Identification of Histone H1 as a Cognate Antigen of the Ulcerative Colitis-associated Marker Antibody pANCA

Abstract

Perinuclear anti-neutrophil cytoplasmic antibody (pANCA)4is a predominant serum marker of ulcerative colitis (UC), and a familial trait associated with disease susceptibility and disease associated MHC haplotypes. This study characterizes the pANCA antigen defined by representative UC-pANCA human monoclonal antibodies, Fab 5–3 and 5–2. Western blot analysis probed with Fab 5–3 revealed specific binding to a nuclear protein doublet (apparent MW=32–33kDa) expressed in several cell types. Purification and tryptic peptide sequencing identified the protein as histone H1, and this specificity was confirmed by Fab 5–3 binding to purified H1. Rabbit anti-histone H1 immunostaining and Western blot analysis confirmed that the pANCA epitope is preferentially immunoaccessible in polymorphonuclear neutrophils (PMN). The epitope was localized to the COOH-terminal region by site-specific proteolysis, and recombinant deletants further localized binding activity for both Fab 5–2 and 5–3 to two non-overlapping segments (AA 69–171 and 172–226) associated with a recurring PKKAK motif. Serum IgG binding was detectable to these segments, but was not significantly correlated with pANCA titer or disease status. These findings indicate that histone H1 bears a recurring COOH-terminal epitope recognized by monoclonal ulcerative colitis-associated pANCA marker antibodies, but this epitope is not a predominant specificity of serum pANCA.