Proteasome-independent HLA-B27 Ligands Arise Mainly from Small Basic Proteins

Miguel Marcilla,José A. López de Castro,Juan J. Cragnolini

doi:10.1074/mcp.m600302-mcp200

Abstract

Many of the constitutive peptide ligands of HLA-B27, a molecule strongly associated with spondyloarthritis, are proteasome-independent. Stable isotope tagging, mass spectrometry, and epoxomicin-mediated inhibition were used to determine their percentage, structural features, and parental proteins. Of 104 molecular species examined, 29.8% were proteasome-independent, paralleling the level of HLA-B27 re-expression in the presence of epoxomicin after acid stripping. Proteasome-dependent and -independent ligands differed little in peptide motifs, flanking sequences, and cellular localization of the parental proteins. In contrast, whereas the former set arose from proteins whose size and isoelectric point distribution largely reflected those in the human proteome, proteasome-independent ligands, other than a few matching signal sequences, were almost totally derived from small (about 6-16.5 kDa) and basic proteins, which account for only 6.6% of the human proteome. Thus, a non-proteasomal proteolytic pathway with strong preference for small proteins is responsible for a significant fraction of the HLA-B27-bound peptide repertoire.

Highlights

Many of the constitutive peptide ligands of HLA-B27, a molecule strongly associated with spondyloarthritis, are proteasome-independent
Most of these ligands are produced in the cytosol. They or their immediate precursors are introduced into the lumen of the endoplasmic reticulum (ER) by the transporter associated with antigen processing (TAP) where they bind to the nascent class I molecule in a process of assisted loading involving calreticulin, ERp57, protein-disulfide isomerase, and tapasin, which together with TAP and the Major histocompatibility complex (MHC) molecule form the peptide-loading complex [1,2,3,4,5,6]
Proteasome-dependent and -independent Ligands Can Be Distinguished by Isotopic Labeling of the B27-bound Peptide Repertoire—To determine the role of the proteasome in generating the B*2705-bound peptide repertoire an approach based on stable isotope tagging of the HLA-B27 ligands was developed (Fig. 1). 15N-Tagged Arg was used to achieve the labeling of virtually every single peptide

Summary

Introduction

Many of the constitutive peptide ligands of HLA-B27, a molecule strongly associated with spondyloarthritis, are proteasome-independent. Because there was a close correlation between the expression level of HLA-B27 in the presence of epoxomicin after acid stripping and the percentage of proteasome-independent ligands and because these came mostly from small basic proteins, we wondered whether the parental proteins of known HLA-B27 ligands reflected the size distribution of the human proteome or that observed in our study.

Results

Conclusion