Use of phage surface expression to analyze regions of human V4-34(VH4-21)-encoded IgG autoantibody required for recognition of DNA: no involvement of the 9G4 idiotope.
The V4-34 gene encodes the majority of autoanti-red cell Abs of I/i specificity. It also encodes a proportion of autoanti-DNA Abs found in patients with systemic lupus erythematosus. Nucleotide sequence analysis of mAbs that use this gene has indicated a role for CDR3 in discrimination between these autoantigens. Specifically, anti-DNA activity may require basic amino acids, such as arginine, found in this region. To investigate this requirement, we have expressed VH and VL sequences from a patient's IgG anti-DNA mAb, as Fab molecules at the surface of phage. Expressed Fab bound strongly to DNA, whereas control VH and VL pairs from an anti-red cell mAb did not. Replacement of the homologous mutated V4-34 sequence by germ-line sequence did not affect binding, indicating that somatic mutations in VH did not contribute significantly. In contrast, replacement of the basic CDR3 by an anti-red cell CDR3 abrogated anti-DNA activity, confirming its major role. However, an influence of VL was revealed by replacing homologous mutated V kappa IIIb by an unmutated V kappa IIIb sequence, reducing binding by approximately 50%. This influence was apparent only with homologous VH since the mutated V kappa was unable to generate anti-DNA activity when combined with anti-red cell VH. The 9G4 idiotope, which arises from FWR1, was expressed by all constructs. Substitution of Trp by Ser at position 7 in FWR11 caused complete loss of idiotope expression, with no effect on recognition of DNA, indicating no influence of idiotope expression on anti-DNA activity. Phage surface expression provides a powerful and rapid technique for assessing sequences relevant for Ab specificity or idiotope expression.
Mockridge
C I
CI
Molecular Immunology Group, Tenovus Laboratory, Southampton University Hospitals, United Kingdom.
Chapman
C J
CJ
Spellerberg
M B
MB
Isenberg
D A
DA
Stevenson
F K
FK
D000595:Amino Acid Sequence; D000974:Antibodies, Antinuclear / Q000737:chemistry / Q000235:genetics*; D017104:Bacteriophage M13 / Q000276:immunology* / Q000378:metabolism; D001667:Binding, Competitive / Q000187:drug effects / Q000235:genetics; D004247:DNA / Q000276:immunology* / Q000494:pharmacology; D005803:Genes, Immunoglobulin; D005822:Genetic Vectors / Q000276:immunology; D006801:Humans; D007140:Immunoglobulin Fab Fragments / Q000096:biosynthesis / Q000737:chemistry; D007074:Immunoglobulin G / Q000737:chemistry / Q000235:genetics*; D007143:Immunoglobulin Heavy Chains / Q000737:chemistry / Q000235:genetics*; D007130:Immunoglobulin Idiotypes / Q000737:chemistry / Q000235:genetics*; D007135:Immunoglobulin Variable Region / Q000737:chemistry / Q000235:genetics*; D008969:Molecular Sequence Data
D000974:Antibodies, Antinuclear; D007140:Immunoglobulin Fab Fragments; D007074:Immunoglobulin G; D007143:Immunoglobulin Heavy Chains; D007130:Immunoglobulin Idiotypes; D007135:Immunoglobulin Variable Region; D004247:DNA