The nature of the MHC class I peptide loading complex

Peter CresswellNaveen BangiaTobias DickGundo Diedrich

The nature of the MHC class Ipeptide loading complex

ALJthors' address

Peter Cresswell, Naveen Bangia, Tobias Dick, Gundo Didrich,

Section of Immunohioiogy, Howard Hughes

Medical Institute, Yale University School of

Medicine, New Haven, Connecticut, USA.

Correspondence to:

Peter C/esswdl

Section of Immimobiology

Howard Hughes Medical Institute

Yale University School of Medicine

PO. Box 208011

New Haven CT 06S20-80H

USA

Fax: 1 203 737 1764

e-mail: [email protected]

Ac kn owledgements

We thank Nancy Dometios for invaluahle help in

preparing this manuscript. G. D. is supported hy

a grant from the German Research Foundation

and T. D. is supported by a DFG Fellowship

(Germany). The work de.scribed was supported

by the Howard Hughes Medical Institute.

/mmunologjcol Reviews 1999

Vol 172:21-28Printed in Denmark. All rights reserved

Copyrighl © Mtinksgaard 1999

Immunological ReviewsISSN 0105-2896

Summary: Peptide binding to major histocompatibility complex (MHC)dass I molecules occurs in the endoplasmic reticulum (ER). Efficient pep-tide binding requires a number of components in addition co the MHCclass I-P; microglobulin dimer (pjm). These include tbe two subunits ofthe transporter associated with antigen presentation (TAPl and TAP2),which are essential for introducing peptides into tbe ER from the cytosol,and tapasin, an MHC-encoded membrane protein. Prior to peptide bind-ing, MHC class I-p^m dimers form part of a large multisubnnit ER com-plex which includes TAP and tapasin. In addition to these specialized com-ponents two soluble 'house-keeping' proteins, the chaperone calreticulinand the tbiol oxidoreductase ERp57, are also components of this complex.Our current understanding of the nature and function of the MHC class Ipeptide loading complex is the topic of this review.

Introduction

The assembly of major histocompatibility complex (MHC)class I molecules has much in common with the folding andassembly of other multisubimit glycoproteins in the endoplas-mic reticulum (ER). All such processes involve transient associ-ations of the assembling glycoproteins with a variety of ERchaperones. These facilitate folding and assembly, largely byinteracting reversibly with exposed hydrophobic regions ofnewly synthesized molecules normally buried in the interior ofthe mature, properly folded and assembled structure. Suchinteractions serve two functions: to prevent aggregation and toretain the glycoproteins in the ER until assembly is correct andcomplete. The process has been termed quality control (1, 2).A characteristic of quahty control is that unassembled subunitsoften are degraded without leaving the ER. For example, inmyeloma cells which do not synthesize immunoglobulin lightchains the unpaired heavy chains are degraded (3-5). In con-trast, unpaired Hght chains are usually secreted. Presumablythey can fold individually, perhaps by dimerizing, in such a waythat aggregation or prolonged chaperone interaction is avoided(6-8).

Cresswell et al • The MHC class I peptide loading complex

In the case of MHC class I molecules, escape from the ERand surface expression requires the proper assembly of a threesubunit structure. The subuuits are the polymorphic MHC-encoded transmembrane glycoprotein or heavy chain, p,microglobulin (P^m), and a small peptide, usually 8-10 aminoacids in length. Without p m or peptide MHC class I heavychains are degraded by proteasomes after retrotranslocationinto the cytosol (9, 10). In addition to the 'house-keeping'chaperones also involved in folding of other giycoproteins, spe-ciahzed ER molecules participate in MHC class I assembly Inthis review, we will present recent work that has enhanced ourunderstanding of this process, as well as raise new questions.

