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The Journal of Immunology, 2000, 164: 1906-1915.
Copyright © 2000 by The American Association of Immunologists

The Murine Liver-Specific Nonclassical MHC Class I Molecule Q10 Binds a Classical Peptide Repertoire1

Francesca Zappacosta2,3,*, Piotr Tabaczewski2,{dagger}, Kenneth C. Parker4,*, John E. Coligan5,* and Iwona Stroynowski6{dagger},{ddagger}

* Laboratory of Allergic Diseases, National Institute of Allergy and Infectious Diseases, National Institutes of Health, Rockville, MD 20852; and {dagger} Center for Immunology and {ddagger} Departments of Microbiology and Internal Medicine, University of Texas Southwestern Medical Center, Dallas, TX 75235-9093


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
The biological properties of the nonclassical class I MHC molecules secreted into blood and tissue fluids are not currently understood. To address this issue, we studied the murine Q10 molecule, one of the most abundant, soluble class Ib molecules. Mass spectrometry analyses of hybrid Q10 polypeptides revealed that {alpha}1{alpha}2 domains of Q10 associate with 8–9 long peptides similar to the classical class I MHC ligands. Several of the sequenced peptides matched intracellularly synthesized murine proteins. This finding and the observation that the Q10 hybrid assembly is TAP2-dependent supports the notion that Q10 groove is loaded by the classical class I Ag presentation pathway. Peptides eluted from Q10 displayed a binding motif typical of H-2K, D, and L ligands. They carried conserved residues at P2 (Gly), P6 (Leu), and P{omega} (Phe/Leu). The role of these residues as anchors/auxiliary anchors was confirmed by Ala substitution experiments. The Q10 peptide repertoire was heterogeneous, with 75% of the groove occupied by a multitude of diverse peptides; however, 25% of the molecules bound a single peptide identical to a region of a TCR V ß-chain. Since this peptide did not display enhanced binding affinity for Q10 nor does its origin and sequence suggest that it is functionally significant, we propose that the nonclassical class I groove of Q10 resembles H-2K, D, and L grooves more than the highly specialized clefts of nonclassical class I Ags such as Qa-1, HLA-E, and M3.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
Molecular and biochemical analyses of class I MHC molecules led to the identification of two major subgroups of these proteins. The classical class I (class Ia) Ags are highly polymorphic, nearly ubiquitously expressed polypeptides that associate with self- and nonself 8–10 residue-long peptides (1, 2). They play key roles in T cell and NK cell-mediated elimination of virally infected and/or malignantly transformed cells. The nonclassical class I (class Ib) Ags are a heterogeneous group of ß2-microglobulin (ß2m)7-associated proteins that display little polymorphism and frequently exhibit low level expression and/or unique tissue distributions (3, 4). Furthermore, many of the class Ib molecules exist in soluble forms that are secreted into the serum and body fluids (5, 6).

Recent studies of rodent and human members of class Ib families revealed remarkable diversity of their ligands, Ag-presenting capacities, and immune as well as nonimmune functions (7, 8, 9, 10, 11, 12). Some of the membrane-bound class Ib proteins are dedicated to presentation of structurally unique forms of ligands. For example, M3 Ag widely expressed on murine tissues, binds selectively N-formylated peptides of mostly prokaryotic origin (13). This property allows M3 to be recognized as a restriction element during CD8+ T cell-mediated clearance of bacterial infections. Another ubiquitously expressed murine Ag, Qa-1, as well as its proposed human homologue HLA-E, associate preferentially with a limited set of hydrophobic leader peptides from class I MHC Ags (14, 15). The resulting class Ib complexes serve as targets for alloreactive cytotoxic T cells, as shown for Qa-1 (16), and as recognition elements for NK receptors (17, 18, 19).

Not all of the known class Ib proteins bind structurally unique ligands. Some, such as murine Qa-2 and human HLA-G, associate with diverse repertoires of peptides reminiscent of class Ia peptides (20, 21). The biological significance of these types of classes Ib complexes is still poorly understood (22). Additionally, very little is currently known about Ag-presenting properties or function(s) of soluble class Ia or Ib molecules reported to exist in a wide range of species, including mouse (23) and human (24, 25).

To address these issues, we performed analysis of ligands associated with the soluble Q10 class Ib protein. This 38- to 40-kDa ß2m-associated molecule is detectable in serum as a multivalent complex of 200–300 kDa, at concentrations ranging from 20 to 60 µg/ml, depending on the mouse strain (26, 27). The Q10 proteins are encoded in the Q region of the H-2 complex, which also contains Qa-2 genes (3), and a cluster of several other class Ib sequences. In common with other Q region class Ib genes, Q10 shows >80% homology with the classical H-2K, D, and L loci (28). Structurally, the protein is truncated at the C terminus and carries several substitutions in the hydrophobic region corresponding to the transmembrane segments of class Ia heavy chains. These features account for the inability of Q10 to insert into the plasma membrane and explain why Q10 is secreted (26, 28).

The Q10 locus exhibits two hallmarks of class Ib genes: it is well conserved, with >99.4% homology between different sequenced alleles (28), and it is expressed in tissue-specific fashion. In adult mice, the protein is synthesized mainly by liver and, in trace amounts, by kidney and stomach (26, 29). During early development, Q10 transcripts are detectable in major organs of fetal hematopoiesis: visceral yolk sac and fetal liver (30). This expression pattern led to the speculation that Q10 participates in the induction of T cell tolerance and/or regulation of embryonic hematopoiesis. We demonstrate here that the peptide-binding ({alpha}1{alpha}2) domains of Q10 associate with eight and nine residue-long self-peptides similar to the class Ia ligands and discuss this finding in the context of potential T cell and NK cell recognition.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Cloning of Q10 cDNA and construction of hybrid genes

The nonpolymorphic Q10 cDNA was isolated from the NOD/Lt (H-2g7) cDNA liver library derived by Girgis et al. (31). The cDNA fragment encoding the N-terminal portion of Q10 (exons 1–3) was amplified by PCR, subcloned into pIC20H plasmid (American Type Culture Collection (ATCC), Manassas, VA), and sequenced. It was found to be identical to the genomic sequence of Q10 from C3H mouse (32) and cDNAs amplified from C57BL/6 and C57BL/10 mice (data not shown).

