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The Journal of Immunology, 2003, 170: 6090-6098.
Copyright © 2003 by The American Association of Immunologists

Biochemical Features of the MHC-Related Protein 1 Consistent with an Immunological Function1

Michael J. Miley2,*, Steven M. Truscott2,{dagger}, Yik Yeung Lawrence Yu{dagger}, Susan Gilfillan*, Daved H. Fremont*, Ted H. Hansen3,{dagger} and Lonnie Lybarger{dagger}

Departments of * Pathology and Immunology and {dagger} Genetics, Washington University School of Medicine, St. Louis, MO 63110


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
MHC-related protein (MR)1 is an MHC class I-related molecule encoded on chromosome 1 that is highly conserved among mammals and is more closely related to classical class I molecules than are other nonclassical class I family members. In this report, we show for the first time that both mouse and human MR1 molecules can associate with the peptide-loading complex and can be detected at low levels at the surface of transfected cells. We also report the production of recombinant human MR1 molecules in insect cells using highly supplemented media and provide evidence that the MR1 H chain can assume a folded conformation and is stoichiometrically associated with {beta}2-microglobulin, similar to class I molecules. Cumulatively, these findings demonstrate that surface expression of MR1 is possible but may be limited by a specific ligand or associated molecule.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
The structure of classical, or class Ia, MHC class I (MHC-I)4 molecules is specifically adapted for promiscuous presentation of peptide Ags to cytolytic CD8+ T cells during infection by intracellular pathogens (1). Although they share the same protein fold, nonclassical, or class Ib, MHC-I molecules are quite diverse in their biology. Some class Ib molecules are highly specialized in terms of Ag presentation (2). For example, mouse Qa-1 and human HLA-E bind signal peptides of other class I molecules and present them to NK cell receptors to prevent destruction of normal cells (3, 4) whereas murine H2-M3 provides immune surveillance against bacterial infection by binding and presenting N-formylated peptides (5, 6, 7). Furthermore, CD1 binds and presents mycobacterial lipid and glycolipid Ags to a unique T cell subset implicated in fighting Mycobacterium (2). Not all immunologically effective class I-like molecules present Ag; for instance, the UL16-binding protein (8), retinoic acid early inducible and H60 (9, 10), MHC-I-related gene A/B (11), and murine UL16-binding protein-like transcript 1 (12) molecules serve as ligands for NKG2D, an activating receptor expressed on NK cells, macrophages, {gamma}{delta}, and CD8+ T cells, while T10 and T22 are ligands for subsets of {gamma}{delta} T cells (13). Though a number of nonclassical MHC-I molecules contribute to immunological functions, others do not. For example, hemochromatosis protein (HFE) is a ligand for the transferrin receptor and functions to regulate the uptake of iron-bound transferrin (14, 15). Additionally, zinc {alpha}-glycoprotein has been shown to bind fatty acids and function in lipid store homeostasis (16, 17).

MHC-related protein 1 (MR1), is a very intriguing nonclassical class I molecule in the infancy of characterization. Discovered by Hashimoto et al. (18) using degenerate PCR, MR1 is encoded outside of the MHC in humans, mouse, and rat (18, 19, 20, 21). Although it is expressed at the mRNA level in multiple tissues (20), surface expression of MR1 protein has not been demonstrated (22), and a function for MR1 has not been defined. However, because the MR1 gene is highly conserved among mammals, MR1 is likely to perform a conserved function. Indeed, the 90% sequence identity between the human and mouse MR1 putative ligand binding ({alpha}1/{alpha}2) domains (20) far exceeds the 70% similarity shared by this region of human and mouse classical class I molecules.

Using a structure-based sequence comparison, human and mouse MR1 amino acid sequences can be evaluated in the context of known class I structures and sequence. Supporting the prediction of a class I protein fold, most of the conserved residues shared by all vertebrate MHC-I molecules are present in MR1. However, of all classical class I conserved residues involved in anchoring peptide termini (Ref.23 ; A pocket Y7, Y59, Y159, W167, Y161 and F pocket Y84, T143, K146, W147), only two are conserved in the A pocket (Y7, W167) and one in the F pocket (W147) of MR1, although the differences observed are in large part conservative (five of six). No significant similarity was found between the residues lining the ligand binding grooves of nonclassical class I molecules and residues predicted to form the binding groove of MR1. This analysis highlights the unique nature of the MR1 {alpha}1/{alpha}2 domain, as well as illustrating that prediction of MR1 ligand binding potential from sequence data alone is problematic.

Upon examining {beta}2-microglobulin ({beta}2m) association in existing class I structures, several {alpha}3 sequence-dependent motifs were observed. MR1 does not conform to any of these motifs, suggesting that MR1 either does not bind {beta}2m or does so in a unique way. Relevant to this issue, recent studies have shown that nascent mouse MR1 (mMR1) molecules are associated with {beta}2m, although {beta}2m-dependent cell surface expression and stoichiometry were not reported (22).

CD8/class I interactions are highly dependent on two loops (AB and CD) in the {alpha}3 domain of class I molecules (24, 25). The mouse and human loops differ in sequence, but are conserved among classical class I molecules within each species, resulting in differential recognition by mouse and human CD8. In contrast, these loops are nearly identical in amino acid sequence between human MR1 (hMR1) and mMR1, with each differing by only one conservative mutation. These data would argue against MR1 being engaged by CD8 in a classical manner.

