Identification of histone H1 as a cognate antigen of the ulcerative colitis-associated marker antibody pANCA

Perinuclear anti-neutrophil cytoplasmic antibody (pANCA)(4) is a predominan t serum marker of ulcerative colitis (UC), and a familial trait associated with disease susceptibility and disease associated MHC haplotypes. This stu dy 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 =32-33 kDa) expressed in several cell types. Purification and tryptic pepti de sequencing identified the protein as histone H1, and this specificity wa s confirmed by Fab 5-3 binding to purified H1. Rabbit anti-histone H1 immun ostaining and Western blot analysis confirmed that the pANCA epitope is pre ferentially immunoaccessible in polymorphonuclear neutrophils (PMN). The ep itope was localized to the COOH-terminal region by site-specific proteolysi s, and recombinant deletants further localized binding activity for both Fa b 5-2 and 5-3 to two non-overlapping segments (AA 69-171 and 172-226) assoc iated with a recurring PKKAK motif. Serum IgG binding was detectable to the se segments, but was not significantly correlated with pANCA titer or disea se status. These findings indicate that histone H1 bears a recurring COOH-t erminal epitope recognized by monoclonal ulcerative colitis-associated pANC A marker antibodies, but this epitope is not a predominant specificity of s erum pANCA. (C) 2000 Academic Press.