We designed two hybrid Q10/Qa-2 molecules. The first, MQ10, encodes the N-terminal portion of Q10 (leader peptide, {alpha}1 and {alpha}2) and the C-terminal portion of Qa-2 ({alpha}3 and the glycosylphosphatidylinositol (GPI) moiety linking Qa-2 product to the cell surface). MQ10 is membrane bound. The second hybrid molecule, SQ10, consists of the same N-terminal domains of Q10 linked to the {alpha}3 domain of the soluble form of Qa-2, followed by six additional histidines (6xHis-tag), and is secreted. The C-terminal domains of MQ10 and SQ10 were derived from different isoforms of Qa-2 genes, Q9m and Q7s, respectively (5).

The following pairs of primers were used to amplify parts of H-2 molecules: for the {alpha}1{alpha}2 region of Q10, (P1) 5'-AAACCCGTCGACGATCCCAGATGGGGGCGATGGCG-3' (signal peptide sequence in bold, SalI site underlined) and (P2) 5'-AAACCCAGATCTGTGCGCAGCAGCGTCT-3' (C-terminal part of {alpha}2 domain in bold, BglII site underlined); for the {alpha}3 region of Q9m, (P3) 5'-GCGCACGGATCCCCCAAAGGCACATGTGACCCATC-3' (Q9m {alpha}3 N-terminal region in bold, BamHI site underlined), and (P4) 5'-CTGCAGCTCGAGTCATGCTGGAGCTGGAGCACAGTCCCC-3' (Q9m C terminus in bold, stop codon in bold italics, and XhoI site underlined); for the {alpha}3 region of Q7s (P3) and (P5) 5'-CCAATCGAATTCGCTGGAGCTGGAGCACAGTCCCC-3' (Q7s C terminus in bold, EcoRI site underlined). The Q9m and Q7s fragments were cloned into plasmid pIC20H and the Q10 fragment into Bluescript II KS(-) (Stratagene, San Diego, CA), respectively, using the indicated underlined sites. To add sequences encoding six histidines followed by a stop codon (in italics = 6xHis-tag), we inserted a synthetic linker 5'-AGCGAATTCACATCACCATCACCATCACTGACTGCAC-3' at the C terminus of the Q7s fragment (in bold) using the underlined EcoRI and XhoI sites. The DNA structures of all fragments were verified by sequencing. Recombinant MQ10 and SQ10 clones were constructed by combining the Q10 fragment with Q9m or Q7s6xHis-tag DNAs in vector pIC20H in the following configuration: SalI–Q10–BglII/BamHI–Q9m–XhoI or SalI–Q10–BglII/BamHI–Q7s6xHis-tag–XhoI. SalI/XhoI fragments containing full-size hybrid Q10 genes were cloned subsequently into the XhoI site of vector pBJ5 behind the ubiquitous SR{alpha} promoter (33) (pBJ5/MQ10 and pBJ5/SQ106xHis-tag). These plasmids were used to transfect various cell lines.

Antibodies

The mAbs 46 (anti-{alpha}3 of Qa-2) (34) and S19.8 (anti-mouse ß2mb) (35) used in Q10-affinity purification and ELISA were purified from mouse ascites fluid or from {gamma}-globulin-free tissue culture supernatants with protein A-Sepharose CL-4B (Pharmacia, Piscataway, NJ) using standard protocols (36). Secondary mAbs used in ELISA were biotinylated with N-hydroxysuccinimidobiotin (Sigma, St. Louis, MO) as described previously (36).

Cell lines and tissue culture

The murine cell lines RMA and its TAP2-deficient mutant, RMA-S (37), were transfected with linearized Q10 constructs and pHEKneo vector (G418 resistance marker) by electroporation as described previously (38). Transfectants expressing the highest levels of MQ10 were selected by flow cytometry with mAb 46. Clones secreting the highest levels of SQ10 were identified by a two-Ab sandwich ELISA (see below) and further characterized by immunoprecipitation with mAb 46. Transfectants were propagated in the presence 0.15 mg/ml of active G418 (Fisher Scientific, Pittsburgh, PA). Large scale cultures of RMA/MQ10-positive cells were grown in the Laboratory of Cellular and Developmental Biology, National Institute of Diabetes and Digestive and Kidney Diseases, National Institutes of Health (Rockville, MD) under the direction of Dr. J. Shiloach. Large scale cultures of RMA/SQ10-positive cells were grown to saturation (2 x 106/ml) in Fenwal Lifecell TC flasks of 3-liter capacity (Baxter Scientific Products, McGaw Park, IL) at the University of Texas Southwestern Medical Center, Dallas. The rat YB2/0 (39) and human C1R (40) cell lines were obtained from ATCC (ATCC CRL 1662) and Dr. J. Forman (University of Texas Southwestern Medical Center, Dallas), respectively. The RMA and RMA-S transfectants expressing MQa-2 and SQa-2 were described elsewhere (38, 41).

Flow cytometry

Cells from subconfluent cultures were stained by indirect immunofluorescence using FITC-conjugated goat-anti mouse IgG as the secondary Ab (Cappel, Durham, NC). The acquisition was performed by FACScan (Becton Dickinson, Mountain View, CA). Data were analyzed with the Lysis program (Becton Dickinson). Dead cells were excluded by a combination of gates set on forward/side scatter and by exclusion of cells staining positive with propidium iodide dye.

Radiolabeling and immunoprecipitation

Radiolabeling and immunoprecipitations were conducted by a modification of a standard method described previously (5, 36). Briefly, RMA transfectants (107 cells) were harvested at the logarithmic phase of growth (8 x 105/ml), washed twice in ice-cold PBS, and resuspended in labeling medium: 1 ml of methionine/cysteine-deficient RPMI 1640 medium (ICN Pharmaceuticals, Costa Mesa, CA) supplemented with 10% dialyzed FBS and 0.5 mCi of [35S]methionine and cysteine (Trans 35S-label; ICN Pharmaceuticals). For phosphatidylinositol-specific phospholipase C (PI-PLC) treatment, tissue culture media were supplemented with 0.3 U of PI-PLC (American Radiolabeled Chemicals, St. Louis, MO). After incubation for 4 h at 37°C, cell supernatants were harvested and precleared with 50 µl of normal rabbit serum. Recombinant Q10 and control proteins were precipitated with saturated amounts of mAb 46 Ab. Ag-mAb 46 complexes were bound to protein A-Sepharose CL-4B (Sigma), washed six times with PBS, denatured, reduced, and analyzed by one-dimensional SDS-PAGE. Gels were stained with Coomassie blue. Radioactively labeled proteins were detected by autoradiography.

