High-affinity binding of short peptides to major histocompatibility complex class II molecules by anchor combinations.

Abstract

We have previously identified four anchor positions in HLA-DRB1*0101-binding peptides, and three anchors involved in peptide binding to DRB1*0401 and DRB1*11O1 molecules, by screening of an M13 peptide display library (420 million independent nonapeptides) for DRbinding activity. In this study, high stringency screening of the M13 library for DRB1*0401 binding has resulted in identification of three further anchor positions. Taken together, a peptide-binding motif has been obtained, in which six of seven positions show enrichment of certain residues. We have demonstrated an additive effect ofanchors in two different ways: (i) the addition of more anchors is shown to compensate for progressive truncation of designer peptides; (ii) the incorporation of an increasing number of anchors into 6-or 7-residuelong designer peptides is shown to result in a gradual increase of binding aMnity to the level of 13-residue-long hig-affinity epitopes. The anchor at relative position 1 seems to be obligatory, in that its substitution abrogates binding completely, whereas the elmination of other anchors results only in partial loss ofbinding affinity. The spacing between anchors is critical, since their effect is lost by shifting them one position toward the N or C terminus. The information born out of this study has been successfully used to identify DR-binding sequences from natural proteins.Each allelic form of the highly polymorphic major histocompatibility complex (MHC) molecules can bind a large array of different peptides to ensure T-cell-mediated immunity to the antigenic universe. Thus, provided that peptides bind to a given MHC molecule in similar or identical conformation (1-4), the sequencing of a sufficiently large pool of bound peptides should reveal the rules of MHC/peptide interaction.Recently, two different approaches have been described for the isolation and characterization of MHC-bound peptide pools. (i) Endogenously processed peptides were eluted from MHC molecules and sequenced in pool or individually. This approach resulted in binding motifs for class I MHC molecules (5-7). However, its application for MHC class II molecules proved to be difficult, because of the limited number of independent sequences identified and the high degree of length heterogeneity of bound peptides (8-11). (ii) Phage-expressed large (=20 million independent sequences) random peptide libraries were screened for MHC binding. Sequencing ofthe bound peptides has revealed allele-specific binding motifs for three different HLA-DR molecules (12, 13). Here, we report a further refinement of the phage library screening, which allowed us to identify additional DRB1*0401-binding anchors. We also demonstrate that the anchor residues in class II binding peptides are additive and position specific, and, in combination, enable short peptides to bind tightly to DR molecules. MATERIALS AND METHODSPreparation of HLA-DR Molecules. DR molecules isolated from cell lines BSM, PREISS (DRB1*0401), HOM-2, LG-2 (DRB1*0101), and SWEIG (DRB1*1101) wer...

High-throughput engineering and analysis of peptide binding to class II MHC

“…Relative display levels of HLA-DR1 with the different peptides, presumably reflecting relative binding affinity between the MHC-II protein and peptide, follow the order Phe ≈ Tyr≈ Trp > Met > Leu ≈ Ile > Val, whereas all other amino acids tested yield very low binding to HLA-DR1. These results show striking agreement with binding preferences previously determined using quantitative in vitro peptide-binding assays (21), qualitative phage display and bioinformatics methods (23,31), and structure-based computational prediction approaches (32). Most importantly, the yeast codisplay method appears to demonstrate quantitative ability paralleling that of competitive binding assays with purified proteins and soluble peptides (21) while retaining the ease of rapid recombinant expression without component purification and compatibility with high-throughput screening approaches.…”

“…A number of studies have assessed peptide-binding to MHC-II on the surface of intact APCs (12,13), whereas a routinely used in vitro approach entails purifying soluble recombinant MHC-II molecules from different expressing systems such as B cell lines (14), insect cells (15,16), yeast (17), or Escherichia coli (18)(19)(20) and then characterizing binding of these molecules to different peptides generated either chemically by solid phase synthesis or genetically by phage display (4,21). The labor-intensive preparation of soluble proteins, lengthy binding assays, or nonquantitative data generated (e.g., peptide abundance) limits the efficiency and throughput of these methods for mapping MHC-II binding specificities across the large number of existing alleles.…”

A Three-Step Kinetic Mechanism for Peptide Binding to MHC Class II Proteins

“…For the complex with MAT peptide, no structure is available, but the peptide register and orientation appear to be the same as observed in the DR1-HA and DR3-CLIP complexes, based on studies of the effects of peptide substitution on the binding affinity (47)(48)(49). To minimize any interaction of the AMCA probe with the DR1-peptide binding reaction, we attached the probe to side chains at positions oriented away from the binding site, at position P2 (MAT), P3 (HA), and P8 (CLIP), numbered relative to the large hydrophobic side chain at P1 that binds into MHC binding site pocket 1 (13).…”

Breaking T cell tolerance against self type II collagen in HLA–DR4–transgenic mice and development of autoimmune arthritis

“…We therefore generated T cell clones from CII-immunized B10.DR4.Ncf1* mice in order to test these against CII peptides in which K 264 and/or K 270 had been substituted for arginine (R), since this has been found to be the preferred amino acid at position P2 for peptides binding to DRB1*0401 (29). We also performed binding studies, which confirmed that substitutions of lysine for arginine residues had minimal consequences for class II MHC peptide binding ( Figure 4A).…”