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Substrates for Class I Processing and Misfolded Full-Length Proteins Are EfficientEndoplasmic Reticulum Demonstrates That Regulated Folding of Tyrosinase in the

EngelhardMarina Ostankovitch, Valentina Robila and Victor H.

http://www.jimmunol.org/content/174/5/2544doi: 10.4049/jimmunol.174.5.2544

2005; 174:2544-2551; ;J Immunol 

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Print ISSN: 0022-1767 Online ISSN: 1550-6606. Immunologists All rights reserved.Copyright © 2005 by The American Association of1451 Rockville Pike, Suite 650, Rockville, MD 20852The American Association of Immunologists, Inc.,

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Regulated Folding of Tyrosinase in the Endoplasmic ReticulumDemonstrates That Misfolded Full-Length Proteins AreEfficient Substrates for Class I Processing and Presentation1

Marina Ostankovitch, Valentina Robila, and Victor H. Engelhard2

Short-lived protein translation products have been proposed to be the principal substrates that enter the class I MHC processingand presentation pathway. However, the biochemical nature of these substrates is poorly defined. Whether the major processingsubstrates are misfolded full-length proteins, or alternatively, aberrantly initiated or truncated polypeptides still remains to beaddressed. To examine this, we used melanoma in which one-third of wild-type tyrosinase molecules were correctly folded andlocalized beyond the Golgi, while the remainder were present in the endoplasmic reticulum in an unfolded/misfolded state.Increasing the efficiency of tyrosinase folding using chemical chaperones led to a reduction in the level of substrate available tothe proteasome and decreased the expression of a tyrosinase-derived epitope. Conversely, in transfectants expressing tyrosinasemutants that are completely misfolded, both proteasome substrate and epitope presentation were significantly enhanced. Protea-some substrate availability was a consequence of misfolding and not simply due to retention in the endoplasmic reticulum. Thus,the extent of folding/misfolding of a full-length protein is an important determinant of the level of epitope presentation. TheJournal of Immunology, 2005, 174: 2544–2551.

T he nature of the protein substrates that enter the class IMHC Ag processing pathway has been the subject of in-tense investigation over several years. A significant per-

centage of cellular proteins with masses greater than 45 kDa isdegraded by the proteasome within 60 min of synthesis (1). Newlysynthesized proteins have also been shown to be an importantsource of substrates for the TAP (2) and to be the source of onevirally derived epitope (3). It has been proposed that these newlysynthesized and rapidly degraded proteins or peptides could cor-respond to defective protein translation products that were aber-rantly initiated or prematurely terminated (4). In support of this,several epitopes have been identified as short polypeptides trans-lated from alternative reading frames, some of which span exon-intron boundaries or lie entirely within introns, and others of whichresult from the use of nonstandard initiation codons (summarizedin Ref. 5). It has also been shown that the level of epitope pre-sentation is enhanced using truncated forms of a protein (6). How-ever, it is not clear what fraction of the total protein economy ofthe cell they represent.

An alternative source of rapidly degraded substrates for class IAg processing is full-length proteins that are sampled: 1) after theybecome terminally misfolded; 2) while they are still in the processof folding; or 3) because they are short-lived based on N-end ruleor other sequences that directly target them for degradation (7–9).Substitution of the N-terminal residue of proteins with N-end de-

stabilizing residues, such as Arg, decreases the t1/2 of proteins byincreasing the level of ubiquination, which in turn augments in-teraction with and degradation by the proteasome (10). In severalstudies, this maneuver led to an increased level of epitope presen-tation to T cells (11–15), while in others it had no effect (11, 16).However, because ubiquination is dependent on the N-terminalresidue rather than protein-folding state, these studies did not ad-dress the importance of protein-folding state as a determinant ofclass I Ag presentation. The insertion of a sequence consistinglargely of repeated Lys and Glu residues (KEKE) has also beenshown to favor proteasome degradation and epitope presentation(15, 17). Although it has been proposed that the KEKE sequencedirectly augments interaction with the proteasome (18) or that itinduces misfolding (15, 17), no definitive data distinguishing thesepossibilities have been published. Interestingly, however, thiswork also demonstrated that a protein with an intermediate rate ofdegradation generated class I MHC-restricted epitopes more effi-ciently than a more rapidly degraded form (15), and also demon-strated the apparent existence of two distinct pools of substrates,giving rise to epitopes based on kinetic measurements. However,the nature of these substrates was not directly examined in this orother work performed to date.

Taken together, these studies demonstrate that enhanced target-ing of model protein Ags to the proteasome frequently augmentsthe expression of class I-restricted epitopes, but may also make nodifference or actually cause diminished expression. They also sug-gest that this could depend on the exact nature of the protein sub-strate for degradation, of which little is directly known. Indeed,while there is strong evidence that newly synthesized translationproducts are a significant source of class I MHC-restricted epitopesin virally infected cells (1–3, 15), there are little direct data thatsupport this hypothesis for epitopes derived from stably expressedendogenous proteins (2). As has been pointed out elsewhere, it stillremains to be established that full-length proteins that are eitherterminally misfolded or in the process of folding are significantsubstrates for the class I Ag processing pathway (4, 5). It is also

Carter Immunology Center and Department of Microbiology, University of Virginia,Charlottesville, VA 22908

Received for publication August 18, 2004. Accepted for publication December3, 2004.

