Unexpected side reaction of lysine and arginine side chains preceding a photocleavable group in MHC-II UV-cleavable peptides. (P5028)

Abstract

Abstract MHC class II molecules (MHCII) present peptides to CD4+ T cells, and HLA-DM (DM) catalyzes peptide exchange favoring binding of high affinity peptides. The effects of DM on peptide association and dissociation kinetics are not yet clear. In the absence of peptide, DR1 (a MHCII) is in equilibrium between inactive and active conformations. In order to understand the kinetics of this process and how it is affected by DM, we wanted to generate DR1 in the active conformation using a peptide carrying the photocleavable 3-amino-3-(2-nitrophenyl)-propionic acid residue, an approach previously used for other MHCI and MHCII-binding peptides. We used well-characterized variants of a peptide derived from influenza hemagglutinin (HA). The variants bind to DR1 with an affinity similar to the parent HA peptide. Surprisingly, photolysis of peptides did not result in peptide release. Mass spectrometry showed that the main photoproduct had a mass decrease of 2Da, resulting from photocleavage followed by unexpected religation to a rearranged peptide that can still bind DR1. Replacement of peptide lysine (or arginine) residues by methionine yielded the expected cleavage products. The methionine substituted peptides can be used for further studies on DR1-peptide binding kinetics and the effect of DM on the different rates of the reaction. These studies provide a caveat to routine use of photocleavable peptides in MHC-peptide exchange studies.