Type 1 diabetes associated HLA-DQ2 and DQ8 molecules are relatively resistant to HLA-DM mediated release of invariant chain-derived CLIP peptides.

HLA-DM is essential for editing peptides bound to MHC class II, thus influencing the repertoire of peptides mediating selection and activation of CD4(+) T cells. Individuals expressing HLA-DQ2 or DQ8, and DQ2/8 trans-dimers, have elevated risk for type 1 diabetes (T1D). Cells coexpressing DM with these DQ molecules were observed to express elevated levels of CLIP (Class II associated invariant chain peptide). Relative resistance to DM-mediated editing of CLIP was further confirmed by HPLC-MS/MS analysis of eluted peptides, which also demonstrated peptides from known T1D-associated autoantigens, including a shared epitope from ZnT8 that is presented by all four major T1D-susceptible DQ molecules. Assays with purified recombinant soluble proteins confirmed that DQ2-CLIP complexes are highly resistant to DM editing, whereas DQ8-CLIP is partially sensitive to DM, but with an apparent reduction in catalytic potency. DM sensitivity was enhanced in mutant DQ8 molecules with disruption of hydrogen bonds that stabilize DQ8 near the DM-binding region. Our findings show that T1D-susceptible DQ2 and DQ8 share significant resistance to DM editing, compared with control DQ molecules. The relative resistance of the T1D-susceptible DQ molecules to DM editing and preferential presentation of T1D-associated autoantigenic peptides may contribute to the pathogenesis of T1D.

Expression of the mouse MHC class Ib H2-T11 gene product, a paralog of H2-T23 (Qa-1) with shared peptide-binding specificity.

The mouse MHC class Ib gene H2-T11 is 95% identical at the DNA level to H2-T23, which encodes Qa-1, one of the most studied MHC class Ib molecules. H2-T11 mRNA was observed to be expressed widely in tissues of C57BL/6 mice, with the highest levels in thymus. To circumvent the availability of a specific mAb, cells were transduced with cDNA encoding T11 with a substituted α3 domain. Hybrid T11D3 protein was expressed at high levels similar to control T23D3 molecules on the surface of both TAP(+) and TAP(-) cells. Soluble T11D3 was generated by folding in vitro with Qa-1 determinant modifier, the dominant peptide presented by Qa-1. The circular dichroism spectrum of this protein was similar to that of other MHC class I molecules, and it was observed to bind labeled Qa-1 determinant modifier peptide with rapid kinetics. By contrast to the Qa-1 control, T11 tetramers did not react with cells expressing CD94/NKG2A, supporting the conclusion that T11 cannot replace Qa-1 as a ligand for NK cell inhibitory receptors. T11 also failed to substitute for Qa-1 in the presentation of insulin to a Qa-1-restricted T cell hybridoma. Despite divergent function, T11 was observed to share peptide-loading specificity with Qa-1. Direct analysis by tandem mass spectrometry of peptides eluted from T11D3 and T23D3 isolated from Hela cells demonstrated a diversity of peptides with a clear motif that was shared between the two molecules. Thus, T11 is a paralog of T23 encoding an MHC class Ib molecule that shares peptide-binding specificity with Qa-1 but differs in function.

ERAAP and tapasin independently edit the amino and carboxyl termini of MHC class I peptides.

Effective CD8(+) T cell responses depend on presentation of a stable peptide repertoire by MHC class I (MHC I) molecules on the cell surface. The overall quality of peptide-MHC I complexes (pMHC I) is determined by poorly understood mechanisms that generate and load peptides with appropriate consensus motifs onto MHC I. In this article, we show that both tapasin (Tpn), a key component of the peptide loading complex, and the endoplasmic reticulum aminopeptidase associated with Ag processing (ERAAP) are quintessential editors of distinct structural features of the peptide repertoire. We carried out reciprocal immunization of wild-type mice with cells from Tpn- or ERAAP-deficient mice. Specificity analysis of T cell responses showed that absence of Tpn or ERAAP independently altered the peptide repertoire by causing loss as well as gain of new pMHC I. Changes in amino acid sequences of MHC-bound peptides revealed that ERAAP and Tpn, respectively, defined the characteristic amino and carboxy termini of canonical MHC I peptides. Thus, the optimal pMHC I repertoire is produced by two distinct peptide editing steps in the endoplasmic reticulum.

Analysis of hepatic glycogen-associated proteins.

Glycogen particles are associated with a population of proteins that mediate its biological functions, including: management of glucose flux into and out of the glycogen particle, maintenance of glycogen structure and regulation of particle size, number, and cellular location. A survey of the glycogen-associated proteome would be predicted to identify the relative representation of known members of this population, and associations with unexpected proteins that have the potential to mediate other functions of the glycogen particle. We therefore purified glycogen particles from both mouse and rat liver, using different techniques, and analyzed the resulting tryptic peptides by MS. We also specifically eluted glycogen-binding proteins from the pellet using malto-oligosaccharides. Comparison of the rat and mouse populations, and analysis of specifically eluted proteins allow some conclusions to be made about the hepatic glycogen sub-proteome. With the exception of glycogen branching enzyme all glycogen metabolic proteins were detected. Novel associations were identified, including ferritin and starch-binding domain protein 1, a protein that contains both a transmembrane endoplasmic reticulum signal peptide and a carbohydrate-binding module. This study therefore provides insight into the organization of the glycogen proteome, identifies other associated proteins and provides a starting point to explore the dynamic nature and cellular distribution of this metabolically important protein population.