The costs of publication of this article were defrayed in part by the payment of pagecharges. This article must therefore be hereby marked advertisement in accordancewith 18 U.S.C. Section 1734 solely to indicate this fact.1 This work was supported by U.S. Public Health Service Grants AI20963 andAI33134 to V.H.E.2 Address correspondence and reprint requests to Dr. Victor H. Engelhard, BeirneCarter Center for Immunology Research, Box 801386, University of Virginia Schoolof Medicine, Charlottesville, VA 22908. E- mail address: vhe@virginia.edu

The Journal of Immunology

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not clear whether such proteins would be sampled after terminalmisfolding or at early stages during the folding process.

One meaning to assess the impact of protein folding on the levelof epitope presentation is to compare wild-type (WT)3 proteinswith naturally occurring mutant forms that are known to misfold,or to alter other conditions that selectively affect the folding ofindividual proteins within cells. Tyrosinase, which represents arobust source of class I MHC-restricted peptides recognized bymelanoma-specific T cells (19), offers a useful model for this eval-uation. Tyrosinase is a membrane-associated N-linked glycopro-tein that is synthesized and folds in the endoplasmic reticulum(ER), and is subsequently transported via the trans Golgi networkto melanosomes (20, 21). Many natural variants of tyrosinase as-sociated with type I oculocutaneous albinism fail to fold (22–25),and unfolded or misfolded WT tyrosinase molecules frequentlyaccumulate in melanoma cells as a consequence of abnormal cel-lular acidification (26, 27). These unfolded/misfolded mutant ty-rosinase or WT tyrosinase molecules are completely retained in theER through prolonged interaction with the ER chaperones calnexinand calreticulin rather than exiting to melanosomes, and are sub-sequently retrotranslocated to the cytosol and degraded by the pro-teasome (20, 24, 26–29). In contrast, the tyrosinase substrates L-dopamine (L-Dopa) and L-tyrosine act as chemical chaperones toenhance the folding of WT tyrosinase molecules and their exportfrom the ER to the Golgi (23). Work from our laboratory estab-lished that the Tyr369 epitope presented by HLA-A*0201 was pro-duced after retrotranslocation of tyrosinase from the lumen of theER to the cytosol, degradation by the proteasome, and reimport ofpeptides into the ER by TAP (28, 30). In this study, we used WTtyrosinase molecules and tyrosinase mutants that have been dem-onstrated to fail to fold (23–26, 31), together with chemical chap-erones, to evaluate the impact of folding of full-length moleculeson the processing and presentation of the Tyr369 epitope.

Materials and MethodsTyrosinase gene constructs and transfectants

The WT and R402Q tyrosinase genes (gift from M. Marks, University ofPennsylvania, Philadelphia, PA) were subcloned in the vector pEF6 (In-vitrogen Life Technologies). The A206T mutation was introduced intopEF6 WT tyrosinase using the QuickChange XL site-directed mutagenesiskit (Stratagene). The melanoma cell line DM331 (tyrosinase�, HLA-A*0201�) (32) was transfected with plasmids encoding WT, R402Q, orA206T tyrosinase using Fugene (Roche Diagnostics). Bulk stable trans-fectant lines were generated by selection with blasticidin (10 �g/ml) (In-vitrogen Life Technologies) and cloned. All experiments using stable trans-fectants were conducted on cells plated at 5000 cells/cm2 and grown for 3days. Folding of tyrosinase was induced by incubation of cells for 3 daysin medium supplemented with 20 �M L-Dopa (Sigma-Aldrich) or 400 �ML-tyrosine (Sigma-Aldrich). To ensure that the pH of the medium and theconcentration of L-Dopa remain the same during the 3-day experiment, themedium and medium supplemented with L-Dopa or L-tyrosine were re-placed every day. Retention of tyrosinase in the ER was induced in mediumsupplemented with 5 �g/ml brefeldin A (BFA) (Sigma-Aldrich) or by in-cubation at 20°C for 4 h. For experiments analyzing proteasome degrada-tion, transfectants were incubated in presence of 50 �M N-acetyl-Leu-Leu-norleucinal (LLnL) (Calbiochem) for 3.5–4 h.

Cytofluorometry

Expression of tyrosinase was measured by intracellular staining after per-meabilization for 30 min with BD Perm/wash (BD Pharmingen) and se-quential addition of C19 (goat anti-tyrosinase; Santa Cruz Biotechnology),biotinylated donkey anti-goat IgG (Santa Cruz Biotechnology), and allo-phycocyanin-conjugated streptavidin (BD Pharmingen). Expression ofHLA-A*0201 was measured by surface staining using the mAb CR11-351

and FITC-conjugated goat anti-mouse IgG (Jackson ImmunoResearchLaboratories). Data were acquired on a FACS and analyzed usingCellQuest software (BD Pharmingen).

