Competition for Access to the Rat Major Histocompatibility Complex Class I Peptide-loading Complex Reveals Optimization of Peptide Cargo in the Absence of Transporter Associated with Antigen Processing (TAP) Association

Ford, Stuart; Antoniou, Antony N.; Butcher, Geoffrey W.; Powis, Simon J.

doi:10.1074/jbc.m400456200

Cited by 10 publications

(8 citation statements)

References 44 publications

(40 reference statements)

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“…This is in agreement with previous studies demonstrating an increased calnexin binding of mouse L d N86K, L d N86Q, and L d S88W mutants (15,16), but not with studies on human A2S88A and etB27N86Q mutants in which calnexin binding was found to be weak and conserved (35) or had not been tested (22). Similar to L d mutants and etB27N86Q (15,16,22,23), Cw1S88G lost its ability to bind calreticulin, ERp57, and tapasin in peptide loading-sufficient cells, but unlike the above mutants (and remarkably) it did retain partial interaction with TAP.…”

Section: Discussionsupporting

confidence: 81%

“…However, the glycosylation-defective mutants RT-1A a N86K and etB27N86Q (22,23) were assembled in vitro following bulk isolation from non-ionic detergent cell extracts whereas Cw1S88G was isolated at different, carefully selected chase points, providing the opportunity to observe an initial refractoriness to binding peptides (similar to etB27N86Q) followed by the stabilization of abnormal class I folds (possibly reminiscent of RT-1A a N86K). The present observations may in part reconcile the available, conflicting evidence.…”

Section: Discussionmentioning

confidence: 99%

“…Moreover, the only two mutants that have been studied in detail displayed two different phenotypes; the human mutant was completely refractory to peptide assembly, in vivo as well as in vitro (22), whereas the rat mutant failed to stably bind natural ligands in vivo but retained its ability to assemble in vitro with specific peptides (23), an alteration reminiscent of those seen in cells defective in calreticulin, TAP, or tapasin (24 -26).…”

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confidence: 99%

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

Martayan¹,

Sibilio²,

Setini³

et al. 2008

Journal of Biological Chemistry

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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...

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Section: Discussionsupporting

confidence: 81%

Section: Discussionmentioning

confidence: 99%

mentioning

confidence: 99%

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

Martayan¹,

Sibilio²,

Setini³

et al. 2008

Journal of Biological Chemistry

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“…The propensity of newly synthesized B*27:05 to misfold and aggregate has long been suggested to correlate with AS etiology [46][47][48][49].…”

Section: Discussionmentioning

confidence: 99%

F pocket flexibility influences the tapasin dependence of two differentially disease‐associated MHC Class I proteins

et al. 2015

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The human MHC class I protein HLA-B*27:05 is statistically associated with ankylosing spondylitis, unlike HLA-B*27:09, which differs in a single amino acid in the F pocket of the peptide-binding groove. To understand how this unique amino acid difference leads to a different behavior of the proteins in the cell, we have investigated the conformational stability of both proteins using a combination of in silico and experimental approaches. Here, we show that the binding site of B*27:05 is conformationally disordered in the absence of peptide due to a charge repulsion at the bottom of the F pocket. In agreement with this, B*27:05 requires the chaperone protein tapasin to a greater extent than the conformationally stable B*27:09 in order to remain structured and to bind peptide. Taken together, our data demonstrate a method to predict tapasin dependence and physiological behavior from the sequence and crystal structure of a particular class I allotype.Keywords: Ankylosing spondylitis r HLA-B27 r Major histocompatibility complex r Molecular dynamics r Natively unstructured proteins r Protein folding r Simulations Additional supporting information may be found in the online version of this article at the publisher's web-site Introduction MHC class I molecules are heterotrimeric proteins that transport antigenic peptides to the cell surface and present them to cytotoxic T cells. They consist of the transmembrane heavy chain (HC), the noncovalently associated light chain beta-2 microglobulin (β 2 m), and an antigenic peptide of eight to ten amino acids. The Correspondence: Prof. Sebastian Springer e-mail: s.springer@jacobs-university.de extracellular part of the heavy chain comprises the α 1 , α 2 , and α 3 domains. The α 1 and α 2 domains form the peptide-binding groove, a superdomain that consists of an antiparallel beta sheet surmounted by two alpha helices, between which the peptide binds [1,2].In the cell, peptide binding to class I is a multistep process within the ER. It involves the peptide-loading complex, which consists of the peptide transporter associated with antigen processing (TAP) that transports peptides from the cytosol into the ER lumen [3] and several chaperone proteins such as tapasin, which binds both to the class I molecule and TAP [4,5].C 2015 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim www.eji-journal.eu Eur. J. Immunol. 2015. 45: 1248-1257 Molecular immunology 1249The sequence of peptides that can bind to class I is determined by interactions with the amino acid side chains of the peptide-binding groove. The high sequence polymorphism of the peptide-binding groove in human class I molecules (human leukocyte antigens, HLA-A, -B, and -C) means that different allotypes bind different peptides; thus, individual HLA allotypes-such as HLA-B27-support-specific immune responses and are statistically associated with disease [6,7]. Among the subtypes of HLA-B27 [8,9], HLA-B*27:05 (B*27:05) shows a very strong statistical association with spondyloarthropathies such as ankylosing spondylitis (AS), in contr...

