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The Journal of Immunology, 2006, 176: 2942-2949.
Copyright © 2006 by The American Association of Immunologists

Lack of Tyrosine 320 Impairs Spontaneous Endocytosis and Enhances Release of HLA-B27 Molecules

Susana G. Santos1,*,{dagger},{ddagger}, Antony N. Antoniou§, Paula Sampaio*, Simon J. Powis{dagger},¶ and Fernando A. Arosa2,*,{ddagger}

* Institute for Molecular and Cell Biology, Porto, Portugal; {dagger} Division of Cell Biology and Immunology, School of Life Sciences, University of Dundee, Dundee, United Kingdom; {ddagger} Instituto de Ciências Biomédicas Abel Salazar, Porto, Portugal; § Cancer Sciences Division, University of Southampton School of Medicine, Southampton General Hospital, Southampton, United Kingdom; and Bute Medical School, University of St. Andrews, St. Andrews, United Kingdom


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 Disclosures
 References
 
Several lines of evidence suggest that endocytosis of MHC class I molecules requires conserved motifs within the cytoplasmic domain. In this study, we show, in the C58 rat thymoma cell line transfected with HLA-B27 molecules, that replacement of the highly conserved tyrosine (Tyr320) in the cytoplasmic domain of HLA-B27 does not hamper cell surface expression of beta2-microglobulin H chain heterodimers or formation of misfolded molecules. However, Tyr320 replacement markedly impairs spontaneous endocytosis of HLA-B27. Although wild-type molecules are mostly internalized via endosomal compartments, Tyr320-mutated molecules remain at the plasma membrane in which partial colocalization with endogenous transferrin receptors can be observed, also impairing their endocytosis. Finally, we show that Tyr320 substitution enhances release of cleaved forms of HLA-B27 from the cell surface. These studies show for the first time that Tyr320 is most likely part of a cytoplasmic sorting motif involved in spontaneous endocytosis and shedding of MHC class I molecules.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 Disclosures
 References
 
Major histocompatibility complex class I (MHC-I)3 molecules are trimeric structures of a 44- to 49-kDa H chain, a 12-kDa L chain named beta2-microglobulin (beta2m), and a 9- to 11-aa peptide. MHC-I molecules are folded and assembled inside the endoplasmic reticulum (ER) with the aid of several chaperones and accessory molecules such as calnexin, calreticulin, tapasin, and ERp57, and are ultimately transported to the cell surface of almost all nucleated cells (1). In addition to their role in presenting peptides to CD8+ CTLs, cell surface MHC-I molecules have been implicated in receptor-mediated endocytosis (2, 3, 4).

The rate of spontaneous endocytosis of MHC-I molecules is higher in lymphoid T cells and monocytes than in B cells and can be induced by activation of protein kinase C-regulated pathways (5, 6, 7, 8, 9). Zuniga and colleagues (5) were the first to report that endocytosis of MHC-I molecules required the cytoplasmic domain and suggested that a conformational change was required for endocytosis. More recently, studies have demonstrated that MHC-I down-modulation induced by the HIV protein Nef relies upon a conserved motif containing Tyr320 located in the cytoplasmic tail (10, 11). Accordingly, Tyr320 appears to be part of a cryptic sorting signal in HLA-A and -B molecules that is exposed upon conformational changes (11). Amino acids other than Tyr320 seem to be critical for endocytosis of other classical as well as unconventional MHC-I molecules (12).

The YXX{phi} tyosine-based motif is known to be involved in endocytosis and sorting of a number of receptors, such as low-density lipoprotein and the transferin receptor (13). Although the YSQA sequence, present in the MHC-I cytoplasmic tail, is not considered to be a typical YXX{phi} tyrosine-based motif, recent work performed with mouse MHC-I molecules suggests that the cytoplasmic domain contains a tyrosine-based targeting signal required for intracellular trafficking (14). This sorting signal may permit recycling of MHC-I molecules between the cell surface and endosomal compartments, which is thought to result in peptide cross-presentation (14) or in the formation of beta2m-free or misfolded MHC-I molecules at the cell surface (15, 16). Of note, MHC-I misfolding at the cell surface is a normal event in activated normal human T cells, is associated with tyrosine phosphorylation, and allows cis interactions with cell surface receptors and signaling molecules (17, 18).

Our interest in the biology of MHC-I molecules derives from studies by several groups including ours, pointing to these molecules as cell surface receptors that undergo conformational changes (i.e., misfolding) that may underlie the regulation of basic biological processes, including receptor-mediated endocytosis and signaling mediated by cell surface receptors (2, 3, 4, 18, 19) and activation in trans of specific counter receptors (20). Significantly, expression of misfolded beta2m-free HLA-B27 H chains and their possible recognition by specific killer Ig-like receptors have been implicated with a group of arthritic diseases (20, 21). The main goal of this study was to evaluate the importance of Tyr320 in the cell surface expression, misfolding, and fate of MHC-I molecules, using HLA-B27-transfected rat thymoma C58 cells as a model.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 Disclosures
 References
 
DNA constructs B*0702 (a gift from A.-M. Little, Anthony Nolan Trust, Paisley, U.K.) and B*2705 cDNAs were cloned in vector pCR3 (Invitrogen Life Technologies). B*2705 was subjected to site-directed mutagenesis to replace Tyr320 for a phenylalanine (Y320F) using the mutation primers 5'-GGAGGGAGCTTCTCTCAGGCTGCGTGC-3' and its respective reverse complement primer. Mutagenesis was performed using QuikChange (Stratagene) methodology, and products were sequenced for confirmation.