Measurement of expression levels and stability of Q10 molecules by ELISA

RMA, RMA-S cells, and their transfectants were grown to a density of 8 x 105 cells/ml. Caps of tissue culture flasks were tightened, and cells were incubated overnight at room temperature. Cells were harvested and washed three times with ice-cold PBS. Pellets were lysed with 0.5% nonionic detergent Nonidet P-40 (Sigma) in 0.2 M phosphate buffer (pH 7.05) and in the presence of proteinase inhibitors: pepstatin A, 5 µg/ml; leupeptin, 2 µg/ml; benzamidine, 2.5 mM; soybean trypsine inhibitor, 20 µg/ml; PMSF, 100 µM; and EDTA, 4 mM. Cell nuclei were pelleted by centrifugation. The protein concentrations were measured using the bicinchoninic acid protein assay (Pierce, Rockford, IL). Where appropriate, adjustments were made to standardize protein concentrations of lysates. Supernatants containing class I complexes were stored on ice until needed (no more than 16 h). To measure MHC levels, we used a modified semiquantitative two-Ab sandwich ELISA assay (41). For MQ10 and SQ10 measurements, mAb 46 (anti-{alpha}3 of Qa-2) was used as primary Ab and biotinylated mAb S19.8 (anti-ß2mb) as secondary Ab. The assay for Qa-2 was performed using the same protocol on cell lysates of MQa-2 transfectants (41). The assay for H-2Kb was performed similarly with mAb 20-8-4 as a primary Ab and biotinylated mAb Y3 as a secondary Ab (41).

Isolation of endogenous peptides from Q10 complexes

MQ10 and SQ10 complexes and their ligands were purified using two different methods. Endogenous peptides bound to MQ10 molecules were isolated by a modification of a method previously described for other membrane-bound class I complexes (42). A total of 1010 MQ10-transfected RMA cells were lysed in 20 mM Tris-HCl (pH 8.0), 150 mM NaCl, 1% 3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonate, 0.25% sodium deoxycholate, 1 mM PMSF, 100 mM iodoacetamide, 5 µg/ml aprotinin, 10 µg/ml leupeptin, 10 µg/ml pepstatin A, 5 mM EDTA, and 0.04% sodium azide. After centrifugation, the cell lysate was loaded onto a column of inactivated Sepharose CL-4B and subsequently onto a Sepharose CL-4B column to which mAb 46 had been coupled (36). After extensive washing, MQ10 complexes were eluted with 10% acetic acid. The released peptides were isolated by centrifugation through an Ultrafree-CL 5kDa microconcentrator (Millipore, Bedford, MA) and concentrated by lyophilization to 250 µl. Peptides were separated by RP-HPLC as described elsewhere (42). Individual fractions were collected, dried, and stored at -20°C before mass spectral analysis.

SQ10 molecules were purified from 50 liters of supernatant of RMA/SQ10 transfectants (P29-3.4) collected over a 3-wk period. The medium collected from cells was supplemented with 0.2% w/v sodium azide and stored at 4°C. The pooled supernatant was concentrated to 4 liters by ultrafiltration using a hollow fiber cartridge with a 30-kDa cutoff (model UFP-3-C-5; A/G Technology, Needham, MA). The concentrate was spun at 14,000 x g for 2 h and filtered through a 0.2-µm membrane. Tris-HCl was added to a final concentration of 0.1 M, and the pH was adjusted to 8.0. SQ10 proteins containing the 6xHis-tag were purified by metal affinity chromatography. Briefly, 30 ml of Ni-NTA Sepharose beads (Qiagen, Chatsworth, CA) was stirred gently overnight at 4°C with the concentrated sample containing recombinant SQ10. Sepharose beads suspension was transferred to a chromatography column and extensively washed. The SQ10 was eluted from the column with 2 column volumes of PBS and 250 mM imidazole. To achieve prompt neutralization, fractions (5 ml) were collected in tubes containing 0.5 ml of 1 M Tris-HCl (pH 7.0). Fractions containing SQ10 were identified by sandwich ELISA. SQ10 molecules were further affinity purified with a mAb M46 column, as described above, for the membrane-bound isoform of the protein. The SQ10 complexes were eluted from affinity columns and allowed to dissociate by treatment of the slurry with 10% acetic acid. The mixture of released peptides was separated from the high m.w. and RP-HPLC was purified as described above for MQ10.

Peptide sequence analysis

All mass spectrometric data were acquired on an API 300 triple quadrupole mass spectrometer (PE-SCIEX, Toronto, Ontario, Canada) equipped with a MicroIonSpray source as previously reported (42). The program MS-Tag, written by Karl Clauser and Peter Baker, and available on the worldwide web at http://prospector.ucsf.edu, was used to match collision-activated dissociation (CAD) spectra against the protein sequence databases available at the web site. N-terminal amino acid sequence analysis was performed by standard automated Edman degradation.

Peptide synthesis

Peptides were synthesized as described previously (41, 43). Purity and sequence of the synthetic peptides was established by analytical RP-HPLC and mass spectrometry.

Protein analysis

Mass spectrometric analysis of the proteins retained by the ultrafiltration membrane, intact or after deglycosylation, was performed after purification on a narrow-bore Vydac C4 column (150 x 2.1 mm, 5 µm, 330 Å pore size) using the gradient described above for peptide separation. Samples were directly injected into the mass spectrometer ion source by infusion at 1 µl/min.

Deglycosylation of SQ10His heavy chain was conducted in 0.5% 3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonate, 100 mM Tris-HCl (pH 8.0), and 0.1 mM DTT. The sample was incubated with 0.5 U of N-glycosidase F (Boehringer Mannheim, Indianapolis, IN) at 37°C for 16 h and subsequently purified by RP-HPLC for mass spectrometric analysis.

Analysis of peptide binding to class I molecules

ELISA-based peptide-induced stabilization assays were conducted by a modification of a method described earlier (41). Briefly, graded concentrations of synthetic peptides were added to cell lysates of RMA-S cells expressing MQ10 molecules (transfectant P25-1). The mixtures were kept on ice for 16 h, followed by an 80-min incubation at 42°C. The presence of conformationally stable MQ10 serologic epitopes was detected by sandwich ELISA with mAb 46 and S19.8 Abs as described above in "Measurement of expression levels and stability of Q10 molecules by ELISA".