House-keeping proteins and MHC class I assembly

Membrane or secreted proteins are synthesized on membrane-associated ribosomes and inserted co-translationally into the ERthrough a specialized structure known as the translocon. N-linked glycans are added in the ER to appropriate asparagineresidues (i.e. those incorporated into the sequence asn-X-ser orasn-X-thr, where X is any amino acid other than proline) dur-ing the translocation process. Human HLA-A, -B or -C mole-cules have a single N-linked glycosylation site at position 86while murine H-2 molecules have an additional 2 or 3,depending on the allele. The ER chaperone BiP, an Hsp 70homolog, is beheved to interact with proteins during theirtranslation and insertion, with an adenosine triphosphate(ATP)-regulated binding and release mechanism providing theenergy for the unidirectional movement of che protein throughthe ER membrane. BiP has been shown to bind to free class Iheavy chains, which is consistent with a potential role in thetranslocation of class I molecules (11)-

The dominant chaperone associated with newly synthe-sized class I heavy chains is calnexin (12-17). Calnexin isunusual first in that it is a transmembrane chaperone, and sec-ond in that it is a lectin with specificity for an intermediate inthe maturation of N-linked glycans. When added to newly syn-thesized glycoproteins from a dolichol phosphate-bound pre-cursor, one of the branched chains of an N-hnked glycan ter-minates in three glucose residues. These are successivelyremoved in the ER by the action of two glucosidases (I and II).Calnexin binds to N-linked glycans bearing a single glucoseresidue. Precisely how calnexin facilitates folding is not under-stood, but it is postulated to be involved in a binding andrelease cycle together with a specific glucosyl transferase (UDP-glucosyl glycoprotein transferase). In this scheme, followinginteraction with calnexin, the glucose residue on the mono-glucosylated N-linked glycan is removed by glucosidase IL If,

after release from calnexin, the glycoprotein has not achieved anative conformation it is reglucosylated by the giucosyl trans-ferase. This enzyme can discriminate between native and non-native forms of glycoproteins (18. 19), actually a characteristicproperty of chaperones. Reglucosylation leads to a secondinteraction with calnexin and another attempt at refolding.When a native conformation is finally achieved the N-linkedglycan is not reglucosylated and the glycoprotein is releasedfrom further calnexin interactions and can leave the ER.

Studies in the mouse indicate that the MHCclass I-calnexin interaction persists after p^m association (12,20). This is in contrast to results obtained in human cells, andone other study in the mouse, where the dominant chaperoneassociated with class I-p:m dimers is calreticulin rather thancalnexin (21, 22). Calreticulin is a soluble homolog of calnexinwhich shares its specificity for monoglucosylated N-Iinked gly-cans. The differential association of the two chaperones withMHC class I molecules at different stages of assembly implies anadditional mode of interaction with unfolded proteins, perhapswith exposed hydrophobic regions as for other chaperones. Achaperone activity for calnexin independent of glycosylation,defined as the abihty to inhibit the aggregation of denaturedproteins, has recently been demonstrated (D. Williams, per-sonal communication).

The interaction of MHC class I-P2m dimers with calreticu-lin has not been kinetically separated from their interactionwith a number of other components which together form alarge multisnbnnit complex in the ER. These componentsinclude an additional house-keeping molecule called ERp5 7(23-25). ERp57 is a member of the thioredoxin family ofenzymes, a subset of which resides in the ER. The prototypicalER resident member of the family is protein disulfide isomerase(PDI). These enzymes catalyze the isomerisation of disulfidebonds of proteins undergoing folding and assembly in the ER,ensuring that the correct interchain and intrachain disulfidebonding patterns of proteins or assemblies of proteins areachieved. As a group the enzymes are called thiol oxidoreduc-tases, because they can catalyze both reduction and oxidation ofdisulfide bonds depending on the local redox potential. Theredox potential of the ER is such that both reduction and oxi-dation reactions are possible, thus facilitating the isomerizationreaction.

ERp5 7 appears to be adapted to function together with cal-nexin and calreticulin in promoting folding, assembly and dis-ulfide bond formation of glycoproteins in the ER (26-28). Amodel has been proposed in which calnexin or calreticulinbinds to a monoglucosylated N-linked glycan of a newly syn-thesized glycoprotein, and ERp57 associates with the chaper-

22 Immunological Reviews 172/1999


.174 Wild type

ERp57

\ CR!