Antibodies

The following Abs were used in this study: ME1 (a gift from T. Elliott, Southampton, U.K., and J. Taurog, Dallas, TX) is a mouse mAb that recognizes folded HLA-B molecules. HC-10 (a gift from H. Ploegh, Harvard Medical School, Boston, MA) is a mouse mAb that reacts preferentially with unfolded HLA-B and -C H chains not associated with beta2m (22). Cy3-conjugated HC-10 was a gift from S. Damjanovich (University of Debrecen, Debrecen, Hungary). Anti-HLA-B27 FITC (One Lambda) is a mAb that recognizes HLA-B27 molecules. OX-26 is a mouse mAb that recognizes the rat transferrin receptor. HRP-conjugated goat anti-mouse Igs and Alexa Fluor 488- and 568-conjugated goat anti-mouse and goat anti-rabbit were purchased from Molecular Probes. FITC-conjugated rabbit anti-mouse was obtained from DakoCytomation.

Cell lines and transfections

C58 cells, a rat thymoma cell line, were maintained in RPMI 1640 supplemented with 5% FCS and 1% penicillin/streptomycin. C58.B7, C58.B27, and C58.B27.Y320F cells were generated by electroporation of plasmid DNA with selection in 1 mg/ml G418. Human peripheral blood lymphocytes were obtained from buffy coats after centrifugation over Lymphoprep (Nycomed). PBMC were resuspended in complete RPMI 1640 medium (5% FCS, 1% penicillin/streptomycin, 1% glutamine) and either left untransfected or transfected using the human T cell nucleofector kit (Amaxa), according to the manufacturer’s instructions, in a Nucleofector (Amaxa).

Radioactive labeling, immunoprecipitation, and enzymatic treatment

Cells were labeled, and HLA-B27 molecules were isolated, as described before (23). Briefly, cells were labeled for 20 min with 7.4 Mbq Trans label (MP Biomedicals) and returned to normal medium, and samples were removed at the indicated time points. After resuspension of cell pellets in ice-cold lysis buffer (1% Triton X-100, 150 mM NaCl, 1 mM PMSF, 5 mM N-ethylmaleimide, 10 mM Tris (pH 7.5)), postnuclear spun lysates were immunoprecipitated with the indicated Abs, followed by Sepharose beads. Immune complexes were washed three times in lysis buffer and, when indicated, digested with 5 mU of endoglycosidase H (Endo-H; Roche). Samples were analyzed under reducing conditions on 8% SDS-PAGE gels and subjected to autoradiography.

Surface stability assay

Cells were harvested and surface biotinylated using 0.25 mg of EZ-link sulfo-N-hydroxysulfosuccinimide-long chain-biotin (Pierce) per 10 x 106 cells/ml PBS for 10 min at room temperature. Excess biotin was removed by washing with PBS, and cells were lysed, as described above. Lysates were incubated at the indicated temperatures for 1 h before immunoprecipitation with ME1 Abs, followed by protein G-Sepharose beads (Sigma-Aldrich) for 2 h at 4°C in an orbital shaker. Washed immunoprecipitates were boiled for 5 min in sample buffer.

Flow cytometry

Cells were stained, as previously described (17). Briefly, cells were harvested and washed twice with PBS. Staining was performed at 4°C for 30 min in staining buffer (PBS, 0.2% BSA, 0.1% sodium azide) in 96-well round-bottom plates (Greiner Bioscience), with ~0.5 x 106 cells/well. For nonconjugated primary mAbs, cells were first labeled with ME1 or HC-10, followed by F(ab')2 of rabbit anti-mouse FITC. Mouse Igs were used as negative controls to define background staining. Acquisition was made immediately after the staining, without fixation in a FACSCalibur (BD Biosciences). For each sample, 10,000–20,000 events were acquired using forward/side light scatter characteristics and analyzed using CellQuest software.

Papain cleavage

Cells were incubated with 25 U/ml papain from Carica papaya (Roche) in presence of 10 µg/ml brefeldin A for 30 min at 37°C in RPMI 1640. Where indicated, a final concentration of 100 mM cysteine was added to the reaction. Cells were then washed and reincubated at 37°C in RPMI 1640 in the presence of 10 µg/ml brefeldin A for the indicated time periods. Cells were then surface stained with HC-10 or ME1 Abs and FITC-conjugated rabbit anti-mouse as second-step Abs, as described above, acquired in a FACSCalibur, and analyzed using the CellQuest software.