Immunoblotting and analysis of maturation of N-linked glycans

Cell pellets (106 cells) were solubilized in 100 �l of buffer containing 10mM Tris-HCl, pH 7.5, 0.5% deoxycholate, 1% Igepal, 5 mM EDTA, 4 mMPMSF, 10 �g/ml aprotinin, 10 �M pepstatin A, 10 �g/ml leupeptin, and100 �M iodoacetamide, and centrifuged at 21,000 � g for 30 min at 4°C.For analysis of maturation of N-glycans, proteins from 50 �l of supernatantwere precipitated with 400 �l of cold acetone at �20°C overnight. Theprecipitates were resuspended in 0.5% SDS and 0.1 M 2-ME and boiled for5 min. After cooling on ice for 2 min, the samples were digested with 2.5mU endoglycosidase H (Endo H; Roche Diagnostics), according to themanufacturer’s recommendations. Alternatively, proteins from supernatantwere directly boiled in sample buffer for 5 min. A total of 5 � 105 cellequivalents per lane was separated on 10% SDS-PAGE gels, transferred toNitroPure nitrocellulose (Osmonics), and blocked in 5% nonfat dried milkin PBS with 0.05% Tween 20. Blots were probed overnight with C19 goatanti-tyrosinase, washed, probed with HRP-conjugated donkey anti-goatIgG, and developed according to the Amersham ECL protocol (AmershamBiosciences). Films were scanned using Scan Maker III (Microtek), andimages were processed using Adobe Photoshop software. Bands werequantified using NIH Image 1.62f software.

Immunofluorescence microscopy

Transfectants were grown on coverslips, washed with PBS, fixed for 10min at room temperature with 4% paraformaldehyde in PBS, and perme-abilized with BD Perm/wash (BD Pharmingen). Tyrosinase was detectedeither with C19 goat (Santa Cruz Biotechnology) or T3.11 mouse anti-tyrosinase Ab with similar results (25). The cells were incubated for 1 hwith combinations of anti-tyrosinase Ab, rabbit anti-calnexin (StressGenBiotechnologies), or mouse anti-lysosome-associated membrane protein(LAMP)1 (BD Pharmingen). These primary Abs were detected with Alexa488 anti-goat and Alexa 594 anti-rabbit or anti-mouse conjugates (Molec-ular Probes). Samples were mounted on glass slides with Vectashield (Vec-tor Laboratories), visualized using an Olympus confocal microscope, andprocessed with Adobe Photoshop 6.0 software.

T cell assay

Tyr369 epitope-specific T cell lines were generated from C57BL/6 miceexpressing a chimeric HLA-A*0201/H2Dd MHC class I molecule andmaintained, as described (28). Resting T cells (50,000) were incubated withthe indicated number of transfectants or Tyr369 peptide-pulsed DM331cells for 16 h, and the level of IFN-� secreted was measured by ELISA(eBiosciences).

ResultsMaturation, folding, and intracellular localization of WT andmutant forms of tyrosinase in an amelanotic melanoma cell line

To examine the importance of protein folding and subcellular lo-calization on the expression of tyrosinase-derived epitopes, wegenerated transfectants of the amelanotic melanoma cell DM331with cDNA expression vectors encoding either WT human tyrosi-nase, or the tyrosinase mutants R402Q and A206T that have beenpreviously shown to be retained in the ER in consequence of in-trinsic misfolding (31). DM331 does not express detectable tyrosi-nase based on Western blot with anti-tyrosinase Abs (32) or PCR(our unpublished observations), but does express HLA-A*0201.Stable transfectant clones expressing similar levels of each tyrosi-nase construct were identified by intracellular staining using theAb C19, which recognizes a C-terminal epitope of tyrosinase. Ty-rosinase maturation in these transfectants was evaluated by diges-tion of postnuclear supernatants of cell lysates with Endo H, sep-aration by SDS-PAGE, and immunoblotting with C19. In cellsexpressing WT tyrosinase, quantitation of total signal intensities ofthe bands by densitometry demonstrated that about one-third of themolecules were insensitive to Endo H, indicating that they hadmoved out of the ER and beyond the cis-/medial Golgi (Fig. 1A).Similar values have been obtained in five independent experimentsinvolving four different cloned transfectants (data not shown). This

3 Abbreviations used in this paper: WT, wild type; BFA, brefeldin A; Endo H, en-doglycosidase H; ER, endoplasmic reticulum; L-Dopa, L-dopamine; LAMP, lyso-some-associated membrane protein; LLnL, N-acetyl-Leu-Leu-norleucinal.

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observation is consistent with others documenting a relatively lowlevel of mature tyrosinase in melanoma cells as opposed to mela-nocytes (26). However, in agreement with previous work (23), theamount of mature tyrosinase was increased markedly by growth ofthe transfectants in medium containing L-Dopa (Fig. 1A) or ele-vated levels of L-tyrosine (data not shown). In contrast, R402Q andA206T tyrosinase molecules were completely Endo H sensitive(Fig. 1A). L-Dopa (Fig. 1A) and L-tyrosine (data not shown) did notinduce any significant maturation of A206T tyrosinase, and only avery minor increase in the maturation of R402Q. Using immuno-cytochemistry and confocal microscopy, WT tyrosinase was foundto partially colocalize with calnexin, a marker of the ER, and withLAMP1, a marker of late endosomes, lysosomes, and melano-somes (Fig. 1B). In contrast, R402Q and A206T tyrosinase mol-ecules colocalized strongly with calnexin and failed to colocalizewith LAMP1. Taken together, these observations confirmed that asignificant portion of WT tyrosinase molecules in the DM331 mel-anoma transfectants had matured beyond the cis-/medial Golgi,reflecting their capacity to fold, while R402Q and A206T tyrosi-nase molecules were completely immature, retained in the ER,indicating their inability to fold.