Daudi

Peptides

Fig. 1. A schematic view of the MHC class Ipeptide loading complex is shown in thecenter, with suggested modes of interactionof the various components indicated. On the

left is shown the subcomplex containing theMHC class I glycoprotein, p,m, calreticulin,ERpS7 and tapasin which is detectable in theTAP-negative cell hne .174. On the right isshown the tapasin-TAP cotuplex detectable inthe Pjm-negative cell line Daudi, with a weaklyassociated free MHC class I heavy chain. Notindicated in the center diagram is the findingsuggested by analysis of pnrified complexesthat up to four of the subcomplexes shown onthe left may be associated with a single TAPheterodimer.

one. A combination of the binding and release mechanismincorporating the glucosyl transferase. described above, andisomerization of disulfide bonds mediated by ERp57 ultimatelygenerates a properly folded glycoprotein with the correctarrangement of disulfide bonds. Hammerling and co-workerswere able to detect an ERp5 7 interaction with class I heavychain-calnexin complexes in addition to the class I-pjm-cal-reticuhn-ERp57 association which we also observed (25). Thedata suggest that disulfide bond formation is initiated whenclass I heavy chains are associated with calnexin and completedafter p m binding and calreticulin association, with ERp57being the thiol oxidoreductase facilitating their formation.Salter and co-workers (29) have shown that disulfide bond for-mation is only partially complete in calnexin-associated humanMHC class I heavy chains, which is also consistent with thismodel.

Dedicated proteins involved in MHC class I assembly

An essential protein for MHC class I assembly is the transporterassociated with antigen processing (TAP). TAP is a hetero-dimeric protein whose subunits, TAPl and TAP2, are encodedby MHC-hnked genes. Each subunit consists of an N-terminalhydrophobic region containing multiple transmembranedomains and a C-terminal hydrophilic region which is cytoso-lic. Its well-established function is to transport, in a poorlyunderstood ATP-dependent manner, peptides derived fromcytosohc proteins across the ER membrane. Here they bind toMHC class I molecules provided they have the correct lengthand binding motif. Mutant cell lines, or mice, lacking either ofthe TAP subunits do not normally express cell surface class Imolecules because peptide binding is required for stability andefficient export from the ER.

A number of years ago, we and others observed a physicalassociation between MHC class I molecules and TAP (16, 30).This association was maintained after solubilization in thedetergent digitonin and was dependent on the formation ofclass I heavy chain-^jm dimers. Sodium dodecyi sulfate-poly-acrylamide gel electrophoresis (SDS-PAGE) analysis of radiola-beled MHC class I-TAP complexes revealed an additional com-ponent with an apparent Mr of 48 kDj. Subsequently this waspurified, partially sequenced and a cDNA clone isolated (31,32). We named it tapasin, for TAP-associated glycoprotein.Tapasin is a transmembrane glycoprotein which, like MHCmolecules, is a member of the immunoglobulin superfamily. Ithas an ER retention signal in its cytoplasmic domain consistingof lysine residues at the - 3 , -4 and -5 positions. The geneencoding tapasin is at the centromeric end of the human MHC(33). Analysis of a p^m-negative mutant cell line, Daudi,showed that tapasin can associate with TAP in the absence ofassembled class I molecules. In a TAP-negative cell line, .174,we were able to demonstrate that complexes exist containingclass I-pjm dimers and tapasin. This led us to suggest that tapa-sin has independent binding sites for class I-p^m dimers andTAP and that it might form a bridge between them (21). Class1-tapasin complexes, whether or not they were associated withTAP, were found to contain calreticulin and, later, ERp57 (21,23). The make-up of the complexes in wild-type, p^m-negativeand TAP-negative cell lines is schematically indicated in Fig. 1.