Immunofluorescence microscopy

Cells were harvested, washed, and fixed in 4% formaldehyde in PBS for 10 min. Then, cells were stained with the indicated Abs in staining buffer containing 0.2% saponin for permeabilization of the cell membranes for 30 min at room temperature. Mouse and rabbit Igs were used as negative controls to define background staining. For lysosome and Golgi apparatus staining, LysoTracker Red and Bodipy TR C5-ceramide markers (Molecular Probes) were used, according to the manufacturer’s instructions. After staining, cells were resuspended in PBS, and 4',6'-diamidino-2-phenylindole (DAPI) was added to a final concentration of 0.2 mg/ml. Mowiol (Pierce) was used to mount the samples on cover slides. Deconvolution microscopy was done using a widefield optical sectioning microscope: DeltaVision Restoration Imaging System (Applied Precision) or Cell Observer System (Zeiss). For each cell, a z-series of 25–30 images at 0.325-µm intervals was captured and processed using constrained interactive deconvolution via SoftWoRx 3.0 (Applied Precision) or Axiovision 4.1 (Zeiss) software. Between 10 and 50 cells were counted in each experiment.

Microscopy endocytosis assay

Cells were harvested, washed, and cell surface stained either with unlabeled (ME1) or conjugated Abs (HLA-B27 FITC or HC-10 Cy3) in staining buffer for 30 min on ice. After washing, cells were incubated in RPMI 1640 at 37°C for different periods. After incubation at 37°C, cells were harvested, washed, and fixed in 4% formaldehyde in PBS for 10 min. Then, cells previously labeled with unlabeled Abs were stained with fluorochrome-conjugated secondary Abs in staining buffer containing 0.2% saponin, as indicated above. Wherever indicated, cells were intracellularly counterstained with dyes or Abs (e.g., OX-26), as described above. Between 10 and 50 cells were counted in each experiment.

Reversible biotinylation

Reversible biotinylation experiments were performed, as previously described (24). Briefly, cells were harvested, washed three times with ice cold PBS-calcium/magnesium (PBS, 0.1 mM CaCl2, 1 mM MgCl2), and then incubated with 0.4 mg of EZ-link sulfo-N-hydroxysulfosuccinimide-disulfide bond-biotin (Pierce) per 10 x 106 cells/ml PBS-calcium/magnesium for 30 min on ice. Excess biotin was washed out with PBS and quenched with TBS (pH 7.6) supplemented with 5% FCS. Cells were then incubated in RPMI 1640 at 37°C for the indicated periods. After the incubation, cells were washed twice with TBS and, when indicated, treated with 46 mM reduced glutathione (GSH) in reducing buffer (90 mM NaCl, 1 mM MgCl2, 0.1 mM CaCl2, 60 mM NaOH, and 5% FCS) for 30 min on ice. Cells were lysed in lysis buffer (10 mM Tris, 130 mM NaCl, 1 mM PMSF, and 1% Brij) for 30 min on ice. Cell lysates were centrifuged at 13,000 rpm for 10 min to remove cell debris and precleared for 1 h with protein A-Sepharose beads (Pharmacia). Precleared detergent lysates were immunoprecipitated with the indicated Abs, followed by Sepharose beads for 2 h at 4°C in an orbital shaker. Washed immunoprecipitates were boiled for 5 min in sample buffer.

SDS-PAGE and Western blot analysis

Samples were resolved by SDS-PAGE, and gels were blotted onto nitrocellulose membranes (Schleicher & Schuell Microscience). Membranes from biotin-labeled samples were blocked in 5% nonfat dry milk in TBS-T (TBS, 0.1% Tween 20) and incubated for 1 h with a 1/7500 dilution of streptavidin-conjugated HRP (Sigma-Aldrich) in TBS-T. In experiments in which total class I was visualized, membranes were blocked as before and then incubated with HC-10 Ab in 1% nonfat dry milk in TBS-T overnight at 4°C. After washing, membranes were incubated with goat anti-mouse HRP-conjugated secondary Abs in TBS-T, and proteins were visualized using ECL (Pierce).

Shedding of cell surface HLA-B27 molecules

Cells were cultured in 100 µl of serum-free RPMI 1640, and 20-µl aliquots of culture supernatant were harvested at the indicated time points, separated by SDS-PAGE, under reducing conditions, and blotted, as described above, for total class I visualization.

Statistical analysis

Two-tailed Student’s t test was used for analysis of differences between groups. Values of p < 0.05 were considered statistically significant.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 Disclosures
 References
 
Lack of Tyr320 does not influence steady state levels of HLA-B27 molecules

A mutant of the HLA-B27 H chain was generated by site-directed mutagenesis introducing a point mutation that replaced the conserved Tyr320 for phenylalanine (Fig. 1A). To examine whether the conserved Tyr320 was involved in assembly, maturation, and transport of HLA-B27 molecules to the cell surface, pulse-chase studies of HLA-B27 molecules stably transfected in the rat thymoma C58 cell line were performed. Immunoprecipitation with ME1 Abs in combination with Endo-H digestion revealed similar rates of assembly of both wild-type (WT) and mutant molecules (Fig. 1B). The molecules immunoprecipitated corresponded to HLA-B27 H chains because ME1 and HC-10 Abs do not cross-react with rat class I molecules (see Fig. 1D below, and data not shown). As a control, HLA-B7 molecules stably transfected into C58 cells were also immunoprecipitated with ME1 Abs, showing a slightly faster assembly kinetic and complete acquisition of Endo-H resistance after the 60-min chase (Fig. 1B). Use of HC-10 Abs, which recognize misfolded and/or partially folded HLA class I molecules, revealed that the pool of HC-10-reactive molecules staying in the ER during the course of the chase period for both WT and mutant HLA-B27 molecules was similar, even though the loss of HC-10-reactive material observed in HLA-B7-transfected cell did not take place. The relatively slow assembly kinetics of HLA-B27 molecules is in accordance with previous reports (25, 26, 27), and the differences in the signal levels for class I H chains brought down by ME1 and HC-10 Abs are the result of the much lower immunoprecipitation capacity of the ME1 Ab. Finally, cell surface thermostability studies demonstrated that both WT and mutant HLA-B27 molecules reach the cell surface and are stably folded (Fig. 1C).