H chains of classical class I molecules associate with endoplasmic reticulum (ER) molecular chaperones to facilitate their assembly with {beta}2m and peptide, both requirements for ER egress (26, 27). More specifically, classical class I molecules associate with calnexin before assembly with {beta}2m, and calreticulin after assembly with {beta}2m. While associated with calreticulin and awaiting peptides, class I molecules are concomitantly associated with TAP, tapasin, and ERp57. This complex of calreticulin, TAP, tapasin, and ERp57 has been referred to as the peptide-loading complex. In this complex, TAP provides the peptides that bind H chain/{beta}2m heterodimers, tapasin facilitates the binding of high affinity peptides (28), and ERp57 facilitates disulfide bond formation in tapasin or the H chain (29). Association of MR1 with any of the ER proteins implicated in the full assembly of classical class I molecules has not been defined, but would help determine the molecular mechanism of MR1 assembly.

We have used both cellular and biochemical approaches to address the possibility that MR1 has an immunological function. To that end, using a baculovirus-mediated insect cell expression system, we were able to produce the soluble ectodomain of hMR1. Initial biochemical characterization of this protein defined its secretion cleavage site, demonstrated stoichiometric association with {beta}2m, and provided evidence for N-linked glycosylation. Using transfected mammalian cell lines, we also identified nascent mMR1 and hMR1 proteins in association with {beta}2m, calnexin, calreticulin, ERp57, TAP, and tapasin. Furthermore, we detected low levels of MR1 protein on the cell surface after transfection, which were augmented by covalently attaching {beta}2m or by swapping the {alpha}3 domain of MR1 with that of a classical class I molecule. These findings are discussed in the context of recent evidence that MR1 activates a subset of double-negative (DN) mucosal T cells expressing an invariant TCR {alpha}-chain (30).


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
DNA clones and mutagenesis

Site-directed mutagenesis of the wild-type MR1 cDNAs (a gift of Dr. S. Bahram, Centre de Recherche d’Immunologie et d’Hematologie, Strasbourg, France) was performed using the QuikChange mutagenesis kit (Stratagene, La Jolla, CA) essentially as described (31). To introduce the 64-3-7 epitope (which is specific for open forms of Ld and Lq), a single substitution (R46Q for hMR1 and K46Q for mMR1) was required to convert the sequence of MR1 surrounding the epitope to the sequence found in Ld (31). The mMR1/Ld chimeric molecules were generated by PCR, linking residues 1–179 of MR1 to residues 183–338 of Ld (for the {alpha}1-{alpha}2 chimera), or 1–278 of MR1 with 284–338 of Ld (for the {alpha}1-{alpha}3 chimera). Likewise, an H2-M3/Ld ({alpha}1-{alpha}2) chimeric molecule was generated that encodes residues 1–182 of 64-3-7 epitope-tagged M3 linked with residues 183–338 of Ld. The {beta}2m/MR1 construct consists of human {beta}2m followed by a 15-mer linker of (G4S)3 then the mature portion of the hMR1 H chain. The cDNAs were inserted into the expression vectors pIRESneo or pIRESpuro2 (Clontech Laboratories, Palo Alto, CA) for transfection of mammalian cells. For insect cell expression, residues 1–279 of hMR1 containing the 64-3-7 epitope were C-terminally extended with a thrombin cleavage sequence (ASSVLPR/GS) and a six-histidine (6-His) tag. This cDNA was inserted into the pBACPhP10 duel promoter baculovirus transfer vector (32) under the polyhedron promoter. Full-length human {beta}2m was then inserted into this vector under the P10 promoter. DNA sequence analysis was used to confirm the correct sequence of all constructs.

Cell lines and transfections

Mouse L cell transfectants of 64-3-7 epitope-tagged H2-M3 and HeLa cells transfected with 64-3-7 epitope-tagged HLA-B27 have been described (33, 34). Transfections of L cells, HeLa cells (human cervical carcinoma), DLD-1 cells ({beta}2m-deficient human colon adenocarcinoma; Ref.35), and B6/WT-3 cells (murine fibroblasts; Ref.36) were performed using FuGene6 (Roche Diagnostics, Indianapolis, IN). Stable transfectants were selected with 0.6 mg/ml geneticin (Life Technologies, Grand Island, NY), identified by intracellular staining/flow cytometry (see below) and cloned by limiting dilution. All cells were maintained in RPMI 1640 (Life Technologies) supplemented with 10% FCS (HyClone Laboratories, Logan, UT), 2 mM L-glutamine, 0.1 mM nonessential amino acids, 1.25 mM HEPES, 1 mM sodium pyruvate, and 100 U/ml penicillin/streptomycin (all from the Tissue Culture Support Center, Washington University School of Medicine, St. Louis, MO). Where appropriate, geneticin was added to a final concentration of 0.6 mg/ml. In some case, cells were incubated for 2 days with human IFN-{gamma} (R&D Systems, Minneapolis, MN) or mouse IFN-{gamma} (BioSource International, Sunnyvale, CA) at 100 U/ml. Two insect cell lines, Sf9 (Spodoptera frugiperda) and Hi-five (Trichoplusia ni), were used. Sf9 cells were cultured in suspension with supplemented Grace’s medium (10% FCS, yeastolate, and lactalbumin hydrolysate to which 100 U/ml penicillin/streptomycin and 0.1% pluronic F-68 (all from Life Technologies) were added). Hi-five cells were cultured in suspension with ultimate insect select media (Invitrogen, Carlsbad, CA; discontinued) and were switched into the above supplemented Grace’s medium while adhered to plates.