Proteasome-dependent degradation of R402Q and A206Ttyrosinase is enhanced compared with WT tyrosinase

We next evaluated whether unfolded/misfolded R402Q andA206T full-length tyrosinase molecules were preferential sub-strates for the proteasome in comparison with WT tyrosinase. Todo so, we quantified the amounts of degradation intermediates ac-cumulating after incubation of the transfectants with the protea-some inhibitor LLnL. Treatment with 100 �M LLnL for 4 h,which we have previously shown to inhibit 90–97% of differentproteasome activities (33), induced the accumulation of �60-kDatyrosinase-derived bands in WT, R402Q, and A206T tyrosinasetransfectants (Fig. 2). These bands have previously been shown tocorrespond to either unglycosylated and full-length (26, 29) orglycosylated and partially proteolyzed (29) tyrosinase molecules,many of which have been retrotranslocated to the cytosol (28, 29).Bands of the same mass are stabilized by LLnL (26, 28, 29), lac-tacystin (26, 29), MG132 (26), and epoxomycin (our unpublished

results), and are apparently generated by a protease distinct fromthe proteasome. Quantitation by scanning densitometry establishedthat in presence of LLnL, the relative amounts of the �60-kDabands compared with those of full-length tyrosinase were 2.5- to3-fold higher for R402Q and A206T tyrosinase than for WT ty-rosinase. These results demonstrated that the amount of R402Qand A206T tyrosinase available to and degraded by the proteasomeover 4 h is greater than for WT tyrosinase.

Tyr369 epitope expression levels on melanoma transfectantsexpressing equivalent levels of full-length WT, R402Q, A206Ttyrosinase

To evaluate whether intrinsically unfolded/misfolded full-lengthtyrosinase molecules were a preferential proteasome substrate for

FIGURE 2. Proteasome degradation substrates from R402Q and A206Ttyrosinase are present at relatively higher levels than those from WT tyrosi-nase. Cloned transfectants of DM331 melanoma cells stably expressing WT,R402Q, or A206T tyrosinase were incubated in presence or absence of LLnLfor 4 h. Cell lysates were analyzed by SDS-PAGE and immunoblotted with theC19 anti-tyrosinase Ab. Films were exposed for 0.5–1 min to detect the broadbands centered at �70 kDa, which represent immature and mature intact formsof tyrosinase, and for 3 min to detect the bands at �60 kDa, which representproteasome-sensitive deglycosylated or partially proteolyzed tyrosinase mol-ecules. Numbers below each band represent the total signal intensities as quan-tified by scanning densitometry density, and the ratio of the intensities of the�60- to 70-kDa species is indicated. Results represented in this figure and Fig.1A are obtained from experiments conducted in parallel and correspond to thesame aliquots of melanoma cells.

FIGURE 1. A significant fraction of WT tyrosinase is mature and localized in late endosomes, whereas R402Q and A206T tyrosinase are immature andretained in the ER. A, Cloned transfectants of DM331 melanoma cells stably expressing WT, R402Q, or A206T tyrosinase were grown for 3 days in thepresence or absence of L-Dopa, lysed, digested with Endo H, analyzed by SDS-PAGE, and immunoblotted with the anti-tyrosinase Ab C19. EndoH-insensitive mature tyrosinase migrates at �70 kDa, while Endo H-sensitive immature tyrosinase migrates at �60 kDa. The membrane was then strippedand immunoblotted with anti-calnexin. Numbers representing the total signal intensities as quantified by scanning densitometry density and correspondingto mature and immature WT tyrosinase are indicated. Results represented in A and Figs. 2, 5, and 6, experiment 2, were obtained from experimentsconducted in parallel and using duplicate aliquots of melanoma cells. B, Immunofluorescence and confocal microscopy of cloned transfectants of DM331melanoma cells stably expressing WT, R402Q, or A206T tyrosinase using anti-tyrosinase and anti-calnexin or anti-LAMP1.