Figure 1
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  		FIGURE 1. Assembly, stability, and cell surface levels of WT and mutant HLA-B27 molecules. A, Predicted cytoplasmic amino acid sequences of HLA-B27 and the HLA-B27.Y320F mutant with the conservative substitution of Tyr320 for a phenylalanine. B, C58 cells transfected with B27-WT, B27-Y320F, or B7 were metabolically labeled with Trans-label, immunoprecipitated at the indicated time points with ME1 or HC-10 Abs, and digested with Endo-H. Autoradiographs show results of pulse-chase analysis for each cell line. Endo-H-sensitive and resistant H chains are indicated. C, C58.B27, C58.B27.Y320F, and C58.B7 cells were surface biotinylated and lysed. Lysates were then incubated at the indicated temperatures for 1 h before immunoprecipitation with ME1 Abs. Samples were resolved by SDS-PAGE and blotted onto nitrocellulose filters and proteins detected using HRP-conjugated ExtrAvidin and chemoluminescence. D, Untransfected C58 cells and transfected C58.B27 and C58.B27.Y320F cells were stained with ME1 (solid line), HC-10 (dashed line), or mouse Igs (gray curve), followed by F(ab')2 of rabbit anti-mouse FITC-conjugated Abs. E, Human peripheral blood lymphocytes (PBL) transiently transfected with no DNA, B27-WT, or B27-Y320F molecules were stained with anti-HLA-B27 FITC-conjugated Abs. Histograms show cell surface expression of WT and mutant HLA-B27 molecules.

 
Tyr320 mutation does not abrogate cell surface expression of HLA-B27 molecules

To ascertain whether the levels of cell surface expression of the folded and misfolded forms of MHC-I were in any way influenced by the Tyr320 mutation, flow cytometry analysis of untransfected and stably transfected C58 cells was performed. As illustrated in Fig. 1, the level of expression of cell surface beta2m-associated HLA-B27 H chains, as detected with ME1 Abs, observed in C58 cells transfected with WT (Fig. 1D, middle histogram) or mutant (Fig. 1D, lower histogram) molecules was similar. The levels of expression of beta2m-free HLA-B27 H chains at the cell surface were also similar in B27-WT- and B27-Y320F-transfected C58 cells (Fig. 1D). Recognition of WT and mutant HLA-B27 molecules at the cell surface by ME1 or HC-10 Abs was specific because neither Ab labeled untransfected C58 cells (Fig. 1D, upper histogram), a result confirmed by immunoprecipitation studies (data not shown). Additional experiments using transiently transfected primary human peripheral blood lymphocytes showed that WT and mutant HLA-B27 molecules were also expressed at similar levels at the cell surface of normal lymphocytes (Fig. 1E). This lack of difference in the levels of cell surface expression of WT and mutant HLA-B27 molecules was observed in different PBL samples.

Tyr320 mutation does not abrogate de novo expression of HC-10-reactive molecules at the cell surface

The results shown in Fig. 1 indicated that misfolded mutant HLA-B27 molecules exist at the cell surface of transfected C58 cells. Yet, considering previous studies indicating that endocytosis is required for the appearance of misfolded class I H chains (15, 16), we wanted to ascertain further whether replacement of Tyr320 influenced re-expression of HC-10-reactive molecules at the cell surface. To that purpose, transfected C58 cells were subjected to papain cleavage to remove misfolded class I molecules present at the cell surface. In agreement with previous reports (15, 28), papain was shown to cleave preferentially HC-10-reactive B27 molecules (Fig. 2A, upper histograms) in the absence of cysteine, only significantly cleaving ME1-reactive molecules in the presence of added cysteine (Fig. 2A, compare middle and lower histograms). Accordingly, papain was used to selectively remove beta2m-free HLA-B27 H chains from the cell surface of transfected C58 cell and then allow for re-expression of HC-10-reactive molecules at the cell surface in presence of brefeldin A. As shown in Fig. 2B, recovery of HC-10 signal is readily observed upon reculture in the presence of brefeldin A both in C58 cells transfected with WT and mutant HLA-B27 molecules. Treatment with brefeldin A blocks transport of class I molecules to the cell surface, thus ruling out a possible involvement of newly synthesized molecules in the re-expression of misfolded HLA-B27 at the cell surface of C58 cells expressing tyrosine mutant molecules (28 , and data not shown).


