title: Mutational analysis of a sequence-specific ssDNA binding lupus autoantibody.
abstract: 11F8 is a murine anti-ssDNA monoclonal autoantibody isolated from a lupus prone autoimmune mouse. This mAb binds sequence specifically, and prior studies have defined the thermodynamic and kinetic basis for sequence-specific recognition of ssDNA (Ackroyd, P. C., et al. (2001) Biochemistry 40, 2911-2922; Beckingham, J. A. and Glick, G. D. (2001) Bioorg. Med. Chem. 9, 2243-2252). Here we present experiments designed to identify the residues on 11F8 that mediate sequence-specific, noncognate, and nonspecific recognition of ssDNA and their contribution to the overall binding thermodynamics. Site-directed mutagenesis of an 11F8 single-chain construct reveals that six residues within the complementarity determining regions of 11F8 account for ca. 80% of the binding free energy and that there is little cooperativity between these residues. Germline-encoded aromatic and hydrophobic side chains provides the basis for nonspecific recognition of single-stranded thymine nucleobases. Sequence-specific recognition is controlled by a tyrosine in the heavy chain along with a somatically mutated arginine residue. Our data show that the manner in which 11F8 achieves sequence-specific recognition more closely resembles RNA-binding proteins such as U1A than other types of nucleic acid binding proteins. In addition, comparing the primary sequence of 11F8 with clonally related antibodies that differ by less than five amino acids suggests that somatic mutations which confer sequence specificity may be a feature that distinguishes glomerulotrophic pathogenic anti-DNA from those that are benign.
authors.affiliation: Department of Chemistry, University of Michigan, Ann Arbor, Michigan 48109-1055, USA.
authors.forename: Joanne
authors.initials: J
authors.lastname: Cleary
authors.forename: Gary D
authors.initials: GD
authors.lastname: Glick
mesh_terms: D019943:Amino Acid Substitution / Q000235:genetics; D000818:Animals; D000911:Antibodies, Monoclonal / Q000737:chemistry / Q000235:genetics; D000915:Antibody Affinity / Q000235:genetics; D000918:Antibody Specificity / Q000235:genetics*; D001120:Arginine / Q000235:genetics; D001483:Base Sequence; D001666:Binding Sites, Antibody / Q000235:genetics*; D004252:DNA Mutational Analysis / Q000379:methods; D004277:DNA, Single-Stranded / Q000032:analysis* / Q000737:chemistry; D005822:Genetic Vectors; D057927:Hydrophobic and Hydrophilic Interactions; D007143:Immunoglobulin Heavy Chains / Q000737:chemistry / Q000235:genetics; D007147:Immunoglobulin Light Chains / Q000737:chemistry / Q000235:genetics; D007135:Immunoglobulin Variable Region / Q000737:chemistry / Q000235:genetics; D016682:Lupus Coagulation Inhibitor / Q000737:chemistry* / Q000235:genetics*; D008239:Lysine / Q000235:genetics; D051379:Mice; D016297:Mutagenesis, Site-Directed; D011485:Protein Binding / Q000235:genetics; D013816:Thermodynamics; D013941:Thymine / Q000737:chemistry
chemical_list: D000911:Antibodies, Monoclonal; D004277:DNA, Single-Stranded; D007143:Immunoglobulin Heavy Chains; D007147:Immunoglobulin Light Chains; D007135:Immunoglobulin Variable Region; D016682:Lupus Coagulation Inhibitor; D001120:Arginine; D008239:Lysine; D013941:Thymine