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“…4B, middle and bottom panels). The relation between full-length US3 and the truncated isoform was further investigated in 293T cells that express little tapasin (13). The 293T cells were cotransfected with cDNAs as indicated and analyzed by coprecipitation and immunoblot (Fig.…”

Section: Resultsmentioning

confidence: 99%

A Short Isoform of Human Cytomegalovirus US3 Functions as a Dominant Negative Inhibitor of the Full-Length Form

Shin

Park

Lee

et al. 2006

J Virol

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Human cytomegalovirus encodes four unique short (US) region proteins, each of which is independently sufficient for causing the down-regulation of major histocompatibility complex (MHC) class I molecules on the cell surface. This down-regulation enables infected cells to evade recognition by cytotoxic T lymphocytes (CTLs) but makes them vulnerable to lysis by natural killer (NK) cells, which lyse those cells that lack MHC class I molecules. The 22-kDa US3 glycoprotein is able to down-regulate the surface expression of MHC class I molecules by dual mechanisms: direct endoplasmic reticulum retention by physical association and/or tapasin inhibition. The alternative splicing of the US3 gene generates two additional products, including 17-kDa and 3.5-kDa truncated isoforms; however, the functional significance of these isoforms during viral infection is unknown. Here, we describe a novel mode of self-regulation of US3 function that uses the endogenously produced truncated isoform. The truncated isoform itself neither binds to MHC class I molecules nor prevents the full-length US3 from interacting with MHC class I molecules. Instead, the truncated isoform associates with tapasin and competes with full-length US3 for binding to tapasin; thus, it suppresses the action of US3 that causes the disruption of the function of tapasin. Our results indicate that the truncated isoform of the US3 locus acts as a dominant negative regulator of full-length US3 activity. These data reflect the manner in which the virus has developed temporal survival strategies during viral infection against immune surveillance involving both CTLs and NK cells.Human cytomegalovirus (HCMV), a member of the Betaherpesviridae, leads to lifelong persistence in the infected host, followed by causing opportunistic pathogenesis in immunocompromised individuals such as neonates, organ transplant recipients, and AIDS patients. Both innate and adaptive immune systems participate in host defense against the viral infection in which natural killer (NK) cells and cytotoxic T lymphocytes (CTLs) function as the major effector cells. NK cell response plays an important role in initial defense, and the deficiency of NK cells results in susceptibility to severe herpesvirus infections (8). NK cell activation is controlled by the interaction of inhibitory receptors of NK cells with major histocompatibility complex (MHC) class I antigens on target cells (26). The missing-self hypothesis proposes that NK cells recognize and attack target cells that lack an adequate level of cell surface-expressed self-MHC class I molecules (25). Generally, the immune response of the host against a virus, including HCMV, is mediated by CTLs, which recognize and eliminate infected cells expressing viral peptide-loaded MHC class I molecules (41).Since the MHC class I molecule is a key factor in the modulation of the immune response against viral pathogens, it is not surprising that class I molecules are preferred targets of viruses to avoid immune surveillance and clearance (27,32,40). HCM...

show abstract

Competition for Access to the Rat Major Histocompatibility Complex Class I Peptide-loading Complex Reveals Optimization of Peptide Cargo in the Absence of Transporter Associated with Antigen Processing (TAP) Association

Cited by 10 publications

References 44 publications

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

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

F pocket flexibility influences the tapasin dependence of two differentially disease‐associated MHC Class I proteins

A Short Isoform of Human Cytomegalovirus US3 Functions as a Dominant Negative Inhibitor of the Full-Length Form

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