Lipid Composition Alters the Assembly of MHCII in Nanodiscs

Abstract

Class II major histocompatibility complexes (MHCII) are transmembrane glycoproteins found in antigen‐presenting cells (APCs). MHCII displays antigenic peptides on the surface of APCs to CD4+ T‐cells, and peptides eliciting a strong immune response against the pathogen from which the peptide originated are designated as immunodominant.The molecular selection criteria leading to immunodominant antigenic peptides binding to MHCII is a critical step in the antigen processing pathway and is an important focal point in immunological research. Traditionally, binding studies are executed with soluble MHCII by removing the transmembrane portion of the protein. In sharp contrast, this project aimed to utilize full length MHCII in its native, membrane‐embedded form. To this end, we used the synthetic membrane model known as the nanodisc to evaluate the effects of membrane lipid composition on MHCII assembly and ultimately its consequences for peptide binding.We isolated full length membrane bound DR1 (mDR1), the predominant MHCII allele, from a B‐lymphoblastoid cell line via immunoaffinity chromatography. Three types of nanodisc were generated, each with distinct lipid composition of their lipid bilayer: simple (phosphatidylcholine), complex fluid, and complex rigid (phosphatidylcholine, sphingomyelin, cholesterol; 60:1:1 or 1:1:1 respectively). Nanodiscs were purified by size exclusion chromatography, and the fractions were run on native and SDS gel electrophoresis, which showed clear differences in assembly of mDR1 between nanodisc types.We observed that the complex fluid nanodiscs showed DR1 assembly as one major SEC‐generated peak, in which the monomer, dimer and tetramer forms of mDR1 were coming together in a single fraction. In contrast, simple and complex rigid nanodiscs were distinguished by multiple SEC peaks. As DR1 tends to form aggregates, we inferred that, in simple and rigid nanodiscs, DR1 tetramers and dimers form in separate nanodisc assemblies.MHCII is not limited to the cell surface – it is also found in endosomal compartments, which have their own signature membrane characteristics. As such, we predicted that MHCII conformation would be altered from one environment to the next. Based on our observations, we conclude that membrane lipid composition has a strong impact on native MHCII assembly and thus could conceivably exert considerable impact on peptide binding kinetics/thermodynamics.Support or Funding InformationResearch reported in this publication was supported by the National Institute Of General Medical Sciences of the National Institutes of Health under Award Numbers UL1GM118991, TL4GM118992, or RL5GM118990. The content is solely the responsibility of the authors and does not necessarily represent the official views of the National Institutes of Health. UA is an AA/EO employer and educational institution and prohibits illegal discrimination against any individual: www.alaska.edu/titleIXcompliance/nondiscrimination.This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.