Antibodies

mAb 64-3-7 is specific for open forms of Ld and epitope-tagged molecules (31), and was used for immunoprecipitations, immunoblots, and flow cytometry. mAb 28-14-8 detects the {alpha}3 domain of Ld (37, 38) and was used for flow cytometry. mAb 4E3 was derived by immunization of {beta}2m-deficient BALB/c mice with a keyhole limpet hemocyanin-conjugated peptide corresponding to mMR1 residues 130–153 (L A M D Y V A H I T K Q A W E A N L H E L Q Y Q). Hybridomas were produced by standard methods and screened by anti-peptide ELISA. The following Abs were used for immunoblot: rabbit anti-human TAP (39); rabbit anti-human tapasin (34); mAb BBM.1 (anti-human {beta}2m; Ref.40); chicken anti-calreticulin serum (Affinity Bioreagents, Golden, CO); rabbit anti-calnexin (StressGen, Victoria, British Columbia, Canada); rabbit anti-ERp57 (41); rabbit polyclonal IgG anti 6-His HRP conjugate (Santa Cruz Biotechnology, Santa Cruz, CA).

Flow cytometry

All flow cytometric analyses were performed using a FACSCalibur (BD Biosciences, San Jose, CA). Dead cells and debris were excluded from analysis on the basis of forward-angle and side-scatter light gating. A minimum of 10,000 gated events was collected for analysis. Data were analyzed using CellQuest software (BD Biosciences). For surface staining, ~5 x 105 cells per sample were incubated on ice in microtiter plates with culture supernatant from the appropriate hybridoma. After washing, PE-conjugated goat anti-mouse IgG (BD PharMingen, San Diego, CA) was used to visualize class I staining. For intracellular staining with 64-3-7, cells were fixed and permeabilized in PBS containing 1% paraformaldehyde, 1% BSA, and 0.5% saponin (all from Sigma-Aldrich, St. Louis, MO) for 20 min on ice. Permeabilized cells were stained in PBS containing 1% BSA and 0.5% saponin into which FITC-conjugated 64-3-7 was diluted.

Immunoprecipitations

For mAb 64-3-7 coimmunoprecipitations, cells were lysed in PBS + 1.0% digitonin (WAKO, Richmond, VA) containing a saturating concentration of Ab. After lysis for 30 min on ice, postnuclear lysates were incubated with protein A-Sepharose (Amersham Pharmacia Biotech, Uppsala, Sweden) for 1 h. Beads were washed four times in PBS + 0.1% digitonin, and bound proteins were eluted by clearing the precipitating Ab from the samples overnight at 4°C by competition with excess peptide EPQAPWM, which corresponds to the 64-3-7 epitope (42). For denaturation/64-3-7 immunoprecipitation experiments, postnuclear lysates were generated in 1% Nonidet P-40 (in TBS) and each sample was split into two equal aliquots. SDS was then added to one aliquot of each pair to a final concentration of 1%. These samples were boiled and then all samples were diluted 10-fold with 1% Nonidet P-40 in TBS before incubation with protein A-Sepharose containing prebound 64-3-7. After binding for 45 min on ice, protein A pellets were washed four times with 0.1% Nonidet P-40 in TBS, and bound proteins were eluted by boiling in 10 mM TrisCl, pH 6.8 + 0.5% SDS + 1% 2-ME. Eluates were mixed with an equal volume of 100 mM sodium acetate, pH 5.4, and either digested (or mock-digested) at 37°C for >1 h with 1 mU endoglycosidase H (ICN Pharmaceuticals, Costa Mesa, CA) that was reconstituted in 50 mM sodium acetate, pH 5.4.

Immunoblotting

Immunoblotting was performed following SDS-PAGE separation of precipitated proteins and transfer to Immobilon P membranes (Millipore, Bedford, MA). Membranes were blocked (1 h to overnight) with PBS + 10% dried milk + 0.05% Tween 20. Primary Abs were added and incubated for 1 h, followed by washing in PBS + 0.05% Tween 20. As a second step, membranes were incubated for 1 h with biotin-conjugated anti-mouse (Caltag Laboratories, San Francisco, CA), anti-rabbit (Jackson Immunoresearch Laboratories, West Grove, PA), or anti-chicken IgG (Zymed Laboratories, San Francisco, CA). After washing, HRP-conjugated streptavidin (Zymed Laboratories) was added for 1 h, followed by three washes. Specific proteins were visualized by chemiluminescence using the ECL system (Amersham, Boston, MA).

Expression and purification of recombinant hMR1 ectodomain

Recombinant baculovirus was generated by cotransfecting Baculogold linearized AcNPV DNA (BD Biosciences) and the pBACPhP10 construct containing {beta}2m and C-terminally 6-His-tagged hMR1 into Sf9 cells. Recombination was verified by immunoblot analysis of Sf9 cell lysates with rabbit polyclonal anti-6-His HRP Ab. In preparation for the expression run, Sf9 cells were expanded in shaker suspension to a volume of 6 liters and were infected using a high multiplicity of infection (5, 6, 7, 8, 9, 10) of recombinant baculovirus when they reached a density of 2 x 106 cells per ml. After 72 h, cells were centrifuged away from the medium, which contained the soluble hMR1. Using a Centramate tangential flow concentration system with a 30 kDa molecular mass cutoff membrane (Pall Life Sciences, Ann Arbor, MI), medium was concentrated (to ~1 liter) and buffer was exchanged into 50 mM NaH2PO4, 50 mM sodium citrate, pH 8.0, 300 mM NaCl, and 0.01% sodium azide. Soluble hMR1 was then bound to Ni-NTA Superflow beads (Qiagen, Valencia, CA) and eluted with binding buffer containing 250 mM imidazole. The eluted protein was then diluted 10 times with 20 mM Tris buffer, pH 8.0, and subjected to anion exchange chromatography with a MonoQ HR 5/5 column (Pharmacia Biotech, Piscataway, NJ). The fractions containing soluble hMR1 were pooled and subjected to size exclusion chromatography using a Superdex 75 16/60 column (Pharmacia Biotech) pre-equilibrated with 20 mM HEPES, pH 7.4, 150 mM NaCl, and 0.01% sodium azide. hMR1 eluted as a single peak at an elution volume corresponding to a molecular mass of ~45 kDa. This protocol reproducibly yields ~1 mg of pure, monodispersed soluble hMR1. PNGase F treatment was performed per the manufacturer’s suggested protocol using GST-coupled PNGase F (Hampton Research, Laguna Niguel, CA). Briefly 25 µg of GST-PNGase F were added to 1 mg of recombinant hMR1 and incubated for 48 h at 20°C in 20 mM HEPES, pH 7.4, 150 mM NaCl. Residual GST-PNGase F was removed using glutathione sepharose. Deglycosylation was monitored by SDS-PAGE.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
Experimental strategy