Figure 2
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  		FIGURE 2. Re-expression of HC-10-reactive molecules at the cell surface is not affected by the tyrosine mutation. A, C58.B27 and C58.B27.Y320F cells were either left untreated (solid bold line), treated with papain alone (dashed line), or treated with papain in the presence of 100 mM cysteine (solid thin line in lower histograms). Afterward, cells were surface stained with HC-10 (upper histograms) or ME1 (middle and lower histograms) Abs, followed by F(ab')2 of rabbit anti-mouse FITC-conjugated Abs. Mouse Igs were used to define background staining (gray curve). B, C58.B27 and C58.B27.Y320F cells were treated with papain in presence of 10 µg/ml brefeldin A and then reincubated at 37°C in RPMI 1640 in the presence of brefeldin A for the indicated time periods. Cells were then surface stained with HC-10, followed by F(ab')2 of rabbit anti-mouse FITC-conjugated Abs (black line). Mouse Igs were used as control to define background staining (gray curve).

 
Lack of Tyr320 impairs spontaneous endocytosis of HLA-B27 molecules

Next, we wanted to ascertain whether replacement of Tyr320 in the cytoplasmic tail of HLA-B27 molecules influenced spontaneous endocytosis in C58 cells. Previous studies have pointed out the importance of Tyr320 in regulating endocytosis and intracellular trafficking of MHC-I molecules upon induction by the HIV viral protein Nef (10, 11). We used immunofluorescence microscopy in combination with reversible biotinylation experiments to monitor endocytosis of B27-WT and B27-Y320F molecules. As illustrated in Fig. 3A, folded B27-WT molecules were spontaneously internalized by C58 cells at 37°C, showing a clear intracellular distribution after 90 min as detected by ME1 Abs (Fig. 3A, compare a, c, and e). In marked contrast, mutant B27-Y320F molecules remained at the cell surface, showing virtually no intracellular staining (Fig. 3A, compare b, d, and f). Comparable results were observed when anti-HLA-B27 FITC-conjugated Abs were used (Fig. 3A, compare g and h; see also Fig. 4). To corroborate those results, C58 cells expressing WT or mutant HLA-B27 molecules were labeled with a cell-impermeable biotin reagent containing a disulfide bond that can be cleaved after treatment with a reducing agent such as GSH, thus allowing us to visualize only those molecules that have been endocytosed (24, 29). As depicted in Fig. 3B, spontaneous endocytosis of HLA-B27 H chains could be already observed after incubation of B27-WT-transfected C58 cells for 30 min at 37°C, but was undetectable at shorter time points (data not shown). After 90 min, a significant fraction of B27-WT molecules was endocytosed (Fig. 3B, top panel). On the contrary, no detectable endocytosis of HLA-B27 H chains was observed in B27-Y320F-transfected C58 cells even after 90 min of incubation at 37°C (Fig. 3B, bottom panel).


Figure 3
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  		FIGURE 3. MHC-I tyrosine mutant displays impaired endocytosis. A, C58.B27 and C58.B27.Y320F cells were surface labeled with ME1 Ab for 30 min on ice and then fixed, permeabilized, and stained with goat anti-mouse Alexa Fluor 488 secondary Ab (green), before (a and b) and after 30 (c and d) or 90 min (e and f) of incubation at 37°C. g and h, Show cells that were surface labeled with anti-HLA-B27 FITC-conjugated Abs, and then incubated at 37°C for 90 min. Nuclei were stained with DAPI (blue). The bar represents 2 µm. B, C58.B27 cells (top panel) and C58.B27.Y320F cells (bottom panel) were harvested and labeled with EZ-Link sulfo-NHS-SS-biotin for 30 min on ice, as described in Materials and Methods, and then incubated at 37°C for 30 or 90 min before GSH treatment. Cells were then lysed, and HLA-B27 molecules were immunoprecipitated with ME1 Abs. Samples were resolved by SDS-PAGE, and blotted onto nitrocellulose filters and proteins detected using HRP-conjugated ExtrAvidin and chemoluminescence. Immunoprecipitation from non-GHS-treated samples and samples treated with GSH at time 0 are included as controls.

 

Figure 4
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  		FIGURE 4. Endocytosis of WT and mutant HLA-B27 molecules in C58 cells. C58.B27 and C58.B27.Y320F cells were surface labeled with anti-HLA-B27 FITC Abs for 30 min on ice and then incubated at 37°C for 90 min (A and B, a and d). Afterward, cells were fixed, permeabilized, and stained either for the rat transferrin receptor, with Ox-26 Ab (A, b and e), or with LysoTracker red (B, b and e). Panels of a medial section of a representative cell depicting each fluorochrome and a merged image (A and B, c and f) in which yellow shows colocalization of HLA-B27 (green) and Ox-26 or LysoTracker (red) are presented. Nuclei were stained with DAPI (blue). The bar represents 2 µm.

