John S. Haurum

An improved assembly assay for peptide binding to HLA-B*2705 and H-2Kk class I MHC molecules

The assembly assay for peptide binding to class I major histocompatibility complex (MHC) is based on the ability to stabilise MHC class I molecules from mutant cell lines by the addition of suitable peptides. Such cell lines lack a functional transporter associated with antigen presentation (TAP) and as a result accumulate empty, unstable class I molecules in the ER. These dissociate rapidly in cell lysates unless they are stabilised by the addition of an appropriate binding peptide during lysis. The extent of stabilisation of class I molecules is directly related to the binding affinity of the added peptide. However, some MHC class I molecules, including HLA-B * 2705 and H-2Kk are unusually stable in their peptide-receptive state making them inappropriate for analysis using this assay or assays which depend on the ability of peptides to stabilise MHC class I molecules at the cell surface. Here we present an improved method that permits reliable measurements of peptide binding to such class I MHC molecules that are unusually stable in the absence of peptide. Cells are lysed in the presence of peptide and incubated at 4 degrees C. After 2 h, during which peptide binding to empty MHC molecules occurs, the lysate is heated to a temperature which preferentially destabilises those MHC molecules that remain empty. We have used this technique to assay peptide binding to HLA-B * 2705, as well as to the murine allele H-2Kk which also displays a stable phenotype when transfected into TAP-deficient T2 cells and show that this method represents a marked improvement over previous methods in terms of lower background signal and higher recovery of peptide bound molecules.

Synthesis of major histocompatibility complex class I binding glycopeptides

Four Major Histocompatibility Complex (MHC) Class I binding glycopeptides and two peptide analogues, from a cytotoxic T-lymphocyte (CTL) epitope of Sendai Virus Nucleoprotein, have been prepared using solid-phase peptide synthesis employing the following glycosyl amino acid building blocks: FmocSer(Ac3-β-D-GlcNAc)OH 1, FmocSer(Ac3-α-D-GalN3)OPfp 2, FmocAsn(Ac3-β-D-GlcNAc)OH 3 and FmocAsn(Ac3-β-D-GalNAc)OH 4. Previously, we examined the influence of glycosylation on peptide binding to the MHC Class I molecule and CTL recognition of these peptides. The synthesis and characterization of compounds 1–4 as well as the resulting glycopeptides is described. In addition, results of NMR investigations demonstrating that peptide K3, and glycopeptides K3-O-GlcNAc and K3-O-GalNAc, show two distinct conformations in solution as a result of cis–trans isomerization about a Tyr-Pro amide bond are reported.