Identification of an I-E^d-Restricted T-Cell Epitope ofEscherichia coliOuter Membrane Protein F

Abstract

A predominant T-cell epitope ofat either the C or the N terminus of the sequence was used, the minimal epitope was determined. Critical residues were determined by using alanine-substituted peptide analogues. T-cell hybridomas were only stimulated by peptides that encompassed amino acids 295 to 303 (9-mer), and the core sequence required a minimum of three additional amino acids at either the amino or the carboxy terminus to induce IL-2 secretion. Critical residues were determined to be phenylalanine at position 295, threonine at position 300, and tyrosines at positions 301 and 302. This study is the first to identify a minimal T-cell epitope and major histocompatibility complex restriction element of the OmpF protein and confirms previous observations that there is considerable degeneracy in the length of peptides that can bind I-E d and variability in the amino acid composition of the C and N termini of these peptides. Antigen recognition by CD4ϩ T cells involves the uptake and proteolysis of native proteins by antigen-presenting cells, followed by the association and surface presentation of smaller peptide fragments bound to class II major histocompatibility complex (MHC) molecules. Interaction of this complex with clonotypic receptors on the surface of T cells results in a cascade of intracellular events collectively termed T-cell activation. Acid elution, followed by protein sequencing of naturally processed peptides from affinity-purified human and murine class II molecules (13,14,17,29,41,47,64,65), has shown considerable variability in the length of bound peptides (11 to 17 amino acids in length) and that a single determinant may be presented as multiple peptide species, depending on the length of the amino-or carboxy-terminal flanking residues. Crystallographic analysis of the MHC class II molecule HLA-DR1 (9, 62) provided the structural explanation for the heterogeneity in length of class II-bound peptides since it was observed that peptides are bound in an extended conformation inside the binding groove, which is open at both ends, unlike the class I peptide-binding groove. Additionally, binding studies have revealed that peptide anchor residues (or their respective binding sites within the groove) are more degenerate in their specificity compared to the stringent binding of class I ligands. While allele-specific binding motifs for class II molecules have been more difficult to identify because of the open-ended structure of the peptide-binding groove and the resulting heterogeneity of bound peptides, several allele-specific motifs have emerged (http://syfpeithi.bmi-heidelberg.com/).Porins are a family of heterotrimeric pore-forming molecules present in the outer membranes of gram-negative members of the family Enterobacteriaceae. Monomeric porin molecules associate to form stable trimeric hydrophilic transmembrane channels that allow passive diffusion of solutes, including antibiotics, across the lipid bilayer. The primary amino acid sequence of porins from several species of the family Enterobacteri...

Toward Prediction of Class II Mouse Major Histocompatibility Complex Peptide Binding Affinity: in Silico Bioinformatic Evaluation Using Partial Least Squares, a Robust Multivariate Statistical Technique

“…We used an independent test data set to evaluate the predictivity of our mouse class II models. Sixty class II epitopes from 21 protein sequences were used in the test set, see Table . − The full protein sequences were retrieved from SWISS−PROT . Complete protein sequences were used as input in this test, and the ability of the algorithm to identify epitopes correctly was assessed.…”