 
Endocytosed HLA-B27 molecules are targeted to endosomal compartments

It has been shown previously that MHC-I molecules are endocytosed and targeted to endosomal and trans-Golgi acidic compartments (7, 8, 9, 10, 30, 31). As depicted in Fig. 3A, after 90-min incubation at 37°C, most B27-WT molecules are found in intracellular compartments. To ascertain the nature of those compartments, we performed microscopy endocytosis assays with HLA-B27 FITC Abs recognizing folded molecules and counterstained the labeled cells with markers for endosomal, lysosomal, ER, and Golgi compartments. As shown in Fig. 4A, endocytosed B27-WT molecules were mainly localized in endosomes, as indicated by colocalization with endogenous transferrin receptors (Fig. 4A, upper panels). Colocalization of a fraction of WT HLA-B27 molecules with lysosomal compartments, as determined by LysoTracker staining, was also observed (see Fig. 4Bc). Labeling with Abs against the chaperone protein disulfide isomerase (an endoplasmic reticulum marker) or with Bodipy-Texas Red ceramide (a Golgi marker) showed no noticeable colocalization with these compartments (data not shown). In agreement with results shown in Fig. 3, mutant B27-Y320F molecules were not endocytosed and remained at the cell surface of transfected C58 cells (Fig. 4, A and B, lower panels). Interestingly, some mutant HLA-B27 molecules colocalized with endogenous rat transferrin receptors at the cell surface (Fig. 4A, lower panel). Notably, this colocalization at the cell surface was associated with differences in the amount of intracellular transferrin receptor between WT and mutant C58 cells, namely a reduction of detectable intracellular transferrin receptor in the latter cells (see Fig. 4A, compare b and e). The reduction in the level of intracellular transferrin receptor in C58 cells expressing the mutant HLA-B27 molecule was not artifactual and was observed in >90% of the cells analyzed in the different experiments performed.

Lack of Tyr320 impairs spontaneous endocytosis of beta2m-free HLA-B27 chains

Next, we wanted to ascertain whether beta2m-free HLA-B27 H chains present at the cell surface of transfected C58 cells behaved like beta2m-associated HLA-B27 molecules. We used Cy3-conjugated HC-10 Abs and performed microscopy endocytosis assays. Fig. 5 illustrates the distribution of misfolded HLA-B27 molecules in B27-WT- and B27-Y320F-transfected C58 cells before and after 90 min of incubation at 37°C to induce spontaneous endocytosis. After 90 min of incubation at 37°C, misfolded B27-WT molecules are mainly found in intracellular compartments (Fig. 5A, compare a and d). However, and in contrast with folded HLA-B27 molecules, counterstaining with OX-26 Abs revealed little colocalization with endogenous transferrin receptors (Fig. 5A, c and f). Yet, misfolded B27-Y320F molecules behaved like the folded ones and failed to be endocytosed after the 90-min incubation at 37°C (Fig. 5B, compare a and d), remaining at the cell surface where again partial colocalization with endogenous transferrin receptors was detectable (Fig. 5B, c and f).


Figure 5
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  		FIGURE 5. Internalization of misfolded HLA-B27 molecules in C58 cells. C58.B27 (A) and C58.B27.Y320F (B) cells were labeled with Cy3-conjugated HC-10 Abs for 30 min on ice, and fixed and permeabilized before (a, b, and c) or after (d, e, and f) 90 min of incubation at 37°C, and then fixed, permeabilized, and stained for the rat transferrin receptor with Ox-26. Panels of a medial section of a representative cell depicting each fluorochrome and a merged image in which yellow shows colocalization of HC-10 (red) and Ox-26 (green) are presented. Nuclei were stained with DAPI (blue). The bar represents 2 µm.

 
Endocytosis of endogenous transferrin receptors is impaired in C58 cells expressing mutant HLA-B27 molecules

The results observed in the endocytosis microscopy assays, namely the reduction of intracellular transferrin receptor expression in C58 cells expressing the mutant HLA-B27 molecule, raised the possibility that impairment in the spontaneous endocytosis of mutant molecules was interfering with the normal endocytosis of the rat transferrin receptor itself. To investigate this possibility further, we performed reversible biotinylation experiments, as above. The immunoprecipitation results showed an apparent impairment in the amount of transferrin receptor internalized in C58 cells expressing the mutant HLA-B27 molecule (Fig. 6A). Densitometry measurements from five separate experiments revealed a significant reduction in the amount of transferrin receptors internalized in mutant C58 cells when compared with WT cells after 90 min (Fig. 6B). These results are in agreement with the microscopy endocytosis assays and suggest that the tyrosine mutant HLA-B27 molecules transfected into C58 cells interfere with the normal process of endocytosis of transferrin receptors.


Figure 6
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  		FIGURE 6. Endocytosis of rat transferrin receptors is impaired in C58 cells transfected with mutant HLA-B27 molecules. A, C58.B27 (upper panel) and C58.B27.Y320F (lower panel) cells were harvested and labeled with EZ-Link sulfo-NHS-SS-biotin for 30 min on ice, and then incubated at 37°C for 30 or 90 min before GSH treatment. Cells were then lysed, and rat transferrin receptors were immunoprecipitated with Ox-26 Abs and protein A-Sepharose. Samples were resolved by SDS-PAGE, and blotted onto nitrocellulose filters and proteins detected using HRP-conjugated ExtrAvidin and chemoluminescence. Immunoprecipitation from non-GHS-treated samples and samples treated with GSH at time 0 are included as controls. B, Densitometry quantification of the transferrin receptor protein bands using integrated intensity values (integrated density = area x (mean intensity – background) was performed using NIH Image software. Data depicted correspond to the percentage of endocytosed transferrin receptor and are the mean ± SD from five separate experiments. *, Statistically significant differences compared with WT (p < 0.05).

