Fig 2: Identification of anti-Tn CICs in sera of patients with IgAN and healthy controls sera and complexes dissociate with glycomimetic.(A) BN-APAGE analysis of Tn(+)matrix affinity purified anti-Tn antibodies and total purified IgM. Left: Native molecular weight markers for approximation of molecular weight (m.w.). P1, P3, and P4 represent three patients; control IgM. (B) Tn(+)matrix affinity-purified ReBaGs6 antibody resolved by BN-APAGE system and blotted for murine IgM. (C) Similar to (A), BN-APAGE analysis of affinity-purified anti-Tn antibodies and total purified IgM from three healthy controls (C2, C3, and C6) and control IgM immunoblotted for IgM. (D) Coomassie-stained SDS-PAGE gel of affinity-purified anti-Tn antibodies from healthy control (C3) and patient (P3); control antibodies and BSA as indicated. Immunoblots (bottom) of same preparation probed as indicated. VVA and IgA blots performed with appropriate controls: purified Tn(+)IgA1 and Tn(-)IgA1 produced from CosmcKO and WT Dakiki cells, respectively. (E) Anti-Tn CICs purified from serum of patients with IgAN immunodepleted with anti-IgA or isotype control antibodies (Ab); depleted materials analyzed by BN-APAGE WB probed for IgM. IP, immunoprecipitation. (F) Anti-Tn CICs purified from serum of patient with IgAN treated with mock, a-methylGalNAc (aGalNAc), or a-methylGlcNAc (aGlcNAc); samples analyzed by BN-APAGE WB and probed for IgM and IgA. (G) Tn glycopeptide array probed with anti-Tn antibodies (C3) preincubated with 20 mM a-methylGalNAc or 20 mM a-methylGlcNAc and stained with Alexa Fluor 488–labeled anti-human IgM. Error bars represent ±1 SD of four replicates. n = 3 except blot images from (A), (C), and (E); n = 2. Asterisk represents normal pentameric IgM.

Fig 3: Anti-Tn antibodies in human sera: Identification and characterization.(A) Coomassie-stained SDS-PAGE analysis of affinity-purified anti-Tn antibodies from serum using Tn(+)matrix (elution) and control (beads alone); affinity-purified materials (top) immunoblotted as indicated (bottom). FT, flow through. Coomassie brilliant blue (CBB)-stained. (B) Similar analysis to (A) (healthy donors; Table 1), with purified control antibodies and bovine serum albumin (BSA) (4 µg); VVA detects Tn(+)IgA1. (C) Purified anti-Tn antibodies bind to Tn(+) matrix; a-methylGalNAc inhibits binding (Alexa Fluor 488–labeled anti-human IgM antibody, top, green). Four times amount of anti-Tn antibodies mixed with Tn(+)matrix [Alexa Fluor 488–labeled anti-human IgG (middle) and IgA (bottom)]. Beads alone, negative control; Differential Interference Contra (DIC), bead images. Performed in three biological replicates (C1, C6, and C10). (D) Tn glycopeptide array probed with purified anti-Tn antibodies from 10 healthy control sera (C3, representative) and stained with Alexa Fluor 488–labeled anti-human IgM antibody (see fig. S3). Chart ID corresponds to Table 2. (E and F) Data analysis using GLAD (Glycan Array Dashboard), comparing binding preferences between Tn on IgA1 and non-IgA1 peptides, and correlation of binding patterns in C1 to C10. VVA binds Tn glycopeptides (positive control), and anti-Tn (recombinant murine anti-Tn IgM) differs from C1 to C10 binding. (G) Purified Tn(+)IgA1 (from CosmcKO cells) and Tn(-)IgA1 (WT) analyzed by SDS-PAGE Western blot (WB), probed with purified anti-Tn antibodies (bottom), VVA (middle), and loading control (IgA; top), n = 3. (H) Tn glycopeptide array probed with purified anti-Tn antibody (C1) after preincubation with purified Tn(+)IgA1 or Tn(-)IgA1 and stained with Alexa Fluor 647–labeled anti-human IgM. For Tn glycopeptide arrays, error bars represent ±1 SD of four replicates. RFU, relative fluorescence units.