Journal: bioRxiv

Article Title: Symmetric Dimethylarginine Modification Drives Shared and Divergent Antigen Reactivity in Lupus

doi: 10.64898/2026.09.22.753525

Figure Lengend Snippet: Plasma samples from 31 SLE patients and two healthy controls were analyzed using two complementary approaches: (A) immunoblotting of HEp 2 cell lysates separated by SDS PAGE, and (B) indirect immunofluorescence on fixed HEp 2 cells (ANA test; additional images in Supplemental Figure S1 and S2). (A) Immunoblotting revealed distinct antibody-reactive band patterns across patients, with variable band intensities. Several patients (e.g., SLE 01, 06, 07, 17, 24, 28, 29, 30) showed few bands, similar to healthy controls. (B) In the ANA test, selected plasma samples (SLE 01, 10, 13, 27) displayed contrasting nuclear staining patterns (green). A commercial monoclonal anti SDMA antibody (SYM10) was used as a counterstain in a separate channel (red). (C) Four monoclonal antibodies (mAbs) cloned from MRL lpr mice were used to stain HEp-2 cells. These mAbs exhibited distinct staining patterns, some of which resembled those observed with SLE patient plasma. For example, mAb P 1G3D2 resembled SLE 10, and mAb P4A2F4 resembled SLE 27.

Article Snippet: Human epithelial type 2 (HEp-2) cell line (ATCC, CCL-23) was used as the antigen source for immunoblot detection of autoantibodies in plasma samples.

Techniques: Clinical Proteomics, Western Blot, SDS Page, Immunofluorescence, Staining, Bioprocessing, Clone Assay

Journal: bioRxiv

Article Title: Symmetric Dimethylarginine Modification Drives Shared and Divergent Antigen Reactivity in Lupus

doi: 10.64898/2026.09.22.753525

Figure Lengend Snippet: (A) Domain architectures of four representative RNA-binding proteins, each containing Arg-Gly-rich segments (red) of varying length and sequence. (B) Chemical structures of monomethylarginine (MMA), symmetric dimethylarginine (SDMA), and asymmetric dimethylarginine (ADMA), with methyl groups highlighted. (C) Eight synthetic peptides bearing different methylated arginine forms (or combinations thereof) were probed with plasma from SLE-22 and visualized with HRP-conjugated goat anti-human IgG (left panel). Peptides 4, 6, 7, and 8, all contained SDMA, reacted with SLE autoantibodies, whereas native, MMA-, and ADMA-containing peptides did not. (D) A tiling array of 29 peptides derived from human SmD1 (the RG-repeat region is highlighted, lower right). Peptides were designed with an 11-amino acid overlap (top right). Peptides 22-29, spanning the RG-repeats, were spotted in duplicate in either native or SDMA form (bottom). Only SDMA-containing peptides (spots 23, 25, 27, and 29) reacted with SLE-22 autoantibodies. (E) PRMT5 inhibitor EPZ015666 reduces specific immunoblot band intensities recognized by SLE-10 and SLE-22 plasma in HEp-2 cells. Arrows indicate bands with reduced intensity following inhibitor pretreatment. α-Tubulin (αTub) serves as a loading control. (F) Schematic of an array containing 105 peptides from 76 proteins identified in a human methylarginine proteome screen. Each peptide was synthesized in native, MMA, SDMA, or ADMA form. Representative data for SmB (amino acids 170-184) show strong reactivity of the SDMA-modified B1 peptide with SLE-22, weak reactivity for the MMA-modified A2 peptide, and no reactivity for native or ADMA forms. (G) Overview of the full array (420 peptides). SLE-22 autoantibodies reacted broadly with predominantly SDMA-containing peptides, with variable signal intensities. Checkered patches indicate two mistakenly synthesized sequences that were still included in the screen. (H) EBV antigen EBNA-1 contains two Arg-Gly-rich segments (as described in A). A 16-peptide array (divided into 4 groups) covering the longer segment (amino acids 378-426) was probed with healthy control plasma, SLE-22 plasma, or control antibodies SYM10 and ASM24. SLE-22 reacted with all four SDMA-containing peptides, mirroring the reactivity pattern of SYM10. (I) Full-length FLAG-tagged recombinant EBNA-1 was expressed in HEK293 cells (human cells that naturally produce arginine methylation, though with unknown frequency) and E. coli (prokaryotes do not generate arginine side-chain methylation). As expected, HEK293-derived EBNA-1 showed stronger reactivity with plasma from SLE-10 and SLE-22, as well as with a monoclonal antibody from the lupus-prone MRL-lpr mouse strain.

Article Snippet: Human epithelial type 2 (HEp-2) cell line (ATCC, CCL-23) was used as the antigen source for immunoblot detection of autoantibodies in plasma samples.

Techniques: RNA Binding Assay, Sequencing, Methylation, Clinical Proteomics, Autoantibodies, Tiling, Derivative Assay, Western Blot, Control, Synthesized, Modification, Peptide Microarray, Recombinant

Journal: bioRxiv

Article Title: Symmetric Dimethylarginine Modification Drives Shared and Divergent Antigen Reactivity in Lupus

doi: 10.64898/2026.09.22.753525

Figure Lengend Snippet: (A) Sera from MRL lpr mice (n=12, bled weekly from 8 to 13 weeks of age) were analyzed by ELISA using synthetic 10×RG peptides: native sequence (GRGRGRGRGRGRGRGRGRGR) vs. SDMA-modified (lowercase ‘r’ denotes SDMA; GrGrGrGRGRGrGrGrGRGR). All 12 mice developed anti SDMA antibody titers above the corresponding native controls, and the majority (9/12) showed a gradual increase in anti-SDMA titers over time. (B) Immunoblotting of HEp-2 lysates revealed progressive changes in band patterns from 10 weeks onward. To identify SDMA specific reactive proteins, cells were pretreated with the methyltransferase inhibitor EPZ015666 (EPZ) before lysis. A prominent ∼15 kDa band (arrowhead) was markedly reduced by EPZ pretreatment, supporting its recognition by anti SDMA antibodies. (C) Sera from three 12 week old MRL lpr littermates (Lpr 1, 2, 3) each showed distinct band patterns against the cell lysates. EPZ pretreatment of the cells reduced the intensity of several bands in all three mice. (D) Workflow for isolating hybridoma clones from individual plasma cells to obtain monoclonal antibodies (mAbs) specific for SDMA. MRL lpr mice were not immunized with any antigen; thus, the resulting mAbs represent naturally arising autoantibodies. (E) Two antibody clones were tested against the native versus SDMA peptide array shown in Figurè3. Clone P4A2F4 reacted extensively with most SDMA containing peptides, whereas clone P2C1C10 showed no reactivity. (F) The two mAbs produced different band patterns on cell lysates. P4A2F4 recognized bands that were reduced in EPZ pretreated lysates, consistent with SDMA specificity. (G) After implantation of the SDMA-reactive hybridoma clone into the peritoneal cavity of healthy BALB/c mice, the kidneys showed extensive IgG deposition, reminiscent of human lupus nephritis.

Article Snippet: Human epithelial type 2 (HEp-2) cell line (ATCC, CCL-23) was used as the antigen source for immunoblot detection of autoantibodies in plasma samples.

Techniques: Enzyme-linked Immunosorbent Assay, Sequencing, Modification, Western Blot, Lysis, Clone Assay, Clinical Proteomics, Bioprocessing, Autoantibodies, Peptide Microarray, Produced