Functional and modelling studies of the binding of human monoclonal anti-DNA antibodies to DNA.
The relationships between the antigen-binding specificities of four human monoclonal anti-DNA antibodies and the structural aspects of the combining sites of two of these were examined. Competition ELISAs were used to examine the reactivities of two IgM MAbs (WRI-176 and RT-79) and two IgG mAbs (D5 and B3) to a wide range of polynucleotides. The mAbs WRI-176 and RT-79 were found to bind predominantly ssDNA, with a preference for poly (dT), whilst D5 and B3 bound components of both ss- and dsDNA, and Z-DNA. The mAb B3 also exhibited a preference for A(T) rich nucleotides. Computer models were generated for the Fv regions of WRI-176 and B3. Models for RT-79 and D5 were not generated as the structure of the long CDR-H3 loops in these mAbs could not be predicted. The B3 combining site contains a groove flanked by three arginines at positions CDR-L1-27A, CDR-L2-54 and CDR-H2-53. Using interactive molecular graphics, B-DNA was docked into the B3 antigen combining site along the plane of the VH/VL interface, whilst Z-DNA was best-fitted at approximately 90 degrees to this direction. The models provide a hypothesis to explain the ability of a single autoantibody to bind two different antigens. In addition, aspects of the base specificity of B3 may be explained. The model of the WRI-176 Fv region revealed a relatively flat surface, on which a large number of hydrophobic and aromatic residues were present. Trp-H52, in particular, is prominent on the surface. This may participate in ssDNA binding through base stacking interactions. The models allow identification of potential targets for site-directed mutagenesis.
Kalsi
J K
JK
Bloomsbury Rheumatology Unit, Department of Medicine, University College London, U.K.
Martin
A C
AC
Hirabayashi
Y
Y
Ehrenstein
M
M
Longhurst
C M
CM
Ravirajan
C
C
Zvelebil
M
M
Stollar
B D
BD
Thornton
J M
JM
Isenberg
D A
DA
D000595:Amino Acid Sequence; D000974:Antibodies, Antinuclear / Q000276:immunology*; D000911:Antibodies, Monoclonal / Q000737:chemistry / Q000276:immunology*; D000918:Antibody Specificity; D001666:Binding Sites, Antibody; D004247:DNA / Q000276:immunology*; D006801:Humans; D008958:Models, Molecular; D008969:Molecular Sequence Data; D013329:Structure-Activity Relationship
D000974:Antibodies, Antinuclear; D000911:Antibodies, Monoclonal; D004247:DNA