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epitope presentation, we next evaluated the expression levels of theHLA-A*0201-restricted Tyr369 epitope on the different DM331melanoma transfectants by measuring their ability to stimulateIFN-� secretion from Tyr369-specific T cell lines. First, we estab-

lished that the assay conditions would accurately reflect differencesin epitope expression on transfectants expressing different levels ofeither WT tyrosinase or HLA-A*0201. Using melanoma transfec-tants that expressed similar levels of HLA-A*0201 molecules, weobserved a correlation between the tyrosinase expression level indifferent transfectants and the secretion of IFN-� (Fig. 3A, Expt.1). Similarly, in transfectants expressing similar levels of tyrosi-nase, we observed a correlation between IFN-� secretion and thelevel of HLA-A*0201 expression (Fig. 3A, Expt. 2). In parallelexperiments, a 2-fold difference in the secretion of IFN-� wascorrelated to a 2-log difference in the concentration of Tyr369 syn-thetic peptide used to pulse untransfected DM331 melanoma cells(Fig. 3B). Finally, we demonstrated that incubation of transfectantsexpressing WT tyrosinase with exogenous Tyr369 synthetic peptideled to a further increase in IFN-� secretion (Fig. 3C). These ex-periments showed that this assay was sensitive to quantitative vari-ations in Tyr369 epitope expression resulting from either tyrosi-nase, HLA-A*0201, or exogenous peptide expression differences.

We next compared the presentation levels of the Tyr369 epitopeby transfectants expressing WT, R402Q, and A206T tyrosinasemolecules. Using short-term bulk populations in which the meanlevels of tyrosinase expression and the percentages of tyrosinase�

cells were similar, cells expressing R402Q tyrosinase stimulatedthe release of 1.8-fold more IFN-� from Tyr369-specific T cellsthan did cells expressing the WT molecule (Fig. 4, Expt. 1). Sim-ilarly, when two different Tyr369-specific T cell lines were stimu-lated with long-term cloned transfectants that expressed uniformand comparable levels of WT and R402Q tyrosinase, the cellsexpressing R402Q stimulated the release of 1.8- to 2.1-fold moreIFN-� (Fig. 4, Expt. 2). This numerical range has been observed infive different experiments involving three distinct cloned transfec-tants expressing WT or R402Q tyrosinase molecules. Finally, atransfectant expressing A206T tyrosinase still stimulated a 1.6-foldhigher level of IFN-� secretion than did a transfectant expressingalmost one-third more WT tyrosinase (Fig. 4, Expt. 3). In threeindependent experiments involving the same transfectant clones,the release of IFN-� was 1.4- to 3.5-fold higher in the presence ofthe transfectant clone expressing A206T than in presence of theclone expressing WT molecules, with a mean of 2.2-fold. Collec-tively, the results of Figs. 2–4 indicate that the expression of un-folded or misfolded tyrosinase is associated with enhanced degra-dation by the proteasome and leads to a significantly augmentedpresentation of the processed Tyr369 epitope at the cell surface.

Folding, maturation, and export of WT tyrosinase from the ERdiminish tyrosinase processing and presentation of Tyr369

As shown in Fig. 1 and elsewhere (23), growth of melanoma cellswith the tyrosinase substrates L-Dopa or L-tyrosine enhanced fold-ing and exit from the ER of WT tyrosinase, while having a min-imal effect on the tyrosinase folding mutants. Consistent with this,growth of WT tyrosinase transfectants in L-Dopa for 3 days sub-stantially reduced the relative amount of 60-kDa tyrosinase deg-radation intermediates that could be detected after a 4-h incubationof the cells with the proteasome inhibitor LLnL (Fig. 5, lanes 2 and4). In contrast, the relative amount of 60-kDa tyrosinase fragmentswas actually somewhat higher in A206T transfectants treated withLLnL and L-Dopa compared with those treated with LLnL alone(Fig. 5, lanes 6 and 8). We therefore tested the hypothesis thataddition of L-Dopa would lead to a reduction in the processing andpresentation of Tyr369 from WT tyrosinase, but not from A206Ttyrosinase. In three independent experiments, two of which areshown in Fig. 6A, we observed a significant decrease in the pre-sentation of Tyr369 by transfectant expressing WT tyrosinase after

FIGURE 3. The level of expression of WT tyrosinase or HLA-A*0201determines the level of presentation of Tyr369. A, Top panels, Expression ofWT tyrosinase in three independent clones of transfected DM331 was mea-sured by intracellular staining with anti-tyrosinase and flow cytometry.Thin black line, WTcl1a or WTcl1b; thin gray line, WTcl2; thick line,WTcl8a or WTcl8b; gray fill, untransfected DM331. The arithmetic meanfluorescence intensity (MFI) of each distribution after subtraction of theDM331 value is shown in the second row of panels. Middle panels, Ex-pression of HLA-A*0201 in the same clones was measured by surfacestaining with anti-HLA-A*0201 and flow cytometry. Bottom panels, Thepresentation of Tyr369 was evaluated as the level of IFN-� secreted byTyr369-specific T cell lines after incubation with the indicated DM331transfectant clones. B, T cell recognition of DM331 transfectant clonesexpressing different levels of WT tyrosinase and the same number of un-transfected DM331 cells pulsed overnight with different concentrations ofexogenous Tyr369 peptide was evaluated by IFN-� secretion. C, T cellrecognition of untransfected DM331 cells and a DM331 transfectant ex-pressing WT tyrosinase before or after overnight pulsing with 1 �M Tyr369

peptide.

