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The Journal of Immunology, 2005, 175: 6651-6658.
Copyright © 2005 by The American Association of Immunologists

Impaired Assembly Results in the Accumulation of Multiple HLA-C Heavy Chain Folding Intermediates1

Leonardo Sibilio*, Aline Martayan*, Andrea Setini2,*, Rocco Fraioli*, Doriana Fruci{dagger}, Jeffrey Shabanowitz{ddagger}, Donald F. Hunt{ddagger} and Patrizio Giacomini3,*

* Laboratory of Immunology, Regina Elena Cancer Institute Centro della Ricerca Sperimentale, Rome, Italy; {dagger} Research Center Ospedale Bambino Gesù, Rome, Italy; and {ddagger} Department of Chemistry, University of Virginia, Charlottesville, VA 22904


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 Disclosures
 References
 
Class I MHC H chains assemble with {beta}2-microglobulin ({beta}2m) and are loaded with peptide Ags through multiple folding steps. When free of {beta}2m, human H chains react with Abs to linear epitopes, such as L31. Immunodepletion and coimmunoprecipitation experiments, performed in this study, detected a preferential association of L31-reactive, {beta}2m-free H chains with calnexin in {beta}2m-defective cells, and with calreticulin and TAP in {beta}2m-expressing cells. In {beta}2m-defective cells, the accumulation of calnexin-bound H chains stoichiometrically exceeded their overall accumulation, a finding that supports both chaperoning preferences and distinct sorting abilities for different class I folds. No peptide species, in a mass range compatible with that of the classical class I ligands, could be detected by mass spectrometry of acidic eluates from L31-reactive HLA-Cw1 H chains. In vitro assembly experiments in TAP-defective T2 cells, and in cells expressing an intact Ag-processing machinery, demonstrated that L31 H chains are not only free of, but also unreceptive to, peptides. L31 and HC10, which bind nearly adjacent linear epitopes of the {alpha}1 domain {alpha} helix, reciprocally immunodepleted free HLA-C H chains, indicating the existence of a local un-/mis-folding involving the N-terminal end of the {alpha}1 domain {alpha} helix and peptide-anchoring residues of the class I H chain. Thus, unlike certain murine free H chains, L31-reactive H chains are not the immediate precursors of conformed class I molecules. A model inferring their precursor-product relationships with other known class I intermediates is presented.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 Disclosures
 References
 
Class I MHC molecules are formed by a glycosylated, polymorphic H (44-kDa) chain, an L (12-kDa) chain, {beta}2-microglobulin ({beta}2m),4 and a short (8–11 mer) peptide Ag derived from the degradation of intracellular proteins. They undergo a complex maturation process involving multiple biosynthetic intermediates and folding steps (1, 2).

In humans, class I H chains (still free of {beta}2m) cotranslationally bind the endoplasmic reticulum (ER) transmembrane chaperone, calnexin. Subsequently, they bind {beta}2m, and the H chain:{beta}2m heterodimer associates with the so-called peptide-loading complex. This is a supramolecular structure comprising calreticulin (the soluble homologue of calnexin), the thiol oxidoreductase ERp57, the TAP, and the peptide editor/facilitator tapasin (1, 2). The peptide-loading complex tethers H chain:{beta}2m intermediates until they acquire peptides, an event that results in yet another conformational change, and the release of fully assembled, thermally stable complexes ready for transport to the cell surface, where they can be recognized by CTL, and NK cells.

The biosynthesis of murine class I molecules is similar, but not identical. For instance, calnexin participates in the formation of the peptide-loading complex only in murine cells (3).

As shown by crystallographic studies (4, 5), the fully assembled, folded class I MHC H chains form a characteristic structure (a {beta}-pleated floor topped by two {alpha} helices) that delimits the Ag-binding groove. In contrast, the three-dimensional structure of peptide-free H chain:{beta}2m folding intermediates (6), and of class I MHC H chains that have not bound {beta}2m (often referred to as free H chains), is not known, possibly due to their instability and/or a lack of defined conformations at early folding steps. Due to the lack of a direct method of analysis, the reactivity of murine (Ld, Db, and Kb) and human (HLA-A, -B, and -C) H chains with Abs to mapped linear epitopes, such as 64-3-7 (7), KU1, KU2, KU3 (8), HC10, HCA2 (9), LA45 (10), Q1/28 (11), and L31 (12), has become a widely used indicator of local unfolding at short amino acid stretches.

