Volume 139, Issue 5, Pages 1753-1761 (November 2010) α-Enolase Autoantibodies Cross-Reactive to Viral Proteins in a Mouse Model of Biliary Atresia  Brandy R. Lu, Stephen M. Brindley, Rebecca M. Tucker, Cherie L. Lambert, Cara L. Mack  Gastroenterology  Volume 139, Issue 5, Pages 1753-1761 (November 2010) DOI: 10.1053/j.gastro.2010.07.042 Copyright © 2010 AGA Institute Terms and Conditions

Figure 1 Identification of bile duct epithelial cytosolic proteins reactive to IgG within BA sera. (A) Immunofluorescence: Panel 1: The normal mouse cholangiocyte cell line (NMC) was incubated with anti-cytokeratin 19 (CK19)-AF555 (red) and counterstained with nuclear Hoechst dye (blue). Panels 2 and 3: Cultured NMCs were incubated with sera from BA or saline (BSS) control mice, followed by anti-IgG-FITC (green). (B) ELISA: Plates were coated with the NMC cytosolic fraction and incubated with sera followed by anti-IgG HRP and substrate. Quantification of serum IgG reactive to NMC proteins is represented by the mean ± standard error of mean optical density. (C) Western blot analysis: The NMC cytosolic fraction was incubated with sera, and IgG reactive to NMC proteins was visualized by chemiluminescence. The IgG from BA sera reacted to unique proteins at 48 kilodaltons and 33 kilodaltons (boxed bands). Gastroenterology 2010 139, 1753-1761DOI: (10.1053/j.gastro.2010.07.042) Copyright © 2010 AGA Institute Terms and Conditions

Figure 2 Mass spectrometry and peptide fingerprint analysis identified the 48-kilodalton protein band as α-enolase. (A) Protein analysis: Peptide mass of the 48-kilodalton proteins was determined by MALDI-TOF mass spectrometry, entered into the Mascot search engine, and analyzed against the murine genome. Four candidate proteins were identified as significant, with α-enolase demonstrating the highest peptide coverage. (B) α-Enolase peptide coverage within candidate protein: Shown in bold are the peptides from the candidate protein that contribute to 64% of the total peptide coverage of murine α-enolase. Gastroenterology 2010 139, 1753-1761DOI: (10.1053/j.gastro.2010.07.042) Copyright © 2010 AGA Institute Terms and Conditions

Figure 3 Generation of anti-enolase antibodies within BA mice is dependent on both RRV infection and biliary obstruction. (A) ELISA: Plates were coated with purified enolase and incubated with sera followed by anti-IgG horseradish peroxidase (HRP) and substrate. Standard curves for anti-enolase were obtained and results shown as mean ± standard error of mean. Control mice included BSS; noncholestatic, RRV-infected siblings of BA (RRV infected, without [w/o] BA); mothers of BA; and bile duct ligated, cholestatic adult mice. Significant differences in antibody levels were found between each control group and BA (*P < .05). (B) Western blot analysis: The NMC cytosolic fraction or purified enolase was incubated with sera, and IgG reactive to protein was visualized by chemiluminescence. (C) ELISA verification of RRV infection: Plates were coated with RRV and incubated with sera followed by anti-IgG HRP and substrate. Standard curves for anti-RRV were obtained and results shown as mean ± standard error of mean. Gastroenterology 2010 139, 1753-1761DOI: (10.1053/j.gastro.2010.07.042) Copyright © 2010 AGA Institute Terms and Conditions

Figure 4 Elevated serum anti-enolase IgM and IgG antibodies in BA patients. (A) Representative immunofluorescence: Panel 1: The human cholangiocyte cell line was incubated with anti-cytokeratin 7-Cy3 (CK7) (red) and counterstained with nuclear Hoechst dye (blue). Panels 2 and 3: Cultured cholangiocytes were incubated with sera from BA or control patients, followed by anti-human IgG-FITC (green). (B) Sera from BA infants at the time of diagnosis (left panel, IgM), BA children >1 year of age with their native liver (right panel, IgG), and other disease age-matched controls were tested for anti-enolase antibodies by ELISA. Each dot represents the average anti-enolase antibody level from a single patient, and the bar is the mean value of all subjects within the group. Gastroenterology 2010 139, 1753-1761DOI: (10.1053/j.gastro.2010.07.042) Copyright © 2010 AGA Institute Terms and Conditions

Figure 5 Anti-RRV and anti-enolase antibodies cross-reactive with both RRV and enolase proteins. Western blot: Purified RRV, ovalbumin (ova), and enolase (1 μg each) were incubated with either purified anti-RRV or anti-enolase antibody, and reactivity was visualized by chemiluminescence. The ∼120-kilodalton RRV protein band reactive to anti-enolase antibody was identified by mass spectrometry to contain proteins VP2, VP4, and VP6. Gastroenterology 2010 139, 1753-1761DOI: (10.1053/j.gastro.2010.07.042) Copyright © 2010 AGA Institute Terms and Conditions

Figure 6 Sequence homology and ribbon diagrams of α-enolase, VP5, and VP8. (A) BLASTp analysis: Murine α-enolase and RRV protein genome were searched for peptide homology. Three significant matches are shown between α-enolase and VP4 (VP4 subunits are VP5 and VP8). Peptides shown in bold represent an exact match, and a “+” sign between the 2 sequences represents conservative amino acid changes. The underlined peptide segment in VP4 is the VP5 subunit that is a known immunogenic region responsible for generating the neutralizing antibody response. Ribbon diagrams: (B) Enolase and (C) VP5/VP8. Homologous amino acids between α-enolase and VP4 (VP5/VP8 subunits) are highlighted in yellow. The underlined VP5 peptide sequence shown in A that is homologous with enolase is highlighted in purple. Gastroenterology 2010 139, 1753-1761DOI: (10.1053/j.gastro.2010.07.042) Copyright © 2010 AGA Institute Terms and Conditions