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growth in L-Dopa or L-tyrosine. However, no significant change inthe presentation of Tyr369 by a transfectant expressing A206T ty-rosinase was detected under the same conditions. The difference inthe effect of L-Dopa on WT and A206T presentation of Tyr369

from WT and A206T tyrosinase was statistically significant ( p �0.005, Fig. 6B). Altogether, these results demonstrate that in-creased folding and export of tyrosinase from the ER to the Golgidecreased the fraction of tyrosinase molecules undergoing degra-dation and also decreased the magnitude of Tyr369 epitopepresentation.

Proteasome-dependent degradation of tyrosinase is increased bymisfolding regardless of ER retention and by ER retentionseparately of intrinsic misfolding

The results above suggested that the failure of tyrosinase to fold inthe ER led directly to an increase in epitope expression based onenhanced interaction with the ER quality control system. However,they did not exclude the possibility that simple enhanced repre-sentation of the protein in the ER, regardless of its folding state,might also lead to enhanced processing and presentation. To assessthe impact of misfolding on tyrosinase processing independent ofdifferences in ER retention, we used BFA (34) or incubation ofcells at 20°C (35, 36) to block anterograde transport of either WTor R402Q tyrosinase from the ER to the Golgi, and compared theaccumulation of tyrosinase degradation intermediates after inhibi-tion of proteasome function with LLnL. In BFA-treated cells, wefound that the relative amount of 60-kDa tyrosinase degradationintermediates for R402Q tyrosinase was still �2.5-fold higher thanthat for WT tyrosinase (Fig. 7). A similar result was obtained afterincubation of cells at 20°C. This demonstrates that the extent oftyrosinase misfolding determines retrotranslocation from the ERindependently of its impact on retention in the ER. In addition, weobserved that the relative amount of the 60-kDa degradation in-termediates from WT tyrosinase was 3-fold higher in presence ofBFA and LLnL than in presence of LLnL alone, while, as ex-pected, the addition of BFA in the presence of LLnL only slightlyincreased the amount of 60-kDa tyrosinase degradation interme-diates for R402Q tyrosinase (Fig. 7). This suggested that eitherproperly folded WT tyrosinase was a substrate for retrotransloca-tion because of its retention, or residence in the ER led to anunfolding of properly folded molecules. Therefore, while we dem-onstrated that the extent of tyrosinase misfolding influences ex-traction from the ER independently of localization in the ER, wealso provide evidence that retention in the ER independently ofintrinsic misfolding favors exit from the ER and degradation by theproteasome.

??

FIGURE 4. Presentation of Tyr369 from R402Q and A206T tyrosinaseis enhanced compared with that of WT tyrosinase. Analyses were con-ducted using bulk populations (Expt. 1) or clones (Expts. 2 and 3) ofDM331 transfectants expressing WT, R402Q, or A206T tyrosinase. Upperpanels, Expression of tyrosinase was measured by intracellular staining andflow cytometry (thin line, WT tyrosinase transfectant or clone; thick line,R402Q in Expts. 1 and 2 and A206T in Expt. 3; gray fill, untransfectedDM331). The background fluorescence observed for DM331 has been sub-tracted in the bar graphs of MFI. Middle panels, Expression of HLA-A*0201 was measured by surface staining with anti-HLA-A*0201 andflow cytometry. Bottom panels, The presentation of Tyr369 was evaluatedas the level of IFN-� secreted by Tyr369-specific T cell lines after in-cubation with transfectants. In experiment 1, DM331 cells were trans-fected with either WT or R402Q tyrosinase, and 100,000 or 30,000short-term bulk transfectants in which the percentages of tyrosinase�

cells were similar were incubated with tyrosinase-specific T cell line 2.In experiments 2 and 3, 5,000 cloned transfectants were incubated withspecific T cell lines 1 or 2, and the levels of secretion of IFN-� shownare after subtraction of the background level of IFN-� secreted in pres-ence of untransfected DM331.

FIGURE 5. Induction of folding of WT tyrosinase with chemical chap-erones decreases the amount of proteasome degradation substrates. Clonedtransfectants of DM331 melanoma cells stably expressing WT or A206Ttyrosinase were grown for 3 days in the presence or absence of L-Dopa, andthen further incubated for 4 h in the presence or absence of LLnL. Celllysates were analyzed by SDS-PAGE and immunoblotted with anti-tyrosinase. Films were exposed for 0.5–1 min to detect the broad bandscentered at �70 kDa, which represent immature and mature intact forms oftyrosinase, and for 3 min to detect the bands at �60 kDa, which representproteasome-sensitive deglycosylated or partially proteolyzed tyrosinasemolecules. Numbers below each band represent the total signal intensitiesas quantified by scanning densitometry density, and the ratio of the inten-sities of the �60- to 70-kDa species is indicated. Results shown in thisfigure and Fig. 1A are obtained from experiments conducted in parallel andcorrespond to the same aliquots of melanoma cells.