Extensive work has shown (7, 8, 13, 14, 15, 16, 17, 18, 19) (reviewed in Ref.1) that murine free H chains are a heterogeneous group of molecules that meet all or some criteria for overall unfolding (absence of exposed amphipathic residues, thermal instability, as well as a lack of association with {beta}2m, peptides, promiscuous chaperones, members of the peptide-loading complex, etc.). Such a systematic characterization is lacking in the case of human free H chains. We are aware of one study (20) aimed at establishing whether human "free" and {beta}2m-associated H chains have a precursor-product relationship similar to that between murine Ld alt and functional Ld (7). Limited information is also available about the precursor-product relationships between human free H chains and other early occurring (chaperone-bound) conformers. Thus, the evidence itself for multiple conformational changes during class I folding in human systems remains mostly indirect.

In this study, the L31 Ab has been selected as a valuable probe of human H chains, because of the location of its epitope. Binding of L31 is strictly dependent on the presence of aromatic amino acids (Y or F, present in HLA-C and certain HLA-B alleles) at position 67 of the {alpha}1 domain {alpha} helix (12). Residue 67 is buried deep in the binding groove and contributes to delimit the B pocket, a recess that secures bound peptides at their P2 anchor position (5).

Taking advantage of the reactivity of L31 and other selected Abs, we have tested the ability of free H chains to bind class I chaperones, {beta}2m, and/or peptides, and have identified distinguishable subsets of L31-reactive free H chain conformers with an unfolded binding groove.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 Disclosures
 References
 
Cell lines

The TAP-defective 174 x CEM.T2 (T2 hereafter) cell line (21), the HLA-Cw1 transfectant (12) in the HLA-A-, HLA-B-, HLA-C-defective 721.221 (221 hereafter) cell line (22), the tapasin-defective 721.220 (220 hereafter) cell line (23), the HLA-homozygous B cell line LG2 (12), as well as the KJ29 and FO-1 cell lines and their {beta}2m transfectants (24, 25) have been described.

Antibodies

The murine mAbs HC10 and L31 bind linear {alpha}1 domain epitopes, including residues 62 (9) and 67 (12), respectively. Q1/28 binds an {alpha}3 domain epitope (11). F4/326 (26) and W6/32 (27) bind class I MHC H chains associated with {beta}2m. Namb-1 binds {beta}2m (28). Rabbit polyclonal Abs to TAP1, tapasin, and ERp57 are described (29). Polyclonals to calnexin and calreticulin were from StressGen Biotechnologies.

Immunochemical methods

Cells were metabolically labeled with [35S]methionine (9.25 MBq/ml), as described in the figure legends, and solubilized with either 1.0% Nonidet P-40 or 0.5% CHAPS in PBS (0.01 M, pH 7.0, 0.15 M NaCl). For immunoprecipitation, purified Abs were covalently linked to Affigel (Bio-Rad). Human {beta}2m was from Sigma-Aldrich. Isoelectric focusing (IEF) and Western blotting techniques (reducing conditions in all cases) are described (12, 24, 25, 29).

Isolation and synthesis of class I peptide ligands

Briefly, W6/32-reactive and L31-reactive class I molecules were isolated from CHAPS lysates of LG2 (Cw*0102) and 221.Cw*0102 transfectants by affinity chromatography, and submitted to acidic elution. Low molecular mass species were recovered by ultrafiltration, and resolved/sequenced by microbore HPLC/triple quadrupole mass spectrometry (30). The synthetic HLA-Cw*0102 ligand NCPERIITL, the HLA-A*0201 ligand GILGFVFTL from the influenza virus matrix glycoprotein, and the irrelevant nonamer QLLGIWGCS were from Sigma-Genosys.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 Disclosures
 References
 
HLA-Cw1 H chains exist as three distinct conformers

Three distinct, nonoverlapping conformers of class I HLA H chains have been described (16, 31): 1) H chains that have not stably bound {beta}2m, and are reactive with Abs (such as HC10) to free, unfolded H chains; 2) H chains that have already bound {beta}2m, but, still free of peptides, remain temporarily associated with the peptide-loading complex, being coimmunoprecipitated by Abs to TAP; and 3) {beta}2m-associated/TAP-free H chains, reactive with conformational (e.g., W6/32) Abs. W6/32-reactive H chains have also been described to associate with calnexin (16). To determine whether HLA-Cw1 H chains also exist in distinct conformations, immunodepletion experiments were performed in 221.Cw1 transfectants (Fig. 1). Neither the class I H chains directly immunoprecipitated by L31 nor those coimmunoprecipitated with TAP were significantly affected by immunodepletion with W6/32 (Fig. 1A, compare lanes 6 and 7 with lanes 2 and 3). However, L31-reactive H chains were affected, although slightly, by immunodepletion of TAP-associated H chains (Fig. 1B, lane 18 compared with lane 13). Interestingly, tapasin-associated H chains (lanes 16 and 11) were removed by TAP depletion, whereas ERp57-associated H chains (lanes 15 and 10) appeared to be unaffected.