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
Construction of cell lines expressing hybrid Q10 proteins

To perform direct biochemical analysis of endogenously synthesized Q10-binding ligands, it is necessary to isolate sufficient quantities of the relevant class I complexes. Since there are currently no known mAbs that would allow purification of wild-type Q10 from serum, we cloned Q10 cDNA and expressed Q10 molecules as class I hybrid proteins in transfected tissue-cultured cell lines (Fig. 1Go). The two putative ligand-binding domains ({alpha}1 and {alpha}2) of Q10 were fused to the {alpha}3 domain and C-terminal portion of another Q region protein, Qa-2 (44, 45, 46). The Qa-2 proteins exist in two isoforms: membrane-bound Qa-2 attached to cell surface via GPI moiety (MQa-2) and soluble Qa-2 derived from the same gene by alternative splicing (5, 46) (SQa-2). The choice of the {alpha}3 domain in the Q10 hybrids was dictated by the high homology between the Q10 and Qa-2 sequences (28, 45), by availability of multiple mAbs recognizing unique Qa-2 epitopes on the {alpha}3 domain (34, 35, 38), and by previous studies showing that the shuffling of Qa-2 domains with other class I domains does not disturb the conformation of the {alpha}1{alpha}2 portion of hybrid complexes (47, 48).



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  		FIGURE 1. Structure of hybrid MQ10 and SQ10 constructs and proteins. Exons originating from Q10 (black boxes) and Qa-2 genes (open boxes) are denoted as leader (L), {alpha}1, {alpha}2, {alpha}3, transmembrane (TM), and cytoplasmic (cyt). GPI anchor of MQ10 is denoted as GPI and 6xHis-tag of SQ10 is denoted as 6His. Positions of primers used for PCR amplification of Q10 and Qa-2 cDNAs are indicated by P1-P5 arrows. Note that the Q10 promoter has been replaced with the SR{alpha} that allows expression in all cell types.

 
Two forms of Q10 hybrid genes were constructed: SQ10, encoding soluble form of Q10, and MQ10, encoding membrane-bound, GPI-linked Q10 protein. The structure of the predicted hybrid Q10 genes and proteins is depicted in Fig. 1Go. The hybrid constructs were transfected into lymphoid-derived cell lines: murine (RMA), rat (YB2/0), and human (CIR) cell lines. High levels of Q10 proteins were detected in every case, suggesting that assembly of these complexes is not limited by the lack of appropriate ligands or chaperones (data not shown and see below). We were unable to perform any studies with transfected liver cell lines because these cells could not be grown to the densities necessary for biochemical characterization of the hybrid molecules (46).

To verify the integrity of Qa-2 conformational epitopes on the {alpha}3 domain of MQ10, the transfected murine RMA cells were stained with six anti-{alpha}3 Qa-2 mAbs (data not shown). All reacted with MQ10 as well as with control wild-type Qa-2-positive cells. As expected, anti-{alpha}1{alpha}2 Qa-2 Abs did not react with MQ10.

The m.w. of MQ10 and SQ10 hybrids, their association with ß2m, and GPI attachment of MQ10 were tested as follows. RMA cells transfected with hybrid Q10 constructs and control Qa-2 genes were biosynthetically labeled with [35S]methionine and cysteine and, where appropriate, treated with PI-PLC, which specifically cleaves GPI-linked molecules and releases them from the cell surface (Fig. 2GoA). The supernatants containing SQ10, PI-PLC-cleaved MQ10, and control Qa-2 molecules were immunoprecipitated with anti-{alpha}3 Qa-2 Ab MAb 46 (34). As predicted, all of the analyzed Q10 and Qa-2 molecules migrated in SDS-polyacrylamide gels with apparent mass of 39–40 kDa (Fig. 2GoA). The SQ10 heavy chain migrates slightly faster in SDS gel than MQ10 released by PI-PLC treatment, which is in agreement with similar observations for SQa-2 and MQa-2 (5). The 39- to 40-kDa weight estimates are compatible with two carbohydrate moieties attached to the mature Q10 and Qa-2 proteins at the putative N-linked glycosylation positions at residues 86 and 256 (3).



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  		FIGURE 2. Hybrid Q10 proteins have the predicted m.w. and are associated with ß2m. A, Biochemical analysis of Q10 proteins expressed in transfected RMA cells. MQ10- and SQ10-transfected and control cells were biosynthetically labeled for 4 h with [35S]methionine and cysteine, and supernatants collected from an equal number of cells were analyzed by immunoprecipitation with mAb 46. The precipitated molecules in lanes 1–10 originated from supernatants of the following cells: lanes 1 and 2, two independent RMA transfectants of SQ10 (P29-4.4 and P29-4.1); lane 3, RMA cells transfected with soluble Qa-2 carrying the 6xHis-tag at the C terminus (P29-3.4); lane 4, RMA cells synthesizing soluble Qa-2 without the 6xHis-tag (P1-6.60); lane 5, PI-PLC-treated RMA cells expressing MQ10 (M15-3); lane 6, RMA cells expressing MQ10 (M15-3) that were not treated with PI-PLC; lane 7, PLC-treated RMA transfectants expressing membrane-bound Qa-2 (P5-5.6); lane 8, RMA transfectants expressing membrane-bound Qa-2 (P5-5.6) that were not treated with PI-PLC; and lanes 9 and 10, control untransfected RMA cells treated (9 ) and untreated (10 ) with PI-PLC. B, Coomassie blue-stained and SDS-PAGE-resolved purified SQ10. The heavy chain and ß2m are indicated by arrows. The additional bands correspond to heavy and light Ig chains which were also released from the mAb 46 immunoaffinity column. Coomassie blue-stained ß2m may include murine and bovine species, whereas radioactively labeled ß2m in Fig. 2GoA corresponds to endogenously synthesized murine ß2m only.

 
Both SQ10 and PI-PLC-cleaved MQ10 coimmunoprecipitated with biosynthetically labeled murine ß2m (Fig. 2GoA). The relative proportions of radioactively labeled murine ß2m precipitated with Q10 heavy chain were lower than those observed in Qa-2 immunoprecipitates, suggesting that these two types of class I chains differ in their ability to associate with endogenous ß2m. There are no Q10/Qa-2 differences involving the putative interdomain contact residues affecting the interaction of the {alpha}3 domain with ß2m or between the {alpha}3 domain with the {alpha}1{alpha}2 domains, assuming homologous interactions to those found by x-ray crystallography for HLA-A2 (49). Therefore, it is likely that the observed lower affinity of Q10 for murine ß2m and its replacement with bovine ß2m (see high levels of unlabeled ß2m recovered from Q10 complexes in Fig. 2GoB and see below) is caused by Q10 specific residues in the {alpha}1 and {alpha}2 domains that contact ß2m directly. These may involve residues 6 and 9 of {alpha}1 and residue 116 of {alpha}2.