To characterize MR1, we used a previously described epitope-tagging strategy (31). The epitope recognized by mAb 64-3-7 is normally found on Ld H chains, but can be transferred to other classical and certain nonclassical class I H chains with substitution(s) of 1 or 2 aa. mAb 64-3-7 is exquisitely specific for the peptide-empty (or "open") conformation of Ld H chains. Open H chains are found associated with the peptide-loading complex while awaiting peptide in the ER and at the cell surface after peptide dissociation. We have successfully tagged several classical and nonclassical class I molecules with this epitope and, in each case, 64-3-7 remains specific for the peptide-empty forms of the respective H chains (Refs. 31 ,33 ,34 ,42 and data not shown). In contrast to peptide binding class I molecules, epitope-tagged HFE molecules (which do not bind peptide) display little, if any, 64-3-7 reactivity at steady-state (not shown). Thus, the 64-3-7 epitope tag not only provides unequivocal identification of the MR1 protein, but also monitors MR1 H chain folding. We introduced the 64-3-7 epitope into MR1 with a single amino acid substitution (K46Q for mMR1 and R46Q for hMR1) and analyzed tagged MR1 using insect and mammalian cell expression systems.

Production of soluble hMR1 (ectodomain) and its initial biochemical characterization

Using a baculovirus-mediated insect cell expression system we were able to express and secrete the mAb 64-3-7 epitope-tagged ectodomain of soluble hMR1 (Fig. 1). Secretion of this protein was medium-dependent, occurring only in a highly supplemented form of Grace’s medium. This result suggested that hMR1 might acquire a ligand from the supplemented medium, thus facilitating secretion. In support of this notion, recombinant MR1 (inclusion bodies) from Escherichia coli analogous to the secreted insect cell hMR1 (minus the secretion leader sequence) failed to refold not only in standard class I refolding conditions but also in a variety of other conditions (data not shown). This is significant because refolding of recombinant classical class I molecules from purified E. coli inclusion bodies is dependent upon the presence of a peptide ligand. In contrast, nonligand-dependent, nonclassical class I molecules readily refold from inclusion bodies via oxidative refolding conditions (43, 44, 45, 46, 47).



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  		FIGURE 1. A, Immunoblot analysis of lysates from Sf9 cells exposed to hMR1 baculovirus, with the indicated Abs, reveals a positive signal at ~33 kDa confirming intracellular viral-mediated hMR1 ectodomain protein expression. B, hMR1 ectodomain (soluble hMR1) is secreted in a medium-dependent fashion. hMR1 baculovirus-infected Sf9 and Hi-five cells grown in supplemented Grace’s medium show a positive immunoblot signal at ~33 kDa in centrifuged media, confirming secretion. hMR1-virus infected Hi-five cells grown in serum-free media exhibit extremely low levels of secretion. Cells grown in the absence of virus exhibit no signal (not shown). Probing Abs are listed in parentheses. These results are representative of multiple independent infection experiments. Note that the diffuse bands observed with the intracellular hMR1 blots vs the more distinct bands seen with the secreted material very likely reflect different glycosylation states of MR1, with secreted MR1 being more uniform.

 
A multistep purification process produced monodisperse, highly purified soluble hMR1 ectodomain (Fig. 2). The final size-exclusion chromatography step yielded a single peak with an elution volume that corresponds to an approximate molecular mass of 45 kDa (data not shown). A silver-stained gel of protein from this peak shows the presence of two bands in approximately a one-to-one stoichiometry, one at 33 kDa and one at 11 kDa. The 33 kDa band represents hMR1, as it is immunoreactive with two different Abs, 64-3-7 and anti-6-His tag (data not shown). We suspected the 11-kDa band was {beta}2m and verified this by immunoblotting with mAb BBM.1 (data not shown). Importantly, this provided conclusive proof that MR1 forms a heterodimer with {beta}2m, much like classical and many nonclassical class I molecules. Both bands (33 and 11 kDa) were subjected to N-terminal amino acid sequence analysis. This analysis revealed that the secretion cleavage site for hMR1 is between residues 22–23 (SDS/RTH). Analysis of the {beta}2m band showed that both bovine and human forms of {beta}2m were in complex with the purified, soluble hMR1. Because certain class I proteins are known to undergo {beta}2m exchange (e.g., Ref.48) it is likely that some human {beta}2m (h{beta}2m) was exchanged for bovine {beta}2m derived from the FCS-supplemented medium.