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  		FIGURE 1. Immunodepletion of HLA-Cw1 H chains. A, CHAPS lysates of metabolically radiolabeled (2-h) 221.Cw1 transfectants were immunodepleted by incubation with excess amounts of immunoadsorbents bearing either an irrelevant Ab (lanes 1–4) or W6/32 (lanes 5–8). The residual class I H chains were either directly immunoprecipitated with L31 and W6/32, or coimmunoprecipitated with an Ab to TAP1, and resolved on an SDS-PAGE (the 44-kDa area of the fluorography is shown). B, CHAPS lysates from unlabeled 221.Cw1 transfectants were immunodepleted and immunoprecipitated, as indicated. HLA-C molecules were selectively identified following SDS-PAGE and electroblotting by staining filters with L31.

 
Thus, HLA-Cw1 H chains also may exist in the form of at least three distinct molecular pools of conformers, two of which (L31-reacting and TAP-associated conformers) may display limited overlap.

L31-reactive conformers associate with calnexin, TAP, and calreticulin

To systematically test the ability of different H chain conformers to interact with all the known class I chaperones, coimmunoprecipitation experiments (Fig. 2A) were performed at steady state on soluble cell extracts from the 221.Cw1 transfectant (12), and two {beta}2m-defective cell lines from different lineages (FO-1 melanoma and KJ-29 kidney carcinoma cells), together with their {beta}2m transfectants (24, 25). Class I conformers were immunoprecipitated by L31 and an Ab to {beta}2m, Namb-1 (28), because Abs to {beta}2m have been shown to be optimal for the unbiased identification of {beta}2m-associated/peptide-empty/W6/32-unreactive class I H chains bound to the peptide-loading complex (31).



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  		FIGURE 2. Coimmunoprecipitation of class I H chain conformers and their chaperones. A, Immunoprecipitates with L31, Namb-1, and in the absence of specific Ab (–) were prepared from CHAPS extracts (2 mg of proteins per immunoprecipitation) from the indicated cell lines, resolved on replicate SDS-PAGE slabs, and electroblotted to nitrocellulose filters. Multiple strips in different m.w. ranges were cut and probed with the indicated Abs (lanes 1–15, 19–33, 37–51, 55–69, 73–87, 91–105, and 109–123). CHAPS extracts from the indicated cell lines (50 µg per lane) were electrophoresed, blotted, and probed in parallel (lanes 16–18, 34–36, 52–54, 70–72, 88–90, 106–108, and 124–126). All of the gels were cast at an acrylamide concentration of 10%, except the gel used to detect {beta}2m (lanes 19–36), which was cast at 12.5% and contained (*) extracts from FO-1, FO-1-{beta}2m, and 221 cells in lanes 34–36. B, All of the specific bands were quantified by densitometry, and the densitometric value of the chaperone band in each lane divided by the densitometric value of the H chain band identified by L31 in the corresponding control lane (i.e., lane 38 divided by lane 2; lane 39 divided by lane 3, etc.). Normalized values of chaperone:class I interaction (expressed as ratios of chaperone/H chain bands) were displayed as histograms.

 
L31 and Namb-1 immunoprecipitates were Western blotted, and the levels of the immunoprecipitated structural subunits of class I molecules were estimated by probing filters with the same Abs (L31 and Namb-1) used for immunoprecipitation (lanes 1–15 and 19–33). L31-reactive H chains were present at different levels in different {beta}2m-positive cells (compare lanes 2 and 3, 8 and 9, and 14 and 15), and {beta}2m was undetectable, as expected, in L31 immunoprecipitates (lanes 20, 26, and 32).

Replicate L31 and Namb-1 immunoprecipitates were probed with Abs to class I chaperones (lanes 37–51, 55–69, 73–87, 91–105, and 109–123). Two distinct coimmunoprecipitation patterns were observed, consistent with a hierarchical order of interactions. Calnexin was much more abundant in L31 immunoprecipitates from {beta}2m-defective cells (lanes 41 and 47) than in L31 and Namb-1 immunoprecipitates from {beta}2m-expressing cells (lanes 38 and 39, 44 and 45, and 50 and 51). In contrast, calreticulin, TAP, tapasin, and ERp57 were all present and most abundant in Namb-1 immunoprecipitates from {beta}2m-expressing cells (lanes 57, 63, 69, 75, 81, 87, 93, 99, 105, 111, and 123). However, some of these latter chaperones were also present, at lower levels, in L31 immunoprecipitates from {beta}2m-positive cells (lanes 56, 62, 68, 74, 80, 86, and 92). Interestingly, calreticulin and TAP were barely detectable even in some L31 immunoprecipitates from {beta}2m-defective cells (lanes 65, 77, and 83). These results demonstrate the ability of H chains to interact with members of the peptide-loading complex independently of {beta}2m.