Partial TAP dependence of MQ10 membrane expression

Multiple studies have demonstrated that mutations in the TAP genes, that direct synthesis of the peptide transporter molecules in the class I Ag presentation pathway, lead to reduced levels of classical class I Ags on the cell surface (37, 50). This phenotype is thought to result from limiting quantities of peptide ligands delivered to the endoplasmic reticulum in TAP-negative mutants and the resulting instability of "empty" class I-ß2m complexes. In most cases, the decrease in membrane expression can be reversed by low temperature (~26°C), which stabilizes peptide-free class I complexes that reach the cell surface in TAP-negative cells.

To address the question of whether MQ10 associates with TAP-delivered peptides, we introduced MQ10 into TAP2-negative RMA-S cells and compared its expression to the parental RMA cells by FACS staining (Fig. 3Go). The control H-2Kb Ag coexpressed on MQ10 tranfectants displayed classical TAP-dependent behavior: ~4-fold reduced expression in RMA-S vs RMA cells at 37°C and 42°C and ~8-fold induction of H-2Kb levels in RMA-S cells at 26°C (38). Qa-2 Ag showed a more drastic reduction of surface levels in RMA-S compared with RMA cells (12–14-fold) at 37°C and 42°C (38). This expression was enhanced only weakly at 26°C, in agreement with our previous data showing that most of the empty Qa-2 fail to reach the cell surface in TAP2-negative cells and accumulate intracellularly (41). MQ10 expression showed a TAP2-dependent phenotype intermediate between H-2Kb and Qa-2. Surface MQ10 levels were reduced (~5-fold in RMA-S vs RMA cells) and were only weakly inducible at 26°C. Thus, compared with wild-type Qa-2, MQ10 contains a somewhat larger fraction (~20%) of heat-stable molecules that reach the cell surface in a TAP2-independent fashion in RMA-S cells. This phenotype is most likely controlled by the structural properties of the {alpha}1 and {alpha}2 domains of Q10.



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  		FIGURE 3. Staining of MQ10 and control MQa-2 and H-2Kb molecules on transfected RMA (A) and RMA-S (B) cells at 26, 37, and 42°C. MQ10 transfectants correspond to sorted, mixed populations of RMA and RMA-S clones expressing the top 5% of MQ10 levels from each transfected cell line. Fluorescence peak channels for MQ10/RMA (mAb 46) were 1715 (26°C), 1703 (37°C), and 1911 (42°C); for MQ10/RMA-S (mAb 46) were 437 (26°C), 365 (37°C), and 352 (42°C); for MQa-2/RMA (mAb 46) were 1433 (26°C), 1596 (37°C), and 1590 (42°C); for MQa-2/RMA-S (mAb 46) were 154 (26°C), 133 (37°C), and 111 (42°C); for H-2Kb/RMA (mAb Y3) were 1286 (26°C), 626 (37°C), and 523 (42°C); for H-2Kb/RMA-S (mAb 46) were 1197 (26°C), 178 (37°C), and 149 (42°C); FITC only, ~15 (37°C).

 
To confirm that the majority of MQ10 complexes in TAP2-negative cells remain intracellular and behave as heat-unstable empty molecules, we analyzed MQ10 from lysates of RMA-S transfectants using conformation-dependent ELISA. The data in Fig. 4Go show that almost all MQ10 molecules, as well as the control H-2Kb and Qa-2 molecules released from lysates of transfected RMA-S cells, loose conformational epitopes upon incubation at 42°C for 80 min, whereas the majority of RMA-expressed complexes are stable under the same conditions. The partial loss of conformational epitopes (in ~30% of the RMA-derived complexes) may be indicative of empty molecules, which accumulate intracellularly because they have not been loaded with peptides or, alternatively, may reflect the fact that some molecules associate with peptides of low affinity that are released upon heat shock.



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  		FIGURE 4. Intracellular MQ10 molecules are unstable at high temperature in RMA-S cells. The concentrations of folded MQ10, H-2Kb, and MQa-2 molecules were measured in cell lysates of transfected RMA (A) and RMA-S cells (B) using conformation-dependent sandwich ELISA. The prechilled lysates were heat shocked at 42°C for the indicated time intervals. The data were standardized so that the time point 0 (no heat shock) corresponds to 100% expression of the conformational epitopes on folded molecules at 4°C.

 
Taken together, these observations are consistent with the notion that the majority (~80%) of mature MQ10 molecules require a functional TAP pathway for cell surface expression. This property suggests that {alpha}1{alpha}2 of MQ10 molecules are peptide loaded.

Isolation and sequencing of peptides associated with membrane-bound and soluble Q10 proteins

The MQ10 and SQ10 complexes expressed in RMA cells were purified by immunoaffinity chromatography and the sequences of several endogenously bound peptides were determined by tandem mass spectrometry (MS/MS).

Because of the different properties of the secreted and membrane-bound class I Ags, the two Q10 complexes were purified using slightly different approaches (see Materials and Methods).

The SQ10 complexes were purified from tissue culture medium using metal affinity chromatography followed by immunoaffinity chromatography using the anti-{alpha}3-specific mAb 46. In our previous studies with human MHC class I molecules, we routinely quantitated the amount of purified complex by measuring the concentration of ß2m that was retained by the ultrafiltration membrane used to separate the peptides from intact proteins. The intact proteins retained in the high m.w. fraction were separated by RP-HPLC, and the amount of ß2m present was estimated by both Edman degradation and absorbance at 280 nm. In the SQ10 preparation both the ß2m and the SQ10 heavy chain were readily detected, allowing us to estimate that about 4 nmol of complex had been purified. Electrospray ionization mass spectrometry (ESI/MS) analysis of the fraction containing the SQ10 heavy chain yielded a molecular mass of 38,874 Da, about 4850 mass units greater than expected for the unglycosylated molecule (molecular mass, 34,026 Da). This mass difference is most likely due to N-linked carbohydrate moieties at Asn86 and Asn256, and it could be accounted for by two triantennary carbohydrate structures. After digestion with N-glycosidase F, in fact, two components with molecular masses of 36,518 and 34,170 Da, respectively, were detected, most likely corresponding to a partially and a completely deglycosylated form of the protein. When the fraction containing ß2m was analyzed by Edman sequencing, a mixed sequence was obtained, indicating that about 20% murine ß2m and 80% bovine ß2m was present; ESI/MS analysis detected only bovine ß2m (molecular mass, 11,632). The preferential association of the SQ10 heavy chain with bovine ß2m is consistent with the observation that murine ß2m undergoes exchange with other species of ß2m present in the medium (Fig. 2Go). The long period of incubation of the SQ10 complex in the FCS-supplemented tissue culture supernatants before purification may account for the observed high proportion of bovine ß2m in SQ10 complexes.

