Studying MHC class II peptide loading and editing in vitro

Ae Ryon Kim, Isabel Emiko Ishizuka, Isamu Z. Hartman, Yuri Poluektov, Kedar Narayan, Scheheraza Sadegh-Nasseri

Research output: Chapter in Book/Report/Conference proceedingChapter

Abstract

HLA-DM is now known to have a major contribution to the selection of immunodominant epitopes. A better understanding of the mechanisms controlling epitope selection can be achieved by examination of the biophysical behavior of MHC class II molecules upon binding of antigenic peptides and of the effect of DM on the interactions. Using purified soluble molecules, in this chapter we describe several in vitro methods for measuring peptide binding to HLA-DR molecules and the effects of HLA-DM on this interaction. A simple qualitative method, Gentle SDS-PAGE Assay assesses the ability of peptides to form tight complexes with MHC class II molecules. Measuring binding kinetics is among the most informative approaches to understanding molecular mechanisms, and here we describe two different methods for measuring binding kinetics of peptide–MHC complexes. In one method, rates of association and dissociation of fluorescently labeled peptides to soluble MHC class II molecules can be determined using G50 spin columns to separate unbound peptides from those in complex with MHC molecules. In another method, association and dissociation of unlabeled peptides and MHC class II molecules can be determined in real time using BIAcore Surface Plasmon Resonance (SPR). We also describe an intrinsic tryptophan fluorescence assay for studying transient interactions of DM and MHC class II molecules.

Original languageEnglish (US)
Title of host publicationMethods in Molecular Biology
PublisherHumana Press Inc.
Pages343-355
Number of pages13
DOIs
Publication statusPublished - Jan 1 2019

Publication series

NameMethods in Molecular Biology
Volume1988
ISSN (Print)1064-3745

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Keywords

  • BIAcore surface plasmon resonance (SPR)
  • Fluorescent peptide
  • Gentle SDS-PAGE assay
  • HLA-DM editing
  • Kinetic studies
  • Peptide binding to HLA-DR
  • Tryptophan fluorescence assay

ASJC Scopus subject areas

  • Molecular Biology
  • Genetics

Cite this

Kim, A. R., Ishizuka, I. E., Hartman, I. Z., Poluektov, Y., Narayan, K., & Sadegh-Nasseri, S. (2019). Studying MHC class II peptide loading and editing in vitro. In Methods in Molecular Biology (pp. 343-355). (Methods in Molecular Biology; Vol. 1988). Humana Press Inc.. https://doi.org/10.1007/978-1-4939-9450-2_24