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  		FIGURE 2. Eight to 25% SDS-PAGE silver-stained gel of secreted, purified hMR1 from insect cells. Two bands are visible in the purified protein lane, one at 33 kDa and another at 11 kDa. The 33-kDa band was identified as hMR1 by immunoblot and N-terminal sequencing. The 11-kDa band was identified as a combination of human and bovine {beta}2m by immunoblot and N-terminal sequencing.

 
hMR1 is predicted to have one N-linked glycosylation site at residue Asn86, a site common to virtually all classical MHC molecules. After treating the soluble hMR1 molecule with PNGase F, we noted a visible band shift in both SDS and native PAGE (Fig. 3). A single band shift in SDS confirmed complete cleavage of at least one N-linked sugar. On the native gel, undigested hMR1 migrates as four visible bands, all of which shift together upon enzyme treatment. Two of these bands could be accounted for by the two different {beta}2m associated with the purified, soluble hMR1. The other two cannot be explained by N-linked glycosylation, as all four shift upon enzyme treatment and hence may represent different ligand/hMR1/{beta}2m complexes. Regardless, these results confirm the presence of N-linked glycosylation, the likely position being residue Asn86.



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  		FIGURE 3. Secreted, purified hMR1 from insect cells was digested with PNGase F and analyzed by SDS and native PAGE. Band shifts on both gels indicate the presence of N-linked glycosylation.

 
Intracellular and cell surface detection of MR1 in mammalian cells

Our analysis of hMR1 produced in insect cells demonstrated that H chains assemble stably with {beta}2m and are secreted as monomers. These findings prompted us to examine the 64-3-7 reactivity pattern of epitope-tagged MR1 molecules in mammalian cells. As shown in Fig. 4, significant pools of intracellular hMR1 and low levels of surface hMR1 were detected by mAb 64-3-7 following stable transfection of HeLa cells. A very comparable staining pattern was observed with mMR1 expressed in a murine fibroblast line (B6/WT-3). The size of the intracellular pool of MR1 in both cell lines was quite similar to that previously observed with epitope-tagged forms of murine (Kb, Kd, and H2-M3) and human (HLA-B27) class I molecules (33, 34, 42). In contrast, cell surface levels of 64-3-7-reactive classical class I molecules are typically much higher than we observed with hMR1 or mMR1. 64-3-7+ forms of classical class I molecules arise at the cell surface after peptide dissociation, whereas ligand association with MR1 is uncertain. Nonetheless, these findings reveal for the first time that MR1 can be expressed on the cell surface.



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  		FIGURE 4. Surface and intracellular 64-3-7 staining of MR1 on transfected cell lines. HeLa or DLD-1 ({beta}2m-deficient) human cell lines, and murine B6/WT-3 cells were stably transfected with 64-3-7 epitope-tagged hMR1, hMR1 with h{beta}2m covalently linked to the N terminus, or mMR1, as indicated. For surface staining, thin lines indicate staining with the secondary Ab alone and heavy lines indicate staining with mAb 64-3-7. For intracellular staining, thin lines indicate staining of nontransfected parental cells and heavy lines indicate staining of the hMR1 or mMR1 transfectants. In the panel representing staining of DLD-1 cells expressing the {beta}2m-hMR1 construct, the dotted line indicates staining for h{beta}2m with mAb BBM1.

 
{beta}2m is required for surface expression of hMR1

Our finding that {beta}2m stably associates with soluble hMR1 (Fig. 2) raised the possibility that surface expression of MR1 is {beta}2m-dependent. We tested this possibility directly by expressing hMR1 in {beta}2m-deficient human DLD-1 cells (35). As shown in Fig. 4, epitope-tagged hMR1 molecules were abundant intracellularly, but were not detected at the cell surface, confirming that {beta}2m is required for surface MR1 expression. These findings were extended by generating a single-chain construct with {beta}2m attached to the N terminus of hMR1 via a flexible linker. Such constructs have been successfully used to express various classical class I molecules in {beta}2m-deficient cells, and these single-chain molecules retain their functional properties (49, 50, 51). hMR1 H chains with covalently attached {beta}2m were introduced by stable transfection into DLD-1 cells. As shown in Fig. 4, both intracellular and surface 64-3-7+ forms of hMR1 were observed in DLD-1 cells. Interestingly, high levels of {beta}2m were detected by mAb BBM.1 (anti-h{beta}2m) after expression of the {beta}2m/MR1 construct, providing further evidence that MR1 molecules can be expressed on the cell surface.

Replacing the {alpha}3 domain of mMR1 with the {alpha}3 domain of Ld facilitates surface expression

To determine whether domains other than the putative ligand-binding domain ({alpha}1/{alpha}2) of the MR1 protein influence its cell surface expression, chimeric molecules were produced and tested. As shown in Fig. 5, a chimeric molecule that possesses the {alpha}1/{alpha}2 domains of mMR1 and the {alpha}3, transmembrane and cytosolic domains of Ld displayed higher levels of surface expression than did intact mMR1. Furthermore, a chimeric molecule with mMR1 sequence in the {alpha}1-{alpha}3 domains and only the transmembrane/cytosolic domains of Ld resulted in improved cell surface expression relative to intact mMR1, but somewhat lower than the aforementioned ({alpha}1–2) chimeric molecule. These results indicate that the {alpha}3 domain and to a lesser extent the transmembrane and cytosolic domains of MR1 regulate its surface expression. Comparison of the {alpha}3 sequence of MR1 to those of classical class I molecules revealed an extra cysteine at position 261 that could result in aberrant disulfide bond formation. To determine whether the cysteine prevents high levels of MR1 cell surface expression, a C261G mutation of mMR1 was generated. However, as shown in Fig. 5, mMR1 C261G was expressed at minimal levels on the cell surface. Thus, MR1 surface expression appears to be limited, at least in part, by sequences outside its putative ligand-binding groove, but the unique cysteine at position 261 does not impede cell surface expression.