 
Lack of Tyr320 enhances release of HLA-B27 molecules

Previous studies have reported that the fate of beta2m-free MHC-I molecules present at the cell surface is to be released following enzymatic cleavage (32). To ascertain whether the lack of Tyr320 influenced HLA-B27 shedding, the presence of H chains in culture supernatants of B27-WT- and B27-Y320F-transfected cells was studied at different time points. As shown in Fig. 7A, the major cleaved form detected, similar in size to the previously described 37-kDa cleaved form of MHC-I (32), appeared in a time-dependent manner in the supernatants of cultures of transfected, but not untransfected, C58 cells. Of note, cleaved H chains were detected in the supernatants of cells expressing the tyrosine mutant HLA-B27 molecules sooner and in significantly higher amounts than in the supernatants of cells expressing the WT HLA-B27 molecules. Densitometry analysis revealed that the amount of cleaved HLA-B27 forms secreted by Y320F-transfected cells was about 5-fold higher than the amount recovered from supernatants of WT-transfected cells, suggesting that Tyr320 could be involved in the regulation of HLA-B27 shedding from the cell surface (Fig. 7B).


Figure 7
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  		FIGURE 7. Shedding of cell surface HLA-B27 molecules from C58-transfected cells. A, C58, C58.B27, and C58.B27.Y320F cells were cultured in serum-free RPMI 1640. For each cell line, aliquots of culture supernatant were harvested at the indicated time points and then separated by SDS-PAGE under reducing conditions and blotted onto nitrocellulose filters, and proteins were detected using HC-10, followed by goat anti-mouse HRP-conjugated Abs and chemoluminescence. MHC-I cleaved forms are indicated. Molecular mass markers are shown in kDa. B, Densitometry quantification of the cleaved forms of MHC-I protein bands shown was performed using NIH Image software.

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
Disclosures
 References
 
Endocytosis of MHC-I molecules has long been recognized, and occurs in a variety of different cell types (5, 6, 7, 8, 9). The process has biological implications for endosomal and lysosomal peptide loading and display, with the latter event designated as cross-presentation (14, 33). MHC-I endocytosis has also been implicated in the formation of beta2m-free H chains and homodimers at the cell surface (15, 16, 28). Thus, elucidation of the molecular checkpoints that regulate MHC-I endocytosis is of significant importance to understand biological processes in which MHC-I molecules interact with receptors either in cis-, such as hormone receptors, T, and NK receptors (2, 3, 4, 18, 19), or in trans, such as with T and NK cell receptors (21, 34).

A number of studies have pointed to Tyr320 present in the cytoplasmic tail of all classical MHC-I molecules as necessary for Nef-induced down-modulation of MHC-I molecules (10, 11). By generating a single point mutation that replaced the highly conserved Tyr320 for a phenylalanine in human HLA-B27 molecules, we have demonstrated for the first time, and by two different approaches, that spontaneous endocytosis of the mutated human class I allele HLA-B27 is markedly impaired. In contrast, synthesis, assembly, and transport of HLA-B27 molecules into the cell surface as well as stability of the cell surface-expressed molecules were not affected by Tyr320 replacement. The low efficiency rate of assembly of both WT and mutant HLA-B27 molecules, as revealed by pulse-chase combined with Endo-H digestion of HC-10-reactive molecules, is in agreement with previous reports showing that HLA-B27 molecules exhibit slow assembly kinetics (25, 26, 27). However, because both WT and mutant molecules assemble and reach the cell surface at similar rates and levels, it is very unlikely that the observed impairment of endocytosis is in any way related to the slow assembly. Our work is in agreement with studies in which class I down-modulation induced by the viral protein Nef was dependent on a tyrosine-containing motif present in the cytoplasmic tail of classical and nonclassical MHC-I molecules (10, 11, 12). The data presented strongly suggest that Tyr320 is part of an unconventional tyrosine-based sorting motif present in the cytoplasmic tail of MHC-I molecules that is involved in the regulation of endocytosis, which is in accordance with previous studies (14, 35).

Importantly, our studies demonstrated that Tyr320 is not essential for the formation of beta2m-free class I molecules at the cell surface. Thus, expression levels of misfolded HLA-B27 molecules at the cell surface (as determined using HC-10 Abs) were similar between B27-WT- and B27-Y320F-transfected C58 cells. Previous studies have argued that internalization is necessary for beta2m-free H chains to be expressed at the cell surface (15, 16). The results of our studies show that beta2m-free H chains are present at the cell surface even if internalization is impaired, thus indicating that formation of misfolded MHC-I molecules is not entirely dependent on endocytosis. Although the possibility of B27-Y320F molecules being internalized at a much slower rate cannot be ruled out, if internalization would be essential for the expression of beta2m-free H chains at the cell surface, we would expect to see differences in the expression levels of misfolded molecules between B27-WT- and B27-Y320F-transfected C58 cells, which was not the case. Moreover, re-expression of misfolded class I molecules at the cell surface after papain cleavage occurs at similar rate for both WT and mutant Y320F molecules. Thus, Tyr320 appears not to be essential for the conversion of folded to misfolded HLA-B27 molecules to take place at the cell surface, at least in C58 cells.