Genetic evidence for the postulated role of tapasin camefrom the discovery that a human cell line, called .220 and pre-viously shown to lack the class I-TAP interaction, did notexpress detectable tapasin at the protein level (21). In collabo-ration with Spies and Grandea, we examined a . 2 2 0 transfectantexpressing the human class I allele HLA-B8. Re-expression oftapasin in .220.B8 by transfection of tapasin cDNA restored the

Immunoiogical Reviews 172/1999 23


association of HLA-B8 with TAP, confirming the hypothesis

(31). Thus, we postulate that peptide-free class I molecules

associated with calreticuhn and ERp57 form part of a large com-

plex with tapasin connecting them to TAP Quantitative analysis

ofthe components ofthe complex, purified from human B-cell

lines using an anti-TAPl affmity column, suggested that it con-

tains approximately four tapasin molecules per TAP dimer and

that each tapasin molecule associates with a single MHC

class I-p^m dimer (31). Calreticulin, and in retrospect ERp57,

since its involvement was not recognized at the time this analy-

sis was performed, may be substoicbiometric compared to

tapasin and class I. This is not clear because of the potential for

dissociation of these components during purification.

Interactions between the components

ofthe MHC class I-TAP complex

The cumulative data suggest tbat there are a number of separateinteractions between the various components, many of themwith low affinity, which together stabilize the class I-TAP com-plex. As already stated, tapasin and TAP interact in the absenceof assembled class I-Pjm dimers. This interaction is highly sus-ceptible to the detergent used for solubilization. They remainassociated in digitonin and to a lesser extent in CHAPS, but areseparated by Triton X-100 and a number of other detergents,including some considered to be relatively mild, such as N-octyl glucoside (21) (data not shown). This might suggest thatthe transmembrane domain of tapasin is important for theinteraction, and consistent with this a soluble tapasin mutant,lacking the transmembrane domain and cytoplasmic tail, doesnot associate detectably with TAP even in digitonin (34). Ofinterest, in tapasin-negative .220.B8 cells TAP levels are 2- to 4-fold lower tban in the parental cell line, and expression of tapa-sin, but not soluble tapasin, restores TAP levels to normal (34),This implies that tapasin plays an important role in stabihzingTAP in the ER membrane, in spite of tbe fact that tbe tapa-sin-TAP interaction is so easily perturbed.

The interaction of newly synthesized glycoproteins withcalnexin or calreticulin can often be inhibited by agents thatinterfere with the formation of monoglucosylated N-linkedglycans, Castanospermine, which inhibits the glucosidases thatremove tbe two terminal glucose residues from the glycan, hashttle effect on the interaction of calnexin with class I heavychains (22, 35) but significantly impedes the calreticulin inter-action with class I-p^m dimers (21, 22). It also inhibitsclass I-TAP association, most likely because without associatedcalreticulin class I-pjm dimers are unstable and free class Iheavy cbains bind poorly to tapasin-TAP complexes. Perhaps

the most interesting aspect of the calreticulin interaction witbclass I is tbat it is only in this context that the calredcu-lin-ERpS7 interaction can be detected without chemical cross-linking (23, 27), ThusinDaudi cells, which lack p m and showno class I-calreticulin association, antibodies to calreticuhn failto co-precipitate ERp57. Likewise, in ,220,B8 cells, wbich lacktapasin and in which the calreticulin-class I interaction isundetectable, no calreticulin-ERp57 association is observed. In1 74 cells, whicb lack TAP but can form a subcomplex contain-ing class I-p2ni dimers, tapasin, calreticulin and ERp57, tbeinteraction is easily seen. The probable implication of theseobservations is that calreticulin and ERp57 bind independentlyto the class I-Pitn-tapasin complex and to each other with rel-atively low affmity and that the interactions act co-operativelyto stabilize the complex.

An additional weak interaction is detectable between freeclass I heavy chains and TAP-tapasin complexes in the absenceof P^m, i,e, in Daudi cells (36, 37). This was originally missedin experiments using conventional metabolic labeling and co-immunoprecipitation analysis (16, 21), probably because tbetechnique is not sensitive enough. More sensitive approaches,involving immunoprecipitation with antisera to TAP 1 followedby Western blotting with anti-class I heavy chain antibodies(36), or immunoprecipitation of radiolabeled TAP complexesfollowed by SDS denaturation and reprecipitation of associatedclass I heavy chains (37), are required. The level of associationis approximately 5% of that seen in in wild-type cells or inDaudi cells transfected with p;m. Tbe conformation of tbe sub-set of class I beavy chains associated with the TAP-tapasin com-plex may be like that of p^m-associated beavy chains but lessstable. Data from Townsend and co-workers suggested a num-ber of years ago that free D'' heavy chains can bind peptide inthe absence of pjm, but that the interaction is of low affinity,probably because of the inberent instability of the peptidebinding groove without ji m (38).

