Structure of Crohn'S Disease-Related Proteins and their Binding to Class II MHC

Abstract

T cell response to enteric bacteria is important in inflammatory bowel disease. pfiT is a T-cell superantigen associated with human Crohn's disease. The bacterial superantigens are a class of protein toxins that share the capacity to induce massive activation of the human immune system. These molecules simultaneously bind to major histocompatibility complex class II molecules on the surface of antigen-presenting cells and T-cell receptors (TCRs) on T cells to stimulate large numbers of T cells. The aim of this study is to analyze the molecular mechanism of superantigen recognition by host receptors. Here, we report the crystal structure of pfiT. This protein was overexpressed in Escherichia coli and purified though GST-affinity and size exclusion chromatography. The protein is selenomethionine labeled and single wavelength anomalous dispersion method was used for determination of the crystal structure. The superantigen crystallizes in the monoclinic space group P21, with two molecules in asymmetric unit cell. The structure was determined to 2.5A resolution. In addition, we performed radiolabeled competitive binding assays between three superantigens: pfiT, Mycoplasma arthritidis-derived mitogen (MAM), PA2885, a novel open reading frame (ORF) in the Pseudomonas aeruginosa genome. Analyses showed that both the microbial homologuepfit and PA2885, just as potent superantigen MAM, are capable of binding to target mammalian cells. Moreover, we labeled these superantigens with FITC and analyzed them by FACS in PBMC. The statistic results show that antibody against HLA-DR has strong effect to block these SAg' binding ability with PBMC, and antibodies against HLA-DQ and DP can also compete binding site in a much weaker manner. These findings support the concept that pfiT, PA2885, MAM are superantigens and can bind to class II MHC molecule .Research is funded by the Crohn’ & Colitis Foundation of America.