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  		FIGURE 5. A, Surface and intracellular staining of mouse L cells transiently transfected with the indicated mMR1 constructs (heavy lines) or nontransfected control cells (dotted lines). The numbers indicate the percentage of cells detected as positive for MR1 expression. A representative example from three independent experiments is shown. B, Surface expression of M3/Ld and mMR1/Ld molecules. Chimeric molecules consisting of the {alpha}1-{alpha}2 of M3 or mMR1 fused in frame to the {alpha}3 domain (plus transmembrane and cytosolic domains) of Ld were stably transfected into L cells. Cells were stained with the secondary Ab alone (thin gray lines) or mAb 28-14-8 that recognizes the {alpha}3 domain of Ld. The heavy dark line indicates 28-14-8 staining of untreated cells and the dotted line in the left panel indicates staining of cells after overnight incubation of cells with the M3-binding peptide Fr38 (6 ).

 
Expression of the mMR1 ({alpha}1-{alpha}2)/Ld chimeric molecule was examined further using a stably transfected L cell line. Because this chimeric molecule has the {alpha}3 domain of Ld, it reacts with mAb 28-14-8 (37). As shown in Fig. 5B, significant 28-14-8 staining of the mMR1/Ld chimeric molecule was observed. It is important to note that mAb 28-14-8 detects the {alpha}3 domain in a manner independent of the {alpha}1/{alpha}2 domain (52). Thus, 28-14-8 staining of the mMR1/Ld chimeric molecule should be independent of the folding and/or ligand binding status of the putative MR1 groove. For comparison, we also show results obtained with an H2-M3 ({alpha}1-{alpha}2)/Ld chimeric molecule. Unlike classical class I molecules that are constitutively expressed due to the abundance of suitable ligands in the ER, M3 expression is induced by peptides initiated with a formylated methionine, which are not abundant in the ER (5, 53, 54). An M3/Ld chimera similar to the one used in this study was previously shown to retain its ligand-specific surface induction (53). Fig. 5B demonstrates that 28-14-8 surface staining of the M3/Ld molecule is only detected after cells have been incubated with M3-binding peptides, whereas considerable surface expression of mMR1/Ld was revealed by 28-14-8 staining of cells grown in standard culture medium. These findings suggest that Ld sequences in the chimeric molecule alter chaperone interactions and/or intracellular trafficking of MR1 resulting in increased surface expression by a mechanism different from that used by intact MR1 molecules.

Generation of an mMR1-specific mAb

We generated a mAb against a peptide derived from the {alpha}2 domain of mMR1 (residues 130–153). This sequence was chosen because an Ab produced to a peptide derived from a similar region of another class I-like molecule (Qa-1) can detect fully assembled molecules (55). We immunized {beta}2m-deficient mice with carrier-conjugated peptide, reasoning that they might be capable of mounting an anti-mMR1 response, as MR1 requires {beta}2m for surface expression (see above). Antisera from these mice reacted strongly with soluble mMR1 when analyzed by immunoblot or ELISA (not shown) and, following hybridoma production, a mAb specific for mMR1 was derived. As shown in Fig. 6A, this mAb (4E3) reacts with mMR1 by immunoblot but does not cross-react with hMR1. Furthermore, it can detect low levels of surface mMR1 expression on transfected cell lines (Fig. 6B). Indeed, all of the constructs described in this study, when expressed in transfected cell lines, stain with mAb 4E3 with very similar intensity to that observed using mAb 64-3-7 (data not shown). The 4E3 mAb should be capable of recognizing endogenous mMR1 protein, yet we have observed no surface staining using splenocytes, thymocytes, or several murine cell lines. In addition, immunoblot analysis of lysates from cell lines and from various mouse tissues did not reveal the presence of endogenous mMR1 (not shown) even though it readily detects transfected mMR1. Therefore, it appears that MR1 is either expressed at very low levels in most cell types and/or is expressed only by a few cell types in a given tissue.



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  		FIGURE 6. A, Immunoblot of whole cell lysates from the indicated cell types using mAb 64-3-7 (specific for epitope-tagged MR1 molecules) or mAb 4E3 (specific for mMR1). B, Surface staining of HeLa cells (left panels) or B6/WT-3 cells (right panels) with mAb 4E3. Staining of the parental cell lines (upper panels) vs the epitope-tagged mMR1 transfectants (lower panels) is shown. Dotted lines indicate staining with the secondary Ab alone and dark lines indicate staining with mAb 4E3.

 
MR1 is associated with the peptide-loading complex

Classical class I molecules awaiting peptides in the ER are associated with the peptide-loading complex comprised of TAP, tapasin, calreticulin, and ERp57 (27, 56). To determine whether MR1 molecules might also be associated with the peptide-loading complex, coimmunoprecipitation experiments were performed, comparing hMR1 and HLA-B27, a classical class I molecule. As shown in Fig. 7, hMR1 clearly associated with each member of the human peptide-loading complex. Indeed, the level of association of hMR1 with the peptide-loading complex appears to be in the same range as HLA-B27. Treatment of cells with IFN-{gamma}, which up-regulates expression of all members of the peptide-loading complex (26), significantly increased the association of hMR1 (and B27) with the loading complex. Analysis of mMR1 expressed in a murine cell line revealed that mMR1 also associates with the peptide-loading complex (Fig. 7B). Furthermore, the association of both murine and hMR1 with the peptide-loading complex could also be demonstrated by precipitating the loading complex with anti-TAP Abs and blotting for MR1 (data not shown). These findings indicate that MR1 uses the same cellular assembly machinery as do classical class I molecules.