MQ10 hybrid molecules were immunoaffinity-purified using mAb 46 specific for the {alpha}3 domain of Qa-2. In this preparation, to our surprise, no ß2m or MQ10 heavy chain was recovered from the ultrafiltration membrane, making it impossible to quantify the amount of class I complexes that had been purified, even though peptides could easily be detected (see below).

The peptides associated with both MQ10 and SQ10 were acid extracted and separated by narrow-bore HPLC. The HPLC profile of the MQ10-associated peptides is shown in Fig. 5GoA. An enlarged view of the region containing the majority of the eluted peptides is shown in Fig. 5GoB. The anticipated peptide-containing fractions were analyzed by ESI/MS. The ESI/MS analysis showed the presence of at least 50 peptides for MQ10 and 110 peptides for SQ10 (data not shown) whose molecular mass fell into the mass range appropriate for 8–11-mer peptides, many of which were present in both samples. The larger number of peptide signals detected for SQ10 may reflect the ability of the soluble form to bind a larger number of peptides. Alternatively, because we were not able to quantify the amount of MQ10 complexes purified, it might simply indicate that a larger quantity of purified SQ10 complex was available for analysis.



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  		FIGURE 5. RP-HPLC separation of MQ-10 associated peptides. A, RP-HPLC chromatogram of peptides eluted from MQ10 complexes following immunoaffinity purification. B, Enlarged view of the elution profile. Fractions eluted between 20 and 45 min were analyzed by ESI/MS. Superimposed on the profile for the MQ10-associated peptides (top line) is the corresponding region of the chromatogram for material obtained upon elution from a nonspecific adsorbent consisting of glycine coupled to Sepharose (bottom line).

 
CAD analysis (51) performed on selected ions present in both the MQ10- and SQ10-purified material defined plausible amino acid sequences for six peptides (Table IGo). A representative CAD spectrum is shown in Fig. 6Go for the peptide with mass:charge ratio (m/z) of 920.2 (peptide 5, Table IGo). By this means, complete sequences were obtained for several peptides. For peptide QGVQXXDF (peptide 5) assignment of Q vs K (which are of nearly identical mass) was made by MS analysis following acetylation. This derivatization resulted in a single 42 mass unit shift, leading us to conclude that the N-terminal amino group is the only amino group present in the peptide. Four of the six peptides were eight amino acids long, and two were nine amino acids long, suggesting that depending on the sequence, Q10 preferentially binds octamers but occasionally nonamers, similar to H-2Kb, -Kk (2) and HLA-B8 (52). All six of the peptides contained Gly at P2, five of the six peptides contained Lxx at P6, and at P{omega} all peptides contained a hydrophobic residue: either Phe (in four sequences) or Lxx. The other positions of the peptides were more variable.


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  		Table I. Sequences of Q10-binding peptides are homologues to endogenous murine proteins

 


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  		FIGURE 6. CAD mass spectrum of peptide ions at m/z 920.2 (peptide 5). Predicted masses for fragment ions of types b and y (51 ) are shown above and below the deduced sequence, respectively. Ions observed in the spectrum are underlined. Interpretation of CAD spectra is fully explained elsewhere (51 ). Because Ile and Leu are of identical mass, they cannot be differentiated on the triple quadrupole instrument and are specified here as Lxx.

 
One of the largest peaks in the absorbance trace, at 34 min (Fig. 5Go), contained peptide TGTETXYF. Unlike most of the other peaks (some of the larger of which were present both in the nonspecific material eluted from glycine-Sepharose and in the Q10 mAb eluate), the peak at 34 min had an UV spectrum with a maximum at 278 nm, typical for peptides containing tyrosine residues, leading to the conclusion that the large absorbance of the material in this peak is largely due to peptide TGTETXYF and not to unrelated molecules. On the basis of absorbance and Edman degradation data (which matched the MS/MS sequence with Leu at P6), we concluded that there was about 250–300 pmol of peptide TGTETLYF in the MQ10 preparation and about 1 nmol in the SQ10 preparation; all other peptides were 60- to 100-fold less abundant. These estimates suggest that the TGTETLYF peptide may constitute as much as ~25% of the total Q10 ligand pool.

Synthetic peptides corresponding to the sequences of the constitutively bound peptides form complexes with MQ10 in vitro

When gene and protein sequence databases were searched for possible parent proteins of the Q10-specific peptides, four peptides from Table IGo matched murine protein sequences. Interestingly, all of these putative proteins correspond to fairly abundant polypeptides. Peptide 1 is identical to an octameric sequence present within two distinct proteasome subunits: constitutively expressed PSMB5 (53) and IFN-{gamma} regulated LMP7 (54). Peptide 2 is homologous to ribophorin (accession number D31717.1) and peptide 6 to cytochrome c oxidase (accession number P43024). The putative source protein of the most abundant peptide (peptide 3) corresponds to TCR V ß-chain (55) and is the only source protein which would not be normally expressed in liver cells. Its presence in the Q10-transfected RMA cells is consistent with the lymphoma phenotype of the parental line. All of the putative source proteins are expressed intracellularly, suggesting that their peptide components were introduced into the Q10 grooves by the class I Ag presentation pathway and were not incorporated into the complexes during the purification procedure from extracellular sources such as tissue culture medium. The alignment of peptide sequences 1, 2, and 6 with their putative sources allowed us also to assign Leu and Ile for these three peptides.

Peptides corresponding to the six sequences reported in Table IGo were synthesized using Leu as the default amino acid in positions where Leu could not be distinguished from Ile (reported as X in Table IGo). In each case, CAD fragmentation spectra were identical to the spectra derived from the Q10-associated peptides, confirming that the deduced sequences were concordant by this criteria. However, when the RP-HPLC retention times of the six synthetic peptides were compared with those obtained for the endogenous Q10 peptides, peptides 4 and 5 showed a higher retention time than expected, likely due to the presence of Ile instead of Leu at some positions. Due to a large number of potential permutations of Leu and Ile in peptides 4 and 5, we have not synthesized additional candidate peptides.

