Recognition of DNA by VH and Fv domains of an IgG anti-poly(dC) antibody with a singly mutated VH domain

J Mol Recognit. 2001 Jan-Feb;14(1):18-28. doi: 10.1002/1099-1352(200101/02)14:1<18::AID-JMR515>3.0.CO;2-2.

Abstract

Secondary antigen stimulation usually produces IgG antibodies with hypermutated V segments. Studying a strong secondary response to the polynucleotide antigen poly(dC), however, we found a highly selective IgG antibody (mAb dC7) with only one mutation (a conservative Leu to Ileu substitution) throughout the whole VH domain. To investigate the roles of VH and VL domains in selective binding by this mAb, we prepared its VH, VL and single-chain Fv (scFv) fragments. A bacterial expression system produced soluble monomeric V region proteins. CD spectra confirmed that they had the beta-secondary structure expected for Ig domains. Both the scFv and VH fragments bound to single-stranded non-protonated poly(dC) and to ssDNA but not to protonated, more structured poly(dC) or dsDNA. The VL domain alone did not bind to nucleic acids, but VL association modified the VH binding, giving the scFv a 10-fold higher affinity than the VH for poly(dC) and greatly increasing the cytosine-dependent selectivity. Non-ionic interactions were prominent in the Fv reaction with a (dC)( n) sequence. Ionic interactions were revealed in Fv cross-reactions with ssDNA, and were more prominent in binding of either poly(dC) or ssDNA by VH alone, consistent with the lesser base selectivity of the VH. Thus, the Fv and VH alone bind to a single antigen, poly(dC), but mechanistic differences result from additional subsites in the Fv. Generation of a selective IgG with very few CDR mutations in either VH or VL, which was accompanied by IgM antibodies with unmutated V regions, also suggests that nucleic acid binding activity is a property of the B cell repertoire even before immunization.

Publication types

  • Research Support, U.S. Gov't, P.H.S.

MeSH terms

  • Amino Acid Sequence
  • Animals
  • Antibody Affinity
  • DNA, Single-Stranded / immunology*
  • Enzyme-Linked Immunosorbent Assay / methods
  • Hydrogen-Ion Concentration
  • Immunoglobulin Fragments / immunology*
  • Immunoglobulin G / immunology*
  • Immunoglobulin Heavy Chains / immunology*
  • Immunoglobulin Variable Region / immunology*
  • Mice
  • Mice, Inbred C57BL
  • Molecular Sequence Data
  • Osmolar Concentration
  • Poly C / immunology*

Substances

  • DNA, Single-Stranded
  • Immunoglobulin Fragments
  • Immunoglobulin G
  • Immunoglobulin Heavy Chains
  • Immunoglobulin Variable Region
  • immunoglobulin Fv
  • poly(dC)
  • Poly C