers are representative of negative selection. A) Comparison of selection pressures in heavy chains between SS and DNMC controls. B) Comparison of selection pressures in light chains between SS and DNMC controls. C) Comparison of selection pressures in heavy chains with acN-glycs vs those without acN-glycs.

Figure 3

Clonal mAb N-glycosylation analysis and antigen-binding by ELISA. A) Alignment of heavy and light chain amino acids from clonally related mAbs from pSS-1 revealing differences that may affect antigen binding. Bold underlined amino acids are different from consensus residues. Box indicates site of acquired N-glycosylation motif (N-C-T, position 21) in 4 of the 5 clones. Symbols: Cross in circle = identical translated heavy chain V-region sequences; Star = identical translated light chain V-region sequences, “*” (asterisk) indicates fully conserved residues; “:” (colon) indicates conservation between groups with strongly similar properties; “.” (period) indicates conservation between groups with weakly similar properties. B) Mobility shift analysis of mAb heavy chains. After PNGase F digest, followed by Coomassie Blue staining a more modest shift is seen in 4-B03k and Bovine IgG (known to have 1 Fc region N-glyc) as compared to 4-A01k, 2-E04k and 4-G03k. Concanavalin A staining indicates the complete removal of glycans by PNGase F. C) Clonal mAbs binding of Ro, La, Sm, and nRNP by ELISA. D) Antigen ELISA shows loss of antigen binding by mutant N21D-306k (acN-glyc -) as compared to its glycosylated counterpart, 3-C06K (acN-glyc+).

Figure 4

Protein model of 2-E04k mAb variable regions with complex-type FWR1 N-glycan. A) Mono image with heavy chain variable region (V_H) oriented on the left and light chain variable region (V_L) on the right. Heavy chain CDR region residues are indicated by red (CDR1), yellow (CDR2), and blue (CDR3). Light chain CDR region residues are indicated by muted colors, pink (CDR1), light yellow (CDR2) and light blue (CDR3). B) The same model rotated upward approximately 50° about the horizontal axis with the position of the heavy chain acN-glyc motif (N21) shown in magenta. C) Schematic of the representative complex-type N-glycan utilized in the model. D) Stereo image of model in panel A.