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  		FIGURE 7. hMR1 and mMR1 interact with the MHC-I peptide-loading complex. A, HeLa cells transfected with 64-3-7 epitope-tagged hMR1 and HLA-B27, or (B) B6/WT-3 cells transfected with mMR1, were left untreated or treated with IFN-{gamma} for 48 h. Digitonin lysates were immunoprecipitated using mAb 64-3-7, resolved by SDS-PAGE, and blotted with the indicated Abs.

 
Evidence for folding of the hMR1 protein

The data presented thus far indicate that MR1 is expressed at relatively low levels on the cell surface. However, because we used mAb 64-3-7 to detect expression (specific for open class I H chains), it remained possible that there existed a fraction of surface-expressed MR1 that is in a 64-3-7-negative conformation. Indeed, for epitope-tagged classical class I molecules, Abs specific for the folded conformation reveal that most molecules at the cell surface are folded, reflecting occupancy with a high-affinity peptide (31, 33, 34, 42). Thus, we sought to investigate whether MR1 also exists in a 64-3-7-negative conformation. To this end, 64-3-7 immunoprecipitations were performed, either with or without prior denaturation of the samples, followed by immunoblotting to detect H chains. In essence, denaturing the samples before immunoprecipitation reveals the entire pool of H chains in the cell, by converting all conformers to a 64-3-7-reactive state. The increase in recovered H chains after denaturation provides a measure of the amount of folded molecules in the cell. In addition, we also assessed endoglycosidase H (endo H) sensitivity of the H chains to determine whether they had egressed from the ER to post-ER compartments. The results of a typical experiment, including relevant controls, are shown in Fig. 8. As expected, denaturation of lysates from Ld-expressing cells led to an increased recovery of H chains by mAb 64-3-7, some of which were endo H-resistant (post-ER). By contrast, denaturation of epitope-tagged H2-M3 molecules did not lead to an increase in 64-3-7-reactive H chains, as anticipated, because the level of endogenous M3-binding peptides is insufficient to promote significant folding of the H chains (33, 54). Consistent with a lack of M3 assembly with peptide, the preponderance of the H chains were endo H-sensitive (ER-resident) at steady-state. Analysis of hMR1 in this manner revealed a pattern that was quite similar to M3. Denaturation of hMR1-expressing cell lysates did not increase the amount of 64-3-7-reactive H chains, and the majority of the H chains were endo H-sensitive at steady-state. These findings indicate that most MR1 molecules remain in the ER in an unassembled state in this cell line and only a small fraction must transit to the cell surface, consistent with a previous report (22).



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  		FIGURE 8. Effects of denaturation on the recognition of different molecules by mAb 64-3-7. 64-3-7 immunoprecipitations were performed on cell lysates from the indicated cell types (left side) or using purified soluble hMR1 (right side), either with or without prior denaturation or endo H digestion, as indicated. Precipitates were separated by SDS-PAGE and immunoblotted using 64-3-7.

 
Although all class I- and class I-like molecules that we have analyzed to date undergo a transition to a 64-3-7-negative state after synthesis (31, 33, 34, 42), it remains formally possible that MR1 is an exception and does not achieve a 64-3-7-negative conformation, even if it binds a peptide or other specific ligand. In fact, this could explain the result from the denaturation experiment described above. Nonetheless, compelling evidence in favor of a conformational transition for MR1 was provided by denaturation analysis of the soluble hMR1 molecules secreted from insect cells (Fig. 8, right side). These molecules were poorly reactive with 64-3-7 in their native state but became strongly reactive following denaturation. This demonstrates that MR1 H chains are capable of assuming a 64-3-7-negative conformation, thus providing serological evidence that MR1 molecules transition to a folded conformation that is consistent with ligand binding by MR1.


   Discussion
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 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
Although the function of MR1 molecules is not known, recent findings strongly suggest that MR1 serves a novel role in immune surveillance in the gut. Tilloy et al. (57) defined an invariant TCR {alpha}-chain in mammals (encoded by V{alpha}7.2-J{alpha}33 in humans and V{alpha}19-J{alpha}33 in mice and cattle) that is expressed on a subpopulation of CD4-CD8- (DN) T cells. Developmental expression of this invariant TCR was shown to be {beta}2m-dependent, but TAP- and CD1-independent (57). Based on these observations, they speculated that an evolutionarily conserved class Ib molecule such as MR1 might select this invariant TCR. Indeed, very recent findings demonstrate that DN T cells expressing this invariant TCR (now referred to as mucosal-associated invariant T cells (MAIT)) are essentially absent in mice lacking the {alpha}1 and {alpha}2 domains of MR1, implying that MR1 is the selecting/restricting element for MAIT cells (30). Based on their findings, these authors speculate that MR1 might monitor commensal flora or serve as a distress signal in the gut. It is important to note that the epitope-tagged forms of MR1 and the MR1/Ld chimeric molecules used in this report are capable of activating MAIT cells (O. Lantz and E. Treiner, personal communication). Thus, expression of each of these constructs results in the presence of functional MR1 at the surface of the cells.