A number of mutations in the class I heavy chain have beenshown to reduce tbe level of class I-TAP association. Alterationsof residue 13 4 in the a2 domain of HLA-A2, residue 2 2 7 in thea3 domain of H2-L' , and residue 222 or residues 219-233 ofthe a3 domain of H2-Dt' have all been shown to sharply reducethe association of these molecules with TAP-tapasin complexes(39-44). Thus, regions in the al and a3 domains are in:ipli-cated in the interaction, as well as the N-linked giycan asdefmed by tbe castanospermine experiments described above.It seems likely that the N-linked glycan is interacting witb cal-reticulin, but because of tbe number of components involvedin the class I-TAP complex, and the co-operative nature of itsassembly, it is not clear with which component of the complex

24 Immunologicol Reviews 172/1999


the mutated regions of the class I a2 and a3 domains are inter-acting. Tapasin may be the most likely possibility.

The region of tapasin which interacts with class I mole-cules appears to involve the N-terminus (37). Expression of amutant tapasin lacking the N-terminal 50 amino acids in.220.B8 cells failed to mediate the class I-TAP interaction.Although it seems likely, one cannot definitively conclude thatthe class I-tapasin interaction site resides within the N-termi-nal 50 amino acids because such a deletion could result in mis-folding. However, expression of progressively N-terminallytruncated tapasin in .220.B8 cells showed that even a mutantmissing the N-terminal 300 amino acids (out of a total of 428)could associate with TAP (3 7). Such mutants were also as capa-ble as wild-type tapasin of restoring TAP levels to normal. Inter-estingly, the tapasin mutation in .220 results in a splicing defectwhich generates a tapasin variant lacking the C-terminal por-tion of the signal sequence and the N-terminal 49 residues ofthe mature protein (45). The small amount of mutant tapasinwhich is successfully translocated into the ER binds to TAP asdo the in vitro generated truncated tapasin mutants, but isunlikely to bind to class I molecules.

The function of the class I-TAP complex

When the association of MHC class I molecules with TAP wasfirst observed it was natural to assume that the interaction hadevolved to facihtate peptide loading. At a minimum one mightassume that the proximity of class I molecules to TAP wouldenhance peptide binding by minimizing the distance peptideswould have to diffuse in the ER before encountering a class Imolecule. Evidence favoring this came from the observationthat in .220 cells, which lack a functional tapasin molecule,class I-restricted peptide loading is inhibited. Thus, .220 cellstransfected with genes encoding HLA-Al, B8 or B4402 do notexpress their products well on the cell surface, consistent withpoor peptide loading. Also, recognition of virally infected.220.A1 or .220.B8 cells by Al- or B8-restricted virus-specificCD8+ T cells is impaired (31, 46). However, the simple ideathat proximity evolved to limit peptide diffusion was calledinto question by the observation that a soluble mutant versionof tapasin, which failed to bind detectably to TAP but did bindto class I molecules, could restore both surface B8 expressionand CD8" T-cell recognition when expressed in .220.B8 cells(34). HLA-B8 molecules failed to co-precipitate with TAP aftersolubiiization in digitonin, and the restoration of normal TAPlevels seen with intact tapasin was not observed. However, theabsence of evidence for an interaction is not evidence for itsabsence, and it remains possible that soluble tapasin mediates

class I-TAP association but it is disrupted upon solubiiization.The transmembrane/cytoplasmic tail region may be separatelyimportant for stabilizing TAP expression. Restoration of normalTAP levels by the N-terminally truncated tapasin mutants failedto restore class I surface expression (37), indicating that theclass I-tapasin interaction is functionally more important thanthe tapasin-TAP interaction.

