VH3 family antibodies bind domain D of staphylococcal protein A.
Staphylococcal protein A (SpA) is a 45-kDa bacterial membrane protein that can interact with either Fc gamma, a constant region portion of IgG, or with the Fab portion that also mediates conventional Ag binding. In recent reports, SpA has been shown to specifically interact with Fab derived from the VH3 family and is little affected by VH CDR3, JH, or light chain usage. To identify a site on SpA responsible for VH3 Fab binding, we cloned and expressed in Escherichia coli the 61 amino acid sequence of SpA that represents domain D, and this small protein exhibited both the VH3 Fab and Fc gamma binding specificities. Surface plasmon resonance measurements demonstrated that domain D and native SpA had the strongest binding interactions with an IgM-kappa encoded by the germline configuration of the VH3 gene VH26c. In contrast, the apparent affinities for Fc gamma binding were at least fivefold weaker. A variant of domain D was also created that is devoid of the three-codon insertion that distinguishes domain D from all other domains in SpA. Although this deletion did not significantly affect the VH3 Fab-mediated SpA binding activity, it did improve the affinity of Fc gamma binding by an order of magnitude. These observations characterize a site on SpA responsible for binding interactions with B cell Ag receptors that are highly analogous to that of superantigens for T cell receptors.
Roben
P W
PW
Sam and Rose Stein Institute for Research on Aging, University of California, San Diego, La Jolla 92093, USA.
Salem
A N
AN
Silverman
G J
GJ
D000595:Amino Acid Sequence; D001483:Base Sequence; D002352:Carrier Proteins; D003001:Cloning, Molecular; D004269:DNA, Bacterial / Q000235:genetics; D004926:Escherichia coli / Q000235:genetics; D005803:Genes, Immunoglobulin; D014644:Genetic Variation; D006801:Humans; D007143:Immunoglobulin Heavy Chains / Q000235:genetics / Q000378:metabolism*; D007135:Immunoglobulin Variable Region / Q000235:genetics / Q000378:metabolism*; D066298:In Vitro Techniques; D008969:Molecular Sequence Data; D011487:Protein Conformation; D013205:Staphylococcal Protein A / Q000737:chemistry / Q000235:genetics / Q000276:immunology*; D013211:Staphylococcus aureus / Q000235:genetics / Q000276:immunology
D002352:Carrier Proteins; D004269:DNA, Bacterial; D007143:Immunoglobulin Heavy Chains; D007135:Immunoglobulin Variable Region; D013205:Staphylococcal Protein A