It is intriguing to interpret our MR1 biosynthesis and expression data in light of these recent functional data. Our results demonstrate that cell surface expression of MR1 is {beta}2m-dependent. However, multiple lines of evidence suggest that MR1 may have a relatively weak {beta}2m association when compared with most classical class I molecules. First, covalently attaching {beta}2m to hMR1 augmented surface expression (as opposed to the surface expression levels observed in transfectants of native MR1). Second, the improved surface expression of the mMR1 ({alpha}1-{alpha}2)/Ld molecule could be explained by enhanced {beta}2m association afforded by the {alpha}3 domain of Ld. Furthermore, a significant proportion (approximately one-third) of the recombinant hMR1 secreted from insect cells was associated with bovine {beta}2m (derived from the culture medium) instead of h{beta}2m. Regarding this latter observation, it is relevant to note that human classical class I molecules display little, if any, {beta}2m exchange (58), whereas only certain mouse classical class I molecules readily exchange {beta}2m (e.g.,48). Thus, MR1 may be atypical in the manner in which it interacts with {beta}2m, consistent with our structure-based sequence comparisons, which failed to identify any similarity to known {beta}2m association motifs in MR1. Nonetheless, MR1 requires association with {beta}2m for surface expression. Our observation of {beta}2m-dependent cell surface expression of MR1 is consistent with its role as the restriction element for MAIT cells, because they are not found in {beta}2m-deficient mice (57).

Perhaps the most important outstanding question from our studies concerns the identity of the putative MR1 ligand. Although circumstantial, we present multiple lines of evidence consistent with a ligand-presenting function for MR1. For example, secretion of recombinant MR1 by insect cells was dependent upon highly supplemented media, and recombinant MR1 from purified E. coli inclusion bodies failed to refold using a variety of refolding conditions. Furthermore, we present serological evidence consistent with MR1 undergoing ligand-induced folding, similar to classical class I molecules. If MR1 is dependent on ligand binding for folding and surface expression, it could also explain why high levels of surface expression have been difficult to achieve (22 and this report). Finally, MR1 interacts with all known members of the peptide-loading complex, including TAP, even though TAP-deficient mice contain MAIT cells (57). Based on these combined findings, it is intriguing to speculate that MR1 may bind to both TAP-dependent and -independent ligands, the latter being presented to MAIT cells, while MR1 containing TAP-dependent ligands may interact with additional cell types.

It is noteworthy that class Ib molecules that have been reported to bind the peptide-loading complex (HLA-E, HLA-G, and H2-M3; Refs. 33 ,59, 60, 61), also bind peptide ligands. Conversely, we have preliminary data indicating that HFE molecules (which do not bind peptide) do not associate with the peptide-loading complex (unpublished observation). Interestingly, HLA-F molecules, of unknown peptide-binding status, were recently shown to associate with TAP (62, 63). Like MR1, HLA-F molecules remain largely intracellular, consistent with speculation that they bind specialized ligands. The homology between MR1 and HLA-A/B/C class I molecules is {approx}35–40%, whereas HLA-F is {approx}75% identical to classical class I molecules (20). Therefore, we were somewhat surprised to observe an interaction between MR1 and the loading complex. Mutational analyses have identified residues within the {alpha}2 and {alpha}3 domains of classical class I H chains that are required for loading complex interaction (27, 64) and MR1 does not possess all of these consensus residues. Thus, MR1 may interact with the loading complex in a novel manner dictated by its specialized ligand binding requirements. Even if MR1 does not present a ligand, it may still require the loading complex to facilitate assembly with {beta}2m (as monitored by calreticulin; Ref.65) and/or isomerization of the {alpha}2 domain disulfide bonds (as monitored by ERp57; Ref.29). Regardless of the role served by the loading complex during MR1 biogenesis, MR1 represents the most divergent member of the class I family known to interact with this complex. Furthermore, it is the first example of a non-MHC encoded class I-like molecule that interacts with the peptide-loading complex.

The recent finding that MR1 is the restricting element for MAIT cells would suggest that MR1 is expressed on the surface of at least some cells. Consistent with this prediction, we provide initial evidence that MR1 can be detected at the cell surface, albeit in transfected cells and only at low levels. Surface expression of MR1 was clearly augmented 1) by covalent attachment of {beta}2m, suggesting that competition with other class I molecules for {beta}2m may limit MR1 expression; 2) by replacement of the C-terminal domains with those from Ld, suggesting that intracellular sorting may alter MR1 expression; or 3) by treating with IFN-{gamma} (S. M. Truscott, unpublished data) that is known to enhance the expression of diverse immune regulatory molecules. Therefore, our findings are most consistent with a model in which MR1 undergoes a conformational transition that may be coincident with ligand binding, or perhaps, posttranslational modification. However, under normal conditions ligand is limiting and the majority of MR1 H chains remain in the ER in association with the peptide-loading complex. Alterations in ligand availability and/or intracellular trafficking could lead to increased surface expression and, ultimately, interaction with MAIT cells.


   Acknowledgments
 
We thank Drs. Oliver Lantz and Emmanuel Treiner for sharing unpublished data, Dr. Seiamak Bahram for cDNAs encoding mMR1 and hMR1, Dr. Olga Naidenko for technical assistance, and Dr. Marco Colonna for support.


   Footnotes
 
1 This work was supported by National Institutes of Health Grants AI46553 (to T.H.H.) and T32AI07163 (to L.L.). Back

2 M.J.M and S.M.T. contributed equally. Back

3 Address correspondence and reprint requests to Dr. Ted H. Hansen, Department of Genetics, Box 8232, Washington University School of Medicine, 4566 Scott Avenue, St. Louis, MO 63110. E-mail address: hansen{at}genetics.wustl.edu Back

4 Abreviations used in this paper: MHC-I, MHC class I; HFE, hemochromatosis protein; MR1, MHC-related protein 1; {beta}2m, {beta}2-microglobulin; ER, endoplasmic reticulum; hMR1, human MR1; h{beta}2m, human {beta}2m; mMR1, mouse MR1; endo H, endoglycosidase H; MAIT, mucosal-associated invariant T cells; DN, double-negative; 6-His, six-histidine. Back

Received for publication January 31, 2003. Accepted for publication April 3, 2003.


   References
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 Introduction
 Materials and Methods
 Results
 Discussion
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