N-Linked Glycosylation Selectively Regulates the Generic Folding of HLA-Cw1

Aline Martayan,Leonardo Sibilio,Andrea Setini,Elisa Lo Monaco,Elisa Tremante,Doriana Fruci,Marco Colonna,Patrizio Giacomini

doi:10.1074/jbc.m709175200

Abstract

To resolve primary (glycosylation-assisted) from secondary (glycosylation-independent) quality control steps in the biosynthesis of HLA (human leukocyte antigen) class I glycoproteins, the unique N-linked glycosylation site of the HLA-Cw1 heavy chain was deleted by site-directed mutagenesis. The non-glycosylated Cw1S88G mutant was characterized by flow cytometry, pulse-chase, co-immunoprecipitation, and in vitro assembly assays with synthetic peptide ligands upon transfection in 721.221 and 721.220 cells. The former provide a full set of primary as well as secondary chaperoning interactions, whereas the latter are unable to perform secondary quality control (e.g. proper class I assembly with peptide antigens) as a result of a functional defect of the HLA-dedicated chaperone tapasin. In both transfectants, Cw1S88G displayed a loss/weakening in its generic chaperoning interaction with calreticulin and/or ERp57 and became redistributed toward calnexin, known to bind the most unfolded class I conformers. Despite this, and quite unexpectedly, a weak interaction with the HLA-dedicated chaperone TAP was selectively retained in 721.221. In addition, the ordered, stepwise acquisition of thermal stability/peptide binding was disrupted, resulting in a heterogeneous ensemble of Cw1S88G conformers with unorthodox and unprecedented peptide assembly features. Because a lack of glycosylation and a lack of tapasin-assisted peptide loading have distinct, complementary, and additive effects, the former is separable from (and upstream of) the latter, e.g. primary quality control is suggested to supervise a crucial, generic folding step preliminary to the acquisition of peptide receptivity.

Highlights

Quality control in the endoplasmic reticulum (ER)4 ensures the proper folding, oligomerization, and sorting of glycopro
Known as the calnexin cycle, monoglucosylated glycoproteins are the elective substrates of the lectin-like “retention-retrieval” chaperones calnexin and/or calreticulin and become folded and disulfide-bonded through the concerted action of calnexin, calreticulin, the thiol-dependent oxido-reductase ERp57, protein disulfide isomerase, and other members of the quality control machinery [1]
Trimmed, conformed substrates are released for intracellular transport and further processing, but the maturation, assembly, folding, and intracellular transport of some glycoproteins require specialized steps, collectively known as secondary quality control [1]

Summary

Introduction

Quality control in the endoplasmic reticulum (ER) ensures the proper folding, oligomerization, and sorting of glycopro-. The heavy chain binds calnexin, associates with ␤2m, and is loaded with peptide on a unique folding and quality control station called “peptide loading complex,” formed by the supramolecular association of generic and class I-dedicated chaperones. The former include calnexin (only in murine cells), calreticulin, ERp57, and protein disulfide isomerase, recently identified [3]. N-Linked Glycosylation of HLA-Cw1 tide-bound), conformed class I heterotrimers are released from the peptide loading complex, leave the ER, and are exported to the cell surface for functional recognition by CD8 T lymphocytes and natural killer (NK) cells [2]

Methods

Results

Conclusion