To verify that the sequences obtained in this study represent genuine endogenous peptides that can associate specifically with Q10, the synthetic homologues were tested in an in vitro peptide-binding assay. The assay measured the peptide-dependent stabilization of MQ10 epitopes on the {alpha}3 domain (recognized by mAb 46) and murine ß2m (recognized by mAb S19.8) by sandwich ELISA. The signal:background ratio of this assay is lower than the one observed with MQa-2 (data not shown). This effect may be explained by preferential displacement of murine ß2m from MQ10 heavy chain by bovine ß2m and/or by higher background of "temperature-resistant" MQ10 complexes formed in transfected RMA-S cells. Five of the six synthetic peptides stabilized the MQ10/ß2m complexes over a wide range of peptide concentrations: 100 ng to100 µg, as shown in Figs. 7Go and 8. Although the peptide stabilization assay cannot be regarded as a rigorous measurement of peptide affinity, the half-maximal and maximal points of concentration curves in Fig. 8Go do not give any indication that the dominant TGTETLYF peptide binds Q10 better than other titered peptides. Hence, we conclude that the peptide-binding affinity of TGTETLYF is comparable to other peptides tested by ELISA approach. Peptide 4, LGAALLGDL, was consistently negative in our assay (comparable to negative controls in Fig. 7Go). This peptide contains four Leu residues synthesized as default amino acids in positions in which Leu could not be distinguished from Ile in the endogenous Q10 sequence.



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  		FIGURE 7. Stabilization of MQ10 conformational epitopes with Q10 synthetic peptides. Group A peptides correspond to synthetic homologues of Q10 peptides listed in Table IGo, B is a poly(A) nonamer AAAAAAAAA, group C peptides are (left to right): RYWAIRTRS, FRYNGLIHL, RYWATRSGG, and GRIDKPILK. All peptides have been used at saturating concentrations of 20 µg/ml. The values shown correspond to the mean of triplicate experiments and are expressed in arbitrary units relative to the internal ELISA standard.

 


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  		FIGURE 8. Stabilization of conformational epitopes on MQ10 hybrid molecules with titered synthetic peptides. The experiments were performed under the same conditions as those in Fig. 7Go, except that a wide range of peptide concentrations was used to stabilize MQ10 complexes. The values shown correspond to the means of triplicate experiments and are expressed in arbitrary units relative to the internal ELISA standard.

 
Q10-associating peptides display classical peptide-binding motif

MS sequencing of MQ10- and SQ10-eluted peptides suggested that these molecules bind a heterogeneous mixture of diverse, endogenously synthesized ligands, which occupy as much as ~75% of all Q10 receptors. The remaining ~25% of Q10 molecules are filled with a single peptide species TGTETLYF. This dual affinity of Q10 molecules prompted us to examine sequence requirements of the Q10 ligands for binding to Q10 groove. We reasoned that a groove that is severely biased toward accepting peptides with defined sequences will be less efficient in associating with mutant peptides which carry single residue substitutions along the entire length of the peptide. This effect has been observed for Qdm peptide which is the dominant peptide in Qa-1b molecule (14, 56). If, on the other hand, the groove can accommodate many diverse peptides then loss/reduction of binding will be observed only when the test peptide is mutated at the classically defined "anchor" positions.

To address this question, we selected peptide 1, HGTTTLAF (homologous to subunits of proteasome), for the analysis. This peptide, unlike TGTETLYF (homologous to TCR V ß-chain), would be present in liver cells and is identical to TGTETLYF in five of eight positions. A series of synthetic peptides substituted by Ala or Ser at each of the positions was synthesized (see legend to Fig. 9Go), and the peptides were used in the ELISA sandwich peptide-binding assay. The results of the binding experiments indicated that only three peptide residues could not tolerate being replaced with Ala for efficient binding to MQ10: Gly at P2, Leu at P6, and Phe at P8. Substitutions of these residues with Ala led to either reduction or loss of binding comparable to negative control peptides VSV, L19, and NP (Fig. 9Go). The three anchor residues correspond to the conserved residues determined from the MS sequencing (Table IGo). Thus, we conclude that the majority of the peptides associating with the Q10 groove display a classical peptide-binding motif similar to the diverse repertoire of ligands that occupy the H-2K, H-2D, or HLA-A and -B grooves.



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  		FIGURE 9. Identification of anchor positions in HGTTTLAF peptide. The wild-type (WT) and mutant synthetic peptides substituted at different residues by Ala (1A-6A, 8A) or by Ser (7S) were tested in peptide stabilization assay for binding to MQ10. The experiment was performed as in Fig. 7Go. The dark and light bars correspond to ELISA values measured with 20 µg and 4 µg of the synthetic peptides, respectively. The sequences of negative control peptides VSV, L19, and NP are listed elsewhere (41 ). The values shown correspond to the means of triplicate experiments.

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
Recent research into the functions of the nonclassical class Ib MHC Ags led to the recognition of their diversity and heterogeneous properties; however, despite the fact that much has been learned about immunological features of murine M3, Qa-1, and human CD1, HLA-E, and HLA-G Ags, the great majority of the class Ib molecules remain uncharacterized. One such molecule is the soluble, liver-specific Q10 protein that is expressed in a wide variety of inbred and wild mouse strains.

In an attempt to learn about Ag-presenting functions of Q10 proteins, we analyzed peptide ligands constitutively associated with the Q10 {alpha}1{alpha}2 domains. Because of the technical limitations imposed by the necessity to produce large amounts of this protein, we expressed and analyzed hybrid Q10/Qa-2 molecules in lymphoid-derived cells. The results of the MS sequencing of the Q10-associated ligands revealed that they are very similar to the processed protein fragments eluted from classical class I Ags. As is the case with H-2Kb or H-2Kk peptides (2), the majority of the Q10 ligands are octameric (although nonamers were also detected). The Q10 peptides carry a peptide-binding motif typical of the class Ia motifs. The conserved residues include a hydrophobic (Phe or Leu) dominant anchor at P{omega} and two additional invariant residues: Gly at P2 and Leu/Val at P6. All three of these positions influence binding of Q10 synthetic peptide homologues to Q10 groove. The residues found at P2, P6, and P{omega} on Q10 ligands have been reported to serve as anchors in peptides eluted from other class I MHC Ags (2). This is not surprising considering the fact that the predicted geometry of the Q10 groove is very similar to HLA-A2. Although Q10 {alpha}1{alpha}2 domains contain a number of unique substitutions that are not commonly found in other class I MHC proteins (at positions 24, 75, 89, 90, 102, 109, 137, 162, and 176), only one of them, Ile 24, is located at a position predicted to face the peptide-binding groove.

