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 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=32-33 kDa) 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.
Eggena
M
M
Molecular Biology Institute and Department of Pathology and Laboratory Medicine, University of California, Los Angeles, CA, 90095, USA.
Cohavy
O
O
Parseghian
M H
MH
Hamkalo
B A
BA
Clemens
D
D
Targan
S R
SR
Gordon
L K
LK
Braun
J
J
D000595:Amino Acid Sequence; D000818:Animals; D019268:Antibodies, Antineutrophil Cytoplasmic / Q000097:blood / Q000276:immunology*; D000911:Antibodies, Monoclonal; D000937:Antigen-Antibody Reactions; D001324:Autoantigens / Q000737:chemistry / Q000235:genetics; D015551:Autoimmunity; D016022:Case-Control Studies; D003093:Colitis, Ulcerative / Q000276:immunology*; D000939:Epitopes / Q000737:chemistry / Q000235:genetics; D006657:Histones / Q000737:chemistry / Q000235:genetics / Q000276:immunology*; D006801:Humans; D007150:Immunohistochemistry; D008969:Molecular Sequence Data; D009504:Neutrophils / Q000276:immunology; D010446:Peptide Fragments / Q000737:chemistry / Q000235:genetics / Q000276:immunology; D011817:Rabbits; D011993:Recombinant Fusion Proteins / Q000737:chemistry / Q000235:genetics / Q000276:immunology
D019268:Antibodies, Antineutrophil Cytoplasmic; D000911:Antibodies, Monoclonal; D001324:Autoantigens; D000939:Epitopes; D006657:Histones; D010446:Peptide Fragments; D011993:Recombinant Fusion Proteins