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

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. Quality control in the endoplasmic reticulum (ER)4 ensures the proper folding, oligomerization, and sorting of glycoproteins. Primary quality control, common to all glycoproteins, is carried out by successive rounds of glucosylation and de-glucosylation of N-linked glycans. In this process, known as the calnexin cycle, monoglucosylated glycoproteins are the elective substrates of the lectin-like "retention-retrieval" chaperones calnexin (a transmembrane protein) and/or calreticulin (the soluble homologue of calnexin) 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). It is agreed that secondary (protein-specific) quality control has two general features: (a) it takes place after completion of primary quality control, and (b) it involves the formation of dedicated complexes between glycoprotein substrates and ER proteins acting as either chaperones or escorts/ guides for intracellular transport. To the best of our knowle...

Distinct Functions for the Glycans of Tapasin and Heavy Chains in the Assembly of MHC Class I Molecules

“…Cells were lysed and proteins immunoprecipitated with anti-TAP1. As previously reported with other deglycosylated class I molecules (6, 7, 32), HA-HLA-A2(N86Q) showed reduced binding to TAP compared to HA-HLA-A2 (Figure 5A). However, despite impaired recruitment of HA-HLA-A2(N86Q) heavy chains, the HA-HLA-A2(N86Q) mutation had no significant impact on the recruitment of β 2 m, tapasin, calreticulin and ERp57 into the PLC (Figure 5A, lanes 3–4 compared to lanes 5–6 of β 2 m, tapasin, calreticulin and ERp57 blots).…”

Orchestration of secretory protein folding by ER chaperones

“…Only one of seven hemagglutinin glycans, attached to Asn81, created a kinetic barrier to folding [32], and even this glycan effect could not be detected in another set of experiments [34]. Among the highly homologous MHC class I molecules, some are sensitive to the presence of glycans [35], whereas others fold equivalently in the presence or absence of the carbohydrates [36]. Furthermore, even proteins that are normally not glycosylated can sometimes benefit from the inclusion of ectopic glycans [37].…”

Preferential interaction of MHC class I with TAPBPR in the absence of glycosylation

“…Human MHC class I molecules have a single conserved N-linked glycan on an asparagine residue found at position 86 (Parham et al, 1977). The functional relevance of glycosylation of human MHC class I has been explored using a number of approaches including mutation of the NxS/T motif to eliminate N-linked glycosylation (Barbosa et al, 1987; Santos-Aguado et al, 1987; Zhang et al, 1995; Harris et al, 2001; Martayan et al, 2008; Rizvi et al, 2011). Non-glycosylated human MHC class I molecules fail to interact with calreticulin, exhibit weak interaction with tapasin and the PLC, and are consequently expressed at lower levels at the cell surface.…”