VH3 family antibodies bind domain D of staphylococcal protein A.

Abstract

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.

The immune evasion roles of Staphylococcus aureus protein A and impact on vaccine development

“…Radke and colleagues identified antibodies against SpA KKAA from the serum of both healthy individuals and those recovering from S. aureus infections, with SpA-specific memory B cells showing a strong VH3 preference ( Radke et al., 2021 ). This study built on previous work outlining the importance of VH : VL genes in B cells and the preference for VH3 genes when targeting S. aureus ( Roben et al., 1995 ). A single isolate, SA101, was proposed to induce antibodies against a novel site on SpA and was found to be the only isolate to have its binding to VH3 sites inhibited by SpA KKAA.…”

Quenchbodies That Enable One-Pot Detection of Antigens: A Structural Perspective

“…Site-specific fluorescent dye-labeled antibody-binding proteins, such as Staphylococcus protein A (PA), Streptococcus protein G (PG), Mycoplasma genitalium protein M (PM), and mouse IgG1-selective affibody, were generated as universal independent quenching and de-quenching probes to simply convert commercially available or synthesized antibodies to Q-bodies. PA binds to VH and Fc regions of IgG, and PG binds to the Fc and Fab regions of IgG [87][88][89][90][91]. Jeong et al generated a PA-PG fusion protein by conjugating PA and PG using an optimized linker between the two proteins to increase their binding affinity [68].…”