One slightly unusual feature of Q10 peptides is that the invariant Gly at P2 is followed by a variant amino acid at P3. The only other class I molecules for which Gly has been deduced to be critical for binding are H-2Dd, where Gly at P2 is nearly invariably paired with Pro at P3 (57) and HLA-B51, where Gly at P2 is almost always paired with an aromatic residue at P3 (2). Because Gly has no side chain and therefore cannot function as an anchor residue directly, but instead promotes local flexibility and destabilization of the peptide binding, it is possible that all four amino acids found at P3 (Ala, Ile, Val, and Thr) play an important role in anchoring of peptides to Q10 groove. The Gly anchor residue at P2 would be expected to correlate with large side chains in the B pocket of the peptide-binding cleft. The only unusual B pocket residue is Ile-24, which is only found in Q8 (45), whose motif has not been determined, and in Qa-1, which is occupied predominantly with a single peptide species carrying Met at P2 (14).

The nature of the putative source proteins giving rise to Q10 peptides warrants some discussion. All identified sequences matched intracellular murine proteins: the LMP7 and PSMB5 proteasomal subunits, ribophorin, cytochrome c oxidase, and TCR V ß-chain. This is in agreement with the notion that the peptides bound to Q10 groove originated from cytoplasmic proteins and were delivered to the complex by components of classical class I MHC Ag presentation pathway. Consistent with this interpretation we found that mutation of the TAP2 gene led to significant reduction of heat-resistant, peptide-filled Q10 molecules expressed on the surface of RMA-S cells. The small proportion of thermally stable MQ10 on RMA-S cells was comparable to H-2Kb expressed in the same background and may correspond to MHC complexes loaded by TAP1/TAP1 homodimers (58).

The identification of the degraded product of TCR V ß-chain as the most abundant peptide in the Q10 groove in RMA cells was the unexpected finding of this study. Edman degradation and absorbance data suggested that peptide TGTETLYF occupied as much as 25% of RMA-expressed Q10 molecules, whereas the remaining 75% of Q10 grooves were filled with a highly heterogeneous mixture of low abundance peptides. Although it is possible that the homology of this peptide to TCR V ß-chain from EL4 cells (55) is serendipitous, it is more likely that it reflects the precursor-product relationship because RMA is a lymphoma cell line and may express the same TCR V ß-chain as EL4. Peptide-binding studies reported here demonstrated that TGTETLYF has similar binding affinity to Q10 cleft as other peptides examined in this study. Hence, we conclude that overrepresentation of this peptide in MQ10 grooves may have been brought about by preferential processing of this peptide or its enhanced delivery to the endoplasmic reticulum, rather than preferential binding to MQ10. Two other lines of evidence support the conclusion that Q10 does not have a highly "specialized" binding groove. First, the modeling studies of Q10 reported previously (59) suggested that the Q10 groove is very similar to HLA-A2, although it may be somewhat shallow due to the presence of multiple bulky residues (Tyr at 99, 155, 156, and 159 and Trp at 97 and 167). Second, alanine-scanning experiments of TGTETLYF-related peptide identified only three conserved anchor residues, at P2, P6, and P8, that are the only prerequisition for peptide binding. In cases where the cleft is preferentially occupied by a single peptide (56, 60), all peptide positions affect binding efficiency to a detectable degree.

Liver cells, in which Q10 is normally synthesized, do not express TCR. Thus, any potential bias of Q10 groove for TCR-derived peptide cannot be easily rationalized, particularly because the region of homology corresponds to the highly variable CDR3 region embedded within the TCR cleft. Nevertheless, we cannot exclude the possibility that the Q10 groove binds under some circumstances, in lieu of normally processed peptide, a fragment of ß-chain looping of the TCR complex on T cells. A precedent for this interaction was recently reported for class II MHC and the TCR {alpha}-chain (61).

The nature of T cell-mediated recognition of Q10 has been addressed before (59, 62). Since Q10 protein shares structural features with many class I MHC proteins (59, 63), alloreactive CTLs raised against Q10 {alpha}1{alpha}2 domains cross-react on multiple class I MHC proteins (59, 62). This property may allow Q10 to interact with a broader range of TCRs than is normally expected for classical class I Ags. Whether such interactions occur in vivo and whether they lead to apoptosis of T cells, as reported for soluble classical class I proteins interacting with TCRs (64), remains to be established. In this regard, it is of interest that liver is the major organ in which T cell death occurs (65).

Association of the Q10 groove with a diverse array of peptides and the similarity of these complexes with classical class I MHC raises another question, namely, whether these proteins can interact with receptors on NK cells. Many different families of NK, B cell, and monocyte receptors recognizing classical and nonclassical (Qa-1, HLA-E, HLA-G) class I complexes were identified in the recent years (17, 18, 19, 66). Some of these receptors bind class I complexes in peptide-dependent fashion while other associations are peptide independent. Because liver is very rich in NK cells, it is of interest to examine whether Q10 proteins engage in specific interactions with NK cell receptors. New experimental approaches such as tetramer staining (67) may allow us to address those issues in the near future.


   Acknowledgments
 
We thank Maile Henson for tissue culture and help with characterization of the transfectants and Dr. Ming Chen for screening of the protein data banks with Q10 peptide sequences.


   Footnotes
 
1 This work was supported in part by grants from the National Institutes of Health (AI19624 and AI37818). Back

2 F.Z. and P.T contributed equally to this work. Back

3 Current address: Department of Physical and Structural Chemistry, SmithKline Beecham Pharmaceutical, King of Prussia, PA 19406. Back

4 Current address: PE Biosystems, 500 Old Connecticut Path, Framingham, MA 01701. Back

5 Address correspondence and reprint requests to Dr. John E. Coligan, Laboratory of Allergic Diseases, National Institute of Allergy and Infectious Diseases, National Institutes of Health, Twinbrook II, Room 205, Rockville, MD 20852. E-mail address: Back

6 Address correspondence and reprint requests to Dr. Iwona Stroynowski, Center for Immunology, University of Texas Southwestern Medical Center, Dallas, TX 75235-9093. E-mail address: Back

7 Abbreviations used in this paper: ß2m, ß2-microglobulin; GPI, glycosylphosphatidylinositol; CAD, collision-activated dissociation; MS, mass spectrometry; PI-PLC, phosphatidylinositol-specific phospholipase C; ESI, electrospray ionization; m/z, mass:charge ratio. Back

Received for publication September 9, 1999. Accepted for publication December 1, 1999.


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 Introduction
 Materials and Methods
 Results
 Discussion
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