The Origin of Proteasome-inhibitor Resistant HLA Class I Peptidomes: a Study With HLA-A*68:01

Abstract

Some HLA class I molecules bind a significant fraction of their constitutive peptidomes in the presence of proteasome inhibitors. In this study, A*68:01-bound peptides, and their parental proteins, were characterized through massive mass spectrometry sequencing to refine its binding motif, including the nearly exclusive preference for C-terminal basic residues. Stable isotope tagging was used to distinguish proteasome-inhibitor sensitive and resistant ligands. The latter accounted for less than 20% of the peptidome and, like in HLA-B27, arose predominantly from small and basic proteins. Under the conditions used for proteasome inhibition in vivo, epoxomicin and MG-132 incompletely inhibited the hydrolysis of fluorogenic substrates specific for the tryptic or for both the tryptic and chymotryptic subspecificities, respectively. This incomplete inhibition was also reflected in the cleavage of synthetic peptide precursors of A*68:01 ligands. For these substrates, the inhibition of the proteasome resulted in altered cleavage patterns. However these alterations did not upset the balance between cleavage at peptide bonds resulting in epitope destruction and those leading to their generation. The results indicate that inhibitor-resistant HLA class I ligands are not necessarily produced by non-proteasomal pathways. However, their generation is not simply explained by decreased epitope destruction upon incomplete proteasomal inhibition and may require additional proteolytic steps acting on incompletely processed proteasomal products. Molecular &

Functional Interaction of the Ankylosing Spondylitis-associated Endoplasmic Reticulum Aminopeptidase 1 Polymorphism and HLA-B27 in Vivo

“…Mass Spectrometry-Individual HPLC fractions were analyzed via MALDI-TOF MS using a 4800 Proteomics Analyzer (Applied Biosystems), as described elsewhere (27). The mass spectra were acquired in reflector positive mode at 25 kV in the m/z range of 800 -2000, using a signal-to-noise ratio (s/n) cutoff of 3, and processed using Data Explorer software, version 4.9 (Applied Biosystems).…”

“…Peptide sequencing was carried out with MALDI-TOF/TOF MS/MS, as described elsewhere (27). Interpretation of the MS/MS spectra was assisted by various tools.…”

Peptide Handling by HLA-B27 Subtypes Influences Their Biological Behavior, Association with Ankylosing Spondylitis and Susceptibility to Endoplasmic Reticulum Aminopeptidase 1 (ERAP1)

“…Electrospray-Orbitrap Mass Spectrometry Analysis-Peptide sequencing by Electrospray-Orbitrap was performed exactly as described elsewhere (34) by LC-MS/MS using an Orbitrap XL mass spectrometer (Thermo Fisher, San Jose, CA) fitted with a capillary HPLC (Eksigent, Dublin, CA).…”

Translating DRiPs: MHC class I immunosurveillance of pathogens and tumors

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