Structural basis for antibody protection against SEB toxicity
EMSL Project ID
32892
Abstract
Staphylococcal Enterotoxin B (SEB) is a superantigen responsible for food poisoning and, in aerosolized form, this 28 kDa protein also has the potential to be used as a biological warfare agent. SEB toxicity arises through binding major histocompatibility complex (MHCII) and T-cell receptor (TCR). Passive transfer of some anti-SEB antibodies can provide protection from a lethal SEB challenge presumably by competing for binding sites on SEB thus blocking those interactions, though this mechanism has not been investigated. Protection against SEB exposure through this type of passive immunization has practical drawbacks and not all anti-SEB antibodies offer protection. In response to the need for better SEB binding reagents which can be used in diagnostic and therapeutic applications, we have developed and characterized five single chain antibodies (scFv) which bind SEB at at least two distinct epitopes with low nanomolar to picomolar affinity. These anti-SEB scFv have several advantages over full length antibodies; they are much smaller (~30 kDa), can be produced in bacteria and, because they were selected from a human antibody library, have potential as therapeutic agents. In order to better understand the basis of protection by SEB antibodies and guide future alternative scaffold affinity reagent development, we are proposing an investigation of the binding interfaces between SEB and our anti-SEB scFvâ??s. These experiments will involve MS/MS analysis of the chemically crosslinked SEB/scFv complexes in order to identify specific sites of interaction thus mapping the different SEB epitopes of each scFv. Specifically, we will crosslink SEB and scFv using commercially available primary amine reactive crosslinker disuccinimidyl suberate (DSS), which will stabilize the solution phase structure by reacting with proteinsâ?? lysine residues. Resulting products will be separated on SDS-PAGE and the crosslinked complex will be cut out and digested with trypsin. Tryptic peptides will be analyzed using high-resolution MS/MS (e.g. an LTQ-Orbitrap) and bioinformatics tools developed at EMSL. In addition, we will explore the use of a novel primary amine reactive cross-linker, which contains an enrichment tag and therefore facilitates enrichment of cross-linked peptides, for further validation of crosslinked peptides.
The results of the experiments proposed here will provide low resolution structural data on the binding interfaces involved in up to five different scFv/SEB interactions. We hope to correlate this new structural insight with the efficacy of protection offered by each scFv in order to help elucidated the mechanism underlying that protection. These results will be part of a manuscript which a) outlines the design and production of anti-SEB scFvâ??s from a human non-immune library using yeast display (completed); b) characterizes the kinetics of the scFv-SEB binding interaction by surface plasmon resonance (completed); c) maps the scFv-SEB binding interface by chemical crosslinking and FTICR (proposed); d) demonstrates scFv-mediated protection from SEB-induced T-cell activation using a cell line and flow cytometry (in progress).
Note: The proposed interface mapping work will be conducted using an attenuated SEB mutant which does not pose a biological risk to investigators.
Project Details
Project type
Limited Scope
Start Date
2009-02-03
End Date
2009-04-05
Status
Closed
Released Data Link
Team
Principal Investigator
Team Members