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DiscussionShort-lived proteins and defective protein translation products,which include aberrantly initiated, prematurely terminated, and un-folded or misfolded full-length proteins, have been proposed to bethe principal substrates that enter the class I MHC processing andpresentation pathway (1–3, 15). However, it is not clear to whatextent proteasome substrates are represented by aberrant transla-tion products that are inherently unable to fold, or alternatively,full-length proteins that are targeted for degradation either becauseof a conformational abnormality, because they take a longer timeto fold properly, or because they contain signals that confer a shortt1/2 (4). Many studies have shown that Ag presentation can bealtered by the introduction of sequences that enhance proteasome

targeting without regard for protein folding state, or by the creationof truncated proteins (6, 11–15). However, no study to date hasdirectly evaluated the impact of protein folding on the efficiency ofAg processing and presentation, nor evaluated whether full-lengthproteins, as opposed to aberrant translation products, are substratesfor processing (4, 5).

Using tyrosinase as a model, we have now provided direct in-sight into these issues. This is due in part to the fact that the C19Ab detects an epitope located in the C-terminal tail of tyrosinase,which is located on the opposite side of the membrane from thelumenal domain whose folding determines tyrosinase maturationand degradation. By using this Ab, we thus evaluate full-lengthtyrosinase molecules, as opposed to prematurely terminated or ab-errantly initiated products. In addition, the folding behavior of WTtyrosinase and the tyrosinase mutants R402Q and A206T has beenwell established in previous work. Unfolded or misfolded forms ofWT tyrosinase are commonly found in melanoma cells (26) as aconsequence of abnormal cellular acidification (27) and the ab-sence of other melanocyte differentiation proteins (37, 38). Onlyabout one-third of WT tyrosinase expressed in melanoma cellsfolds properly, while the remainder fails to fold, but can be rescuedin presence of the chemical chaperones L-Dopa or L-tyrosine (23).R402Q tyrosinase molecules are completely retained in the ER at37°C, but can be partially rescued and exported to the Golgi at lowtemperature or by addition of chemical chaperones (27, 31).A206T tyrosinase is a mutation leading to oculocutaneous albi-nism. It is completely retained in the ER through prolonged inter-action with the chaperone calnexin (24), and cannot be rescued bylow temperature incubation or chemical chaperones. Neither ofthese molecules exhibits enzymatic activity in the unfolded state,strongly suggesting that misfolding is responsible for their com-plete retention in the ER. Finally, N-glycans are attached to ty-rosinase during synthesis, undergo structural modification as aconsequence of folding and export from the ER, and are removedduring retrotranslocation as a prelude to degradation by the pro-teasome. Based on our ability to distinguish among these forms,we have shown a direct relationship between the levels of theunfolded/misfolded and reverse translocated forms of full-lengthtyrosinase and the amount of epitope presented.

FIGURE 6. Induction of folding of WT tyrosinase decreases presenta-tion of Tyr369. A, Cloned transfectants of DM331 melanoma cells stablyexpressing WT or A206T tyrosinase were grown for 3 days in the absence(�) or presence of L-Dopa (f) or L-tyrosine (u). Upper panels, Expressionof tyrosinase was measured by intracellular staining and flow cytometry.The background fluorescence observed for DM331 has been subtracted inthe bar graphs of MFI. Middle panels, Expression of HLA-A*0201 wasmeasured by surface staining and flow cytometry. ND, not done. Bottompanels, Presentation of Tyr369 was evaluated as the level of IFN-� secretedby a Tyr369-specific T cell line after incubation with cloned transfectants.The levels of secretion of IFN-� shown are after subtraction of the back-ground level of IFN-� secreted in presence of untransfected DM331. Re-sults shown in this figure, experiment 2, and Figs. 5 and 1A are obtainedfrom experiments conducted in parallel and correspond to the same ali-quots of melanoma cells. B, Percentage of inhibition of IFN-� released byTyr369-specific T cells as a consequence of melanoma cell culture in thepresence of L-Dopa, calculated from the data of A, experiments 1 and 2.The p value was calculated using two-sample Student‘s t test and Minitab13.0 software.

FIGURE 7. Proteasome substrate levels generated from WT and R402Qtyrosinase retained in the ER. Clones of transfectants expressing WT orR402Q tyrosinase were incubated at 37°C in presence or absence of BFAfor 30 min, or at 20°C, and then further incubated in the presence or ab-sence of LLnL for 3.5 h. Cell lysates were analyzed by SDS-PAGE andimmunoblotted with anti-tyrosinase. Films were exposed for 0.5–1 min todetect the broad bands centered at �70 kDa, which represent immature andmature intact forms of tyrosinase, and for 3 min to detect the bands at �60kDa, which represent proteasome-sensitive deglycosylated or partially pro-teolyzed tyrosinase molecules. Numbers below each band represent thetotal signal intensities as quantified by scanning densitometry density, andthe ratio of the intensities of the �60- to 70-kDa species is indicated.

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In the study presented in this work, we have directly assessedthe influence of misfolding/unfolding in the case of a full-lengthprotein, regardless of its impact on ubiquination, on the efficiencyof Ag processing and presentation. We showed that increasing thefolding efficiency of WT tyrosinase using chemical chaperones ledto a consequent decrease in the amount of immature WT tyrosinasein the ER and proteasome-sensitive tyrosinase degradation inter-mediates correlated with a decrease in Tyr369 presentation. Con-versely, in transfectants expressing R402Q and A206T, which areunable to fold and are completely retained in the ER, the protea-some-dependent degradation of tyrosinase molecules and the pre-sentation level of Tyr369 were significantly enhanced comparedwith transfectants expressing WT tyrosinase. To exclude the pos-sibility that the availability of tyrosinase molecules for degradationand epitope presentation was strictly a function of representation inthe ER independent of folding state, we evaluated the impact ofmisfolding on degradation after retention of R402Q and WT ty-rosinase in the ER using BFA or low temperature. Proteasomesubstrate availability was a consequence of misfolding state, andnot simply due to retention in the ER. Our results demonstrate thatthe extent of folding or misfolding of full-length protein is animportant determinant of the final level of epitope presentation.

