Comparative Quantitative Mass Spectrometry Analysis of MHC Class II-Associated Peptides Reveals a Role of GILT in Formation of Self-Peptide Repertoire

Abstract

Gamma interferon Inducible Lysosomal Thiol reductase (GILT) is a unique lysosomal reductase that reduces disulfide bonds of endocytosed proteins. Lack of GILT clearly decreases CD4 T cell-antigen specific responses against some epitopes of antigens containing disulfide bonds, but not to proteins with few or no disulfide bridges. Hence, global impact of GILT on antigen presentation is currently not well understood. We used Nano-LC-ESI-MS/MS to investigate how GILT affects diversity of self-peptides presented by MHC class II molecules. Surprisingly, the repertoire of self-peptides in the absence of GILT does not appear to be significantly different, as only few peptide species (∼2%) were found to be the unique indicators of GILT's presence or absence. In the absence of GILT about thirty peptide species (∼5%) were found either uniquely or fourteen to hundred fold more abundantly expressed than in the presence of GILT. Our data indicate that GILT has limited yet unexpected effect on self-peptide species presented by MHC class II antigens.

Diverse cellular and organismal functions of the lysosomal thiol reductase GILT

“…Instead, GILT-independent epitopes tend to be surface exposed, and GILT-dependent epitopes tend to be buried, such that the requirement for GILT appears to depend on whether the epitope requires reduction to be exposed for MHC class II binding (Maric et al, 2001). A mass spectrometric analysis of MHC class II bound peptides from wild-type and GILT−/− resting splenocytes reveals that the bulk of the self peptide repertoire at steady state is GILT-independent with 5.5% of peptides more abundant in wild-type samples, 2% of peptides unique to GILT−/− samples, and 3.5% of peptides at least 10-fold more abundant in GILT−/− samples (Bogunovic et al, 2010). …”

Disulfide Reduction in the Endocytic Pathway: Immunological Functions of Gamma-Interferon-Inducible Lysosomal Thiol Reductase

“…A recent study used mass spectrometry to evaluate the impact of GILT on the overall repertoire of MHC class II-bound peptides eluted from wild-type and GILT À=À resting splenocytes (8). Surprisingly, no unique peptides were identified from GILT-containing APCs, and only 5.5% of peptides are more abundant in wild-type APCs.…”

Multiplexed relative and absolute quantitative immunopeptidomics reveals MHC I repertoire alterations induced by CDK4/6 inhibition

“…Specifically, most common relative quantification pMHC methods lack a normalization strategy to account for variations in sample input and processing. [15][16][17][18][19][20] Peptide losses during processing are estimated to be as much as 99.5% but vary across peptides and samples, underscoring the need for normalization. 21 Absolute quantification of pMHCs to date is most commonly performed by comparing endogenous levels of pMHCs to exogenous peptide standards, again failing to account for sample losses.…”