Monoclonal anti-H1 histone autoantibodies from unimmunized Balb/c mice. Specificity and VH and VL domain sequences.
Examination of the nuclear reactivities of monoclonal IgM kappa autoantibodies, secreted by GFM-5 1B12 and NU-6 1F12 hybridomas derived from germ-free and nude mice, respectively, demonstrated homogeneous nuclear immunofluorescence staining patterns consistent with the recognition of histones. Under these conditions, GFM-5 1B12 and NU-6 1F12 mAbs produced species non-specific binding to components within the nuclei of mouse, human and Drosophila melanogaster cells. Immunoblotting confirmed the binding of these two autoantibodies to autologous H1 histones as well as bovine and insect H1 histones. Identification of the epitopes bound by GFM-5 1B12 and NU-6 1F12 mAbs within the D. melanogaster H1 histones was undertaken using 248 overlapping octapeptides encompassing the entire sequence of D. melanogaster H1 histones. GFM-5 1B12 mAbs bound several octapeptides derived from the amino- and carboxyl-terminal regions of D. melanogaster H1 histones with accessible KT, AT or VT amino acids. NU-6 1F12 mAbs, which stained nuclei within sections of D. melanogaster lavae, failed to bind to any of the 248 linear octapeptides, implying recognition of a conformational H1 histone epitope by this autoantibody. ELISA analysis of the polyspecific binding properties of GFM-5 1B12 and NU-6 1F12 mAbs demonstrated that both antibodies exhibited unique polyspecificity profiles. GFM-5 1B12 mAbs recognized bovine carbonic anhydrase and mouse IgG1, while NU-6 1F12 bound bovine cardiolipin, rat cytochrome c, Escherichia coli beta-galactosidase, toxoid from Clostridium tetani, mouse IgG1 and the haptens, DNP and FITC from the 24 antigen test panel. Comparison of the VH and VL domain sequences of GFM-5 1B12 and NU-6 1F12 mAbs demonstrated that the variations in autoreactivity and polyspecificity profiles resulted from amino acid variations in the CDRs of the VH and VL domains of these autoantibodies. Significantly, major differences in the VH domain sequences of the NU-5 1F12 and GFM-5 1B12 mAbs suggest that the VH domains may preferentially contribute to the unique specificities of the two anti-H1 histone autoantibodies.
Underwood
J R
JR
Department of Pathology, University of Melbourne, Australia.
Cartwright
G A
GA
McCall
A M
AM
Tribbick
G
G
Geysen
M H
MH
Hearn
M T
MT
D000595:Amino Acid Sequence; D000818:Animals; D000974:Antibodies, Antinuclear / Q000096:biosynthesis; D000911:Antibodies, Monoclonal / Q000096:biosynthesis; D000918:Antibody Specificity; D001323:Autoantibodies / Q000096:biosynthesis*; D001483:Base Sequence; D004331:Drosophila melanogaster; D006657:Histones / Q000737:chemistry / Q000235:genetics / Q000276:immunology*; D006801:Humans; D007143:Immunoglobulin Heavy Chains / Q000737:chemistry / Q000235:genetics / Q000276:immunology*; D007147:Immunoglobulin Light Chains / Q000737:chemistry / Q000235:genetics / Q000276:immunology*; D007075:Immunoglobulin M / Q000096:biosynthesis*; D051379:Mice; D008807:Mice, Inbred BALB C; D008819:Mice, Nude; D008969:Molecular Sequence Data; D010449:Peptide Mapping; D010455:Peptides / Q000276:immunology
D000974:Antibodies, Antinuclear; D000911:Antibodies, Monoclonal; D001323:Autoantibodies; D006657:Histones; D007143:Immunoglobulin Heavy Chains; D007147:Immunoglobulin Light Chains; D007075:Immunoglobulin M; D010455:Peptides