MHC-I molecules undergo spontaneous endocytosis in different cell types, although with different kinetics. Importantly, the rate of spontaneous endocytosis of MHC-I molecules has been reported to be much higher in lymphoid T cells and monocytes than in B cells (6, 7, 8, 9). After endocytosis, MHC-I molecules traffic through endosomal, trans-Golgi acidic compartments and even lysosomes, in the case of dendritic cells, in which cross-presentation may take place or molecules can simply be targeted for degradation (7, 8, 9, 10, 14, 30, 31). Using markers for the different internal compartments, we demonstrated that in C58 cells the transfected B27-WT molecules spontaneously traffic through the early endocytic pathway, colocalizing with a pool of endogenous transferrin receptors. B27-WT molecules spontaneously endocytosed did not accumulate in the ER or Golgi. A pool of WT HLA-B27 molecules appears to be targeted for degradation, as shown by the punctual colocalization with lysosomes. Importantly, these data indicate that C58 cells are capable of internalizing human MHC-I molecules like HLA-B27, which agrees with previous reports (8, 30, 31). Therefore, the impaired spontaneous endocytosis of B27-Y320F molecules observed in C58 cells is most likely the result of the tyrosine point mutation and not of the system in which the molecules are being expressed. In this context, the colocalization between mutant B27-Y320F molecules and endogenous transferrin receptors at the cell surface, together with the reverse biotinylation data showing an apparent impairment in the endocytosis of transferrin receptors in C58 cells transfected with tyrosine mutant HLA-B27 molecules, raise important questions regarding the biological implications of an impaired spontaneous endocytosis of MHC-I molecules to the traffic of the transferrin receptor itself. Although our work does not allow us to conclude that HLA-B27 molecules and transferrin receptors are physically close enough to have a biological meaning, there are reports linking classical MHC-I molecules and transferrin receptors both at the physical and functional level (2, 36). In this context, it is worth mentioning that the MHC-I-like molecule Hfe is known to interact with the transferrin receptor and modulate its endocytosis (37).

MHC-I molecules are also found in soluble forms (both beta2m associated and beta2m free) in a number of body fluids and in cell culture supernatants of activated T cells as a result of cleavage and/or shedding, and several authors have described these molecules as immunoregulatory (32, 38, 39). Higher levels of cleaved HLA-B27 H chains were found in culture supernatants of B27-Y320F-transfected C58 cells than in culture supernatants of B27-WT-transfected C58 cells. Although the increase of cleaved forms was observed using HC-10 Abs, we could not ascertain whether the cleaved forms found originated from beta2m-associated or beta2m-free HLA-B27 H chains. Based on previous studies, including ours (18, 32), we favor the scenario in which the higher presence of truncated forms of HLA-B27 molecules observed in culture supernatants of B27-Y320F-transfected C58 cells is due to release of beta2m-associated HLA-B27 chains, with shedding acting as a mechanism to compensate for the impaired spontaneous endocytosis. The increased release of mutant beta2m-associated B27-Y320F molecules could provide an explanation for the similar cell surface expression of B27-Y320F when compared with B27-WT molecules. This assumption is supported by recent work from our lab demonstrating that in activated T cells beta2m-free class I molecules are tyrosine phosphorylated and inhibition of that phosphorylation results in an enhanced release of class I molecules (18).

Taken together, our results support the view that Tyr320 is part of a sorting motif present in the cytoplasmic domain of classical MHC-I molecules. The exact role that Tyr320 itself plays in other processes, such as formation of MHC-I dimers, remains to be characterized. Likewise, whether phosphorylation of Tyr320 determines if class I molecules are going to become misfolded, endocytosed, or cleaved is a subject that requires and deserves further investigation.


   Acknowledgments
 
We thank Hidde Ploegh, Neil Bulleid, and Sándor Damjanovich for providing Abs used in this study, and Dr. Damjanovich for helpful comments.


   Disclosures
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 Disclosures
References
 
The authors have no financial conflict of interest.


   Footnotes
 
The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

1 Current address: Bute Medical School, University of St. Andrews, St. Andrews, U.K. Back

2 Address correspondence and reprint requests to Dr. Fernando A. Arosa, Lymphocyte Biology Group, Institute for Molecular and Cell Biology, Rua do Campo Alegre, 823, 4150-180 Porto, Portugal. E-mail address: farosa{at}ibmc.up.pt Back

3 Abbreviations used in this paper: MHC-I, MHC class I; beta2m, beta2-microglobulin; DAPI, 4',6'-diamidino-2-phenylindole; Endo-H, endoglycosidase H; ER, endoplasmic reticulum; GSH, glutathione; NHS-SS, N-hydroxysulfosuccinimide; WT, wild type. Back

Received for publication April 21, 2005. Accepted for publication November 30, 2005.


   References
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 Introduction
 Materials and Methods
 Results
 Discussion
 Disclosures
 References
 

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