Why is the MHC class I-tapasin interaction so important?One likely answer is that tapasin stabilizes class I-pim dimers inthe absence of peptides. In pulse-chase experiments, class I-p^mdimers were found to be less stable in .220 cells than in TAP-negative .174 cells (47), although they are ultimately degradedin both cell types. Thus, tapasin may be regarded as a class I-spe-cific chaperone, perhaps mediating proper folding, but certainlyprotecting it from degradation and prolonging its peptide bind-ing capacity Some alleles, e.g. HLA-B2705, seem less dependenton tapasin for surface expression (46). They may be more stableas empty class I-pim dimers in the ER.

It is unknown whether tapasin has an additional, moredirect role in peptide binding. Tapasin has been found to bindcovalently, upon irradiation by ultraviolet hght, to photoacti-vatable peptide derivatives translocated by TAP into the ER,leading to the suggestion that it might be an intermediate inpeptide loading (48). However, this remains uncertain. Byanalogy with the MHC class II system, tapasin may function likethe invariant chain, which occupies the class II binding grooveand stabilizes the class II molecule (49, 50), and/or like HLA-DM, which catalyses the exchange of peptides by class II mole-cules, in part by stabilizing them in the absence of associatedpeptides (51, 52).

The role of calreticulin and ERp57 in the class I-TAP com-plex is also tinclear. Calreticulin seems to be important for initi-ating class I-TAP/tapasin association, based on the inhibitory ef-fects of castanospermine (21). The interaction is maintained af-ter assembly of the class I-TAP/tapasin complex, and a majorquestion is whether this is functionally significant. Castanosper-mine also inhibits the release of the heavy chain from TAP (22).Therefore, the deglucosy la tion/reglu cosy lation process de-scribed earlier may be ongoing in the class I-TAP complex. ClassI molecules could disassociate from the complex after degluco-sylation and, if they are not occupied by a peptide, calreticulinmight mediate their reassociation following their reglucosylationby the UDP-glucosyl glycoprotein transferase described above.The transferase may discriminate between peptide-occupied andpepdde-free class I molecules molecules in the same way it dis-criminates between native and non-native forms of other glyco-proteins. Reiteration of this process could 'edit' peptide affinity,as HLA-DM does for MHC class II molecules.

Immunoiogical Reviews 172/1999 25


The continued association of ERp57 with the class I-TAP

complex (23-25) is similarly mysterious. The available evi-

dence suggests that disulfide bond formation in MHC class I

molecules is complete after calnexin dissociation and calreticu-

lin association (29). The prolonged interaction of ERp57 after

this stage may indicate that one of the disulfide bonds in the

class I molecule is subjected to repeated reduction/oxidation

reactions during its association with the complex. Perhaps the

most intriguing possibility is that the bond which connects the

al a-helix to the floor of the binding groove (cys 101 -cys 164)

(Fig. 2) is reversibly reduced and that this process provides a

mechanism for regulating the affinity of peptide binding. If

associated peptide binds with high affinity it would be difficult

to reduce the bond because of strong non-covalent interactions

within the binding groove, whereas a low affinity peptide

might readily permit reduction. MHC class I molecules with

mutations in either cys 101 or cys 164 are poorly expressed on

the cell surface, implying that their ability to bind peptides is

impaired (44).

Clearly there are many unanswered questions regarding the

mechanisms governing peptide binding to MHC class I mole-

cules in the ER. On one level the process appears to represent

an extremely complex folding pathway, with many elements in

common with the assembly of other multisubunit glycopro-

teins. On another it is a carefully regulated and specialized sys-

tem ensuring the surface expression of high affinity

class I-peptide complexes essential for CD8-positive T-cell

a

Fig. 2. A schematic view of the MHC class I peptide binding groovewith the cysteine pair connecting the a2 domain a-helix to the floorof the binding groove shown in yellow. Isomerization of this disulfidebond, mediated by ERp57, may play a role in regulating peptide binding.

immunity The ideas presented above require considerable test-

ing, and the complete rationale for the existence of the MHC

class I-TAP complex, with all of its varied components,

remains to be uncovered.

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The nature of the MHC class I peptide loading complex