Staphylococcal Protein A Binding to V_H3Encoded Immunoglobulins

Abstract

Staphylococcal protein A (SPA) is a B-cell superantigen which binds specifically to the variable region of human VH3 encoded antibodies. We undertook to identify the VH3 regions involved in the interaction with SPA by producing mutant antibodies in the baculovirus expression system. We had previously shown that a single amino acid change at position 57 in the CDR2 of a human SPA nonbinding VH3 encoded rheumatoid factor converted it to an SPA binder, implicating CDR2 in SPA binding. When regions of the mutated binder were exchanged with those from a mouse nonbinding antibody, the pattern of SPA binding indicated that residues in FR1, CDR2 and FR3 are involved in the interaction between VH3 encoded antibodies and SPA. In addition, all three regions are simultaneously required for SPA binding to occur. When any one of the three regions was altered, SPA binding was severely disrupted.

Staphylococcus aureus Protein A Triggers T Cell-Independent B Cell Proliferation by Sensitizing B Cells for TLR2 Ligands

“…Although it is well-recognized that SpA activates V H 3-BCR ϩ B cells (5,13,14,16), its mechanism of action used for sensitization of B cells toward TLR2 ligands remains unclear. The present data only provide evidence that SpA sensitizes human B cells for TLR ligands as seen with anti-Ig stimulation.…”

“…This effect was subsequently shown to be due to the binding and activation of surface Igs belonging to the phylogenetic clone V H 3, a subgroup of BCRs that is abundantly expressed among the murine innate B1 and MZ B cell subsets and is also expressed in 10 -60% of human peripheral blood B lymphocytes (11)(12)(13)(14)(15)(16). The early in vitro studies on human B cells proposed that SpA presentation on bacterial cell walls resulted in BCR activation via the cross-linking of surface Igs.…”

Improving the developability of an anti-EphA2 single-chain variable fragment for nanoparticle targeting

“…The framework of the heavy chain variable region of D2-1A7 is IGHV3-30. VH3s are considered very stable 19 and typically have the ability to bind to protein A, [20][21][22] making them a commonly used framework for therapeutic antibodies. We therefore decided keep the heavy chain within the VH3 family, but to graft the heavy chain of D2-1A7 onto a IGHV3-23 backbone, the most common isotype.…”

Rapid production of specific vaccines for lymphoma by expression of the tumor-derived single-chain Fv epitopes in tobacco plants

“…Bacterially produced 38C13-myc scFv at 300 ng͞ml was used as a positive control and for quantitation (23). After a 1-hr room temperature incubation, plates were washed again five times, and incubated 1 hr at room temperature with 1 g͞ml protein A-horseradish peroxidase (HRP; Sigma), which recognizes a site in the heavy-chain variable (V) region of 38C13 scFv (31,32). Plates were washed and then developed by a 10-min room temperature incubation with 0.15% 2,2Ј-azino-bis(3-ethylbenzthiazoline-6-sulfonic acid) (ABTS; Sigma) in 100 mM sodium citrate buffer, pH 8.5͞0.001% hydrogen peroxide.…”