We cannot fully exclude the possibility that prematurely termi-nated forms of tyrosinase, which we would not detect using C19,represent an additional source of Ag for class I presentation. How-ever, to the extent that these protein forms contain a full-lengthlumenal domain whose folding is augmented by L-Dopa or L-ty-rosine, and disrupted by R402Q and A206T mutations, they arefunctionally equivalent to full-length molecules in their contribu-tion to Ag processing and presentation. Conversely, we would ex-pect that aberrantly initiated tyrosinase molecules would remainlargely unfolded/misfolded, even in the presence of L-Dopa or L-tyrosine, so that the differences in epitope presentation shown inthis study would not have been observed. In addition, aberrantlyinitiated WT tyrosinase molecules would fail to enter the ER be-cause they lack a signal sequence, and would be synthesized in thecytosol. We have previously shown that cells expressing a trun-cated form of tyrosinase in the cytosol present two forms of theTyr369 epitope, one containing a genetically encoded Asn371 andthe other containing a deamidated Asp at the same position (28). Incontrast, cells expressing full-length tyrosinase express only thelatter epitope (28, 39). Collectively, these results indicate that ab-errantly initiated tyrosinase molecules are an insignificant sourceof class I MHC-restricted epitopes.

Somewhat surprisingly, we also found that enforced retention ofWT tyrosinase in the ER using BFA enhanced proteasome-depen-dent degradation independently of intrinsic misfolding, althoughthe level of proteasome substrate observed was still lower than thatarising from R402Q tyrosinase under normal conditions. BFA col-lapses the cis-/medial Golgi into the ER to create a single com-partment (34). This structure would then contain not only imma-ture unfolded or misfolded tyrosinase, but also some moleculesthat are sufficiently well folded to have moved to the Golgi. Theprocessing of immature unfolded/misfolded tyrosinase moleculesshould not be increased by enforced ER retention because theseforms are normally retained in the ER. Properly folded tyrosinasemolecules are also improbable sources for this enhanced retro-translocation and degradation. Previous studies have shown thatcorrect folded glycoproteins are not reglucosylated by the uridinediphosphate-glucose glucosyl transferase (40), and consequently,they do not interact with the calnexin/ER degradation-enhancing�-mannosidase-like protein molecular complex that has recentlybeen shown to play an important role in retrotranslocation fromthe ER (41, 42). Instead, we favor the hypothesis that the increase

in retrotranslocation and degradation observed when ER retentionis enforced results from the processing of a form of tyrosinase thatis partially folded, and has a lower, but still significant probabilityof interaction with uridine diphosphate-glucose glucosyl trans-ferase and calnexin. We hypothesize that this species is normallyable to be exported from the ER to the Golgi, but is retrotranslo-cated and degraded by the proteasome when it is forced to beretained in the ER. Tyrosinase normally complexes with and isstabilized by other melanosomal proteins (37). It is possible thattyrosinase molecules expressed in the absence of these proteins, asis the case in our transfectants, represent such partially foldedspecies.

Our work provides insights into the Ag processing of proteinssynthesized in the ER and establishes a link between quality con-trol in the ER and epitope processing. The mechanisms of Agprocessing of membrane-associated proteins have been poorly in-vestigated in comparison with cytosolic proteins, although a sig-nificant proportion of all cellular proteins is translocated into thelumen of the ER and all enveloped viruses produce excess glyco-proteins in the ER of the host cells (43).

Unfolded/Misfolded proteins that accumulate as a consequenceof cellular stress, viral infection, or cellular transformation may beimportant sources for enhanced presentation of peptides. Hypoxia,which commonly occurs in cancer cells, has been shown to pro-mote accumulation of unfolded/misfolded proteins (44). Physio-logical responses associated with the accumulation of unfoldedproteins have been shown to occur as a consequence of dysregu-lated glucose homeostasis in diabetes (45), and as a consequenceof excess protein production in cells infected with hepatitis C or Bviruses (46, 47). Other cellular insults, such as perturbation in re-dox status or in calcium homeostasis, deprivation of glucose, havebeen associated with the accumulation of unfolded/misfolded pro-teins in the ER (44). Finally, misfolding and aberrant ER retentionof tyrosinase due to dysregulation of pH homeostasis is frequentlyobserved in metastatic melanomas, which are often amelanoticeven when they express WT tyrosinase (26, 27). In the presentwork, we have shown that this leads to enhanced Ag presentation.It remains to be demonstrated that these other physiological stres-sors have a similar effect.

DisclosuresThe authors have no financial conflict of interest.

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