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Autoantibodies to tissue transglutaminase target epitopes dependent on residues in the N-terminal domain

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dc.contributor.author Kozhakhmetova, A
dc.contributor.author Wyatt, R
dc.contributor.author Elvers, K
dc.contributor.author Emery, D
dc.contributor.author Williams, C
dc.contributor.author Annis, P
dc.contributor.author Lampasona, V
dc.contributor.author Gillespie, K
dc.contributor.author Williams, A
dc.date.accessioned 2019-09-09T10:14:37Z
dc.date.available 2019-09-09T10:14:37Z
dc.date.issued 2017-03-24
dc.identifier.citation A Kozhakhmetova, R Wyatt, K Elvers, D Emery, C Williams, P Annis, V Lampasona, K Gillespie, A Williams. Autoantibodies to tissue transglutaminase target epitopes dependent on residues in the N-terminal domain. Coeliac UK Research Conference, 24 March 2017, London, UK en_US
dc.identifier.uri http://nur.nu.edu.kz/handle/123456789/4229
dc.description.abstract INTRODUCTION: Tissue transglutaminase (tTG), is the main autoantigen of coeliac disease (CD). Specific tTG epitopes, including N-and C-terminal sites as well as the catalytic triad, have been reported to be targeted by autoantibodies in CD, but the findings are controversial. We aimed to confirm which of the tTG sites, catalytic core, C- or N-terminus, are critical for antibody binding as this could aid the design of more specific assays and inform studies of disease pathogenesis. METHODS: Plasmid DNA encoding human tTG was mutated at sites in the catalytic core (Cys277, His335, Asp358 to alanine), N- (Arg19, Glu153 to serine) and C-terminal domains (Met659 to serine) using PAGE-purified mutagenic primers. Antibody binding to a panel of 35S-labelled tTG mutant antigens synthesized in vitro was assessed by radioimmunoassay in sera from 111 TGA-positive individuals (mean age 40 years, range 1.1-80 years) out of 445 patients sent for measurement of CD-associated autoantibodies to the Cork University Hospital, Eire. Wilcoxon signed-ranks test (SPSS) was used for statistical analysis. RESULTS: Showed that single mutation of catalytic core amino acids C277A, D358A, H335A had little effect on the relative binding (RB) of antibodies to antigen compared with native tTG (median RB; p-values: 100%; 0.723, 93%; 6.1E-5, 106%; 0.019, respectively for each mutant, while the triple Cys277/His335/Asp358 mutation slightly decreased RB (85%; 3.7E-16). Single mutation of the N-terminal amino acids, Arg19 or Glu153, elicited significantly decreased RB in ≥90% of sera (66%; 4.2E-17, 76%; 1.0E-11, respectively), as well as for the double Arg19/Glu153 (48%; 3.1E-17) and triple Arg19/Glu153/Met659 (48%; 1.1E—15) mutants. Mutation of Met659 alone had a variable effect, decreasing binding in others (102%; 0.004). The combined Cys277/His335/Asp358 and Arg19/Glu153/Met659 tTG mutations reduced binding in 96% of sera (60%; 2.2E-19). DISCUSSION: The N-terminal domain of tTG involving amino acids Arg19 and Glu153 is important to the integrity of tTG autoantibody epitopes in CD. Simultaneous mutation of these two amino acids has the highest impact on antibody-binding to tTG, while modifying catalytic core previously reported as being important, had little effect on binding. Effect of C-terminal mutation is more variable. These findings suggest that a major epitope is located in the N-terminal, but additional regions of the antigen are likely to contribute to antibody-binding. Characterization of mechanisms and epitopes involved in anti-tTG autoimmunity is important for development of targeted therapeutic manipulations in CD patients or at-risk individuals. en_US
dc.language.iso en en_US
dc.publisher Coeliac UK's Research Conference en_US
dc.subject Tissue transglutaminase, autoantibodies, epitopes, site-directed mutagenesis, radioimmunoassay en_US
dc.title Autoantibodies to tissue transglutaminase target epitopes dependent on residues in the N-terminal domain en_US
dc.type Abstract en_US
workflow.import.source science

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