title: Analysis of a nucleic-acid-binding antibody fragment: Construction and characterization of heavy-chain complementarity-determining region switch variants.
abstract: The display of antibody (AB) fragments (Fab) on the surface of filamentous bacteriophage (phage) and selection of phage that interact with a particular antigen (Ag) has enabled the isolation of Fab that bind nucleic acids. Nucleic acid (NA) binding Ab occur in vivo in connective tissue disease patients and certain inbred strains of mice and are thought to be pathogenic. Although there is ample data concerning the amino acid (aa) sequence of murine monoclonal Ab (mAb) reactive with DNA, significantly less is known about how autoAb interact with NA. The complementarity-determining regions (CDR) contained in the Fab contribute to most Ag binding, especially through heavy (H)-chain CDR 3. We have examined the role of individual H-chain CDR of a previously isolated recombinant single-stranded DNA-binding Fab (DNA-1) in nucleic acid interaction using a combination of H-chain CDR switching and solution-binding experiments. The three H-chain CDR of DNA-1 Fab were independently switched with the H-chain CDR of a Fab (D5) with very similar sequence and framework (FR) that binds DNA poorly in order to create all possible H-chain CDR combinations. The chimeric Fab genes were bacterially expressed, and their products were purified and analyzed. Results indicated that the H-chain CDR 3 of DNA-1 Fab, in the context of the remainder of the H-chain of D5 Fab, restored binding to oligo(dT)15 to 60% of DNA-1 levels, whereas H-chain CDR 1 and 3 of DNA-1 with CDR 2 of D5 Fab restored binding to 100% A combination of H-chain CDR 2 and 3 of DNA-1 Fab with H-chain CDR 1 of D5, unexpectedly resulted in the ability of the chimeric Fab to bind RNA preferentially over DNA. These studies demonstrate the importance of both H-chain CDR 1 and 3 in DNA recognition and further suggest that the specificity of the type of NA recognized by a particular Fab can be drastically altered by exchanging CDR.
authors.affiliation: Department of Molecular Microbiology and Immunology, University of Missouri, Columbia, 65212, USA.
authors.forename: M J
authors.initials: MJ
authors.lastname: Calcutt
authors.forename: A A
authors.initials: AA
authors.lastname: Komissarov
authors.forename: M T
authors.initials: MT
authors.lastname: Marchbank
authors.forename: S L
authors.initials: SL
authors.lastname: Deutscher
mesh_terms: D000818:Animals; D004247:DNA / Q000378:metabolism; D004277:DNA, Single-Stranded / Q000378:metabolism*; D004268:DNA-Binding Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D004591:Electrophoresis, Polyacrylamide Gel; D007140:Immunoglobulin Fab Fragments / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D007143:Immunoglobulin Heavy Chains / Q000737:chemistry / Q000235:genetics; D007134:Immunoglobulin Switch Region; D007135:Immunoglobulin Variable Region / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D051379:Mice; D008815:Mice, Inbred Strains; D011071:Poly T / Q000378:metabolism; D011233:Precipitin Tests; D012342:RNA, Small Nuclear / Q000378:metabolism; D016601:RNA-Binding Proteins / Q000737:chemistry / Q000235:genetics; D011993:Recombinant Fusion Proteins / Q000737:chemistry / Q000235:genetics
chemical_list: D004277:DNA, Single-Stranded; D004268:DNA-Binding Proteins; D007140:Immunoglobulin Fab Fragments; D007143:Immunoglobulin Heavy Chains; D007135:Immunoglobulin Variable Region; D012342:RNA, Small Nuclear; D016601:RNA-Binding Proteins; D011993:Recombinant Fusion Proteins; D011071:Poly T; D004247:DNA