Because different immunoprecipitates contain different amounts of H chains, a more accurate quantification of chaperoning interactions required densitometry and normalization (described in the legend to Fig. 2). Densitometric analysis demonstrated that L31 conformers were only 1.5 times more abundant in lane 5 than lane 8, but coprecipitated calnexin was 9 times more abundant in lane 41 than in lane 44. Following normalization (densitometric value in lane 41 divided by that in lane 5, and lane 44 divided by lane 8, Fig. 2B), this resulted in a ~6-fold increase in H chain:calnexin association, suggesting that L31 conformers more tightly interact with calnexin when synthesized in the absence of {beta}2m.

A similar normalization procedure, conducted on Namb-1 immunoprecipitates (Fig. 2B), was expected to be less accurate, because this Ab also reacts with L31-unreactive H chain alleles and, more importantly, a large fraction of {beta}2m-associated H chains (unlike L31 conformers) is expressed on the cell surface, at which location they are not available for interaction with ER-resident chaperones. Consequently, normalization might underestimate the amounts of chaperones coimmunoprecipitated by Namb-1, as compared with those coprecipitated by L31.

Notwithstanding this limitation, we conclude that L31-reacting H chains preferentially interact with calnexin, particularly in {beta}2m-defective cells, whereas H chains associated with {beta}2m display a preference (the magnitude of which cannot be precisely assessed) for interaction with the known members of the peptide-loading complex. These data are consistent with a selective, although not exclusive, interaction of calnexin and the members of the peptide-loading complex with distinct populations of H chains with different degrees of {beta}2m association and folding.

HLA-C H chains reacting with L31 are unfolded substrates of chaperoning

To obtain independent experimental evidence for a dominant interaction between L31 conformers and calnexin in FO-1 cells (Fig. 2B, highest histogram), the above coimmunoprecipitation/blotting experiments performed at steady state were complemented by a direct coimmunoprecipitation experiment from radiolabeled cell extracts.

Calnexin:H chain interactions were detected by a continuous (2-h) metabolic pulse using an Ab to calnexin (Fig. 3A, lanes 5 and 10), as well as two distinct Abs to free H chains (L31 and HC10; lanes 3, 4, 8, and 9), in both FO-1 and FO-1-{beta}2m cells. The observed position of the calnexin band is compatible with its predicted isoelectric point (<http://au.expasy.org/uniprot/P27824>), and was confirmed in separate IEF blotting experiments, by staining filters with the same Ab to calnexin used for coimmunoprecipitation (data not shown). The low intensity of the calnexin band is consistent with the widely accepted notion that calnexin molecules actively involved in the chaperoning of newly synthesized proteins belong to a large, slowly renewing molecular pool (32). Accordingly, calnexin molecules immunoprecipitated in a radiolabeled form may represent a minor fraction in a large pool of molecules synthesized before metabolic pulse.



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  		FIGURE 3. Interactions of L31-reactive HLA-C H chains with calnexin. A, Immunoprecipitates from CHAPS lysates of metabolically labeled (2-h) FO-1 cells and their {beta}2m transfectants were run on an IEF gel under reducing conditions. IEF positions of HLA-A, HLA-B, and HLA-C alleles (arrowheads) and calnexin are indicated. All of the alleles displayed sialylated (s) components exclusively in W6/32 immunoprecipitates from FO-1-{beta}2m transfectants. B, Nonidet P-40 lysates of metabolically labeled (30-min) FO-1 cells were immunodepleted and immunoprecipitated with either the indicated Abs or an irrelevant Ab (–). Immunoprecipitates were run on an SDS-PAGE slab.

 
Possibly due to a more efficient incorporation of radiolabeled precursors, immunoprecipitated calnexin was apparently more abundant in FO-1-{beta}2m than FO-1 cells (compare, for instance, lanes 5 and 10). However, the calnexin band was much less abundant than the coprecipitated H chain bands in the absence of {beta}2m (lane 5), and much more abundant in its presence (lane 10). Densitometric analysis and calculation of the H chain:calnexin ratios in the two lanes estimated an ~8-fold difference in association (data not shown). The same conclusion can be drawn for lanes 3, 4, 8, and 9, although the differences in the ratios, calculated in the same way, were less striking (between 2- and 3-fold). In summary, two independent methods (Figs. 2 and 3A) detected strong and long-lived interactions between L31-reactive H chain substrates and calnexin, particularly in the absence of {beta}2m.

In agreement with previous studies (16), a calnexin band was present in W6/32 immunoprecipitates from {beta}2m-expressing cells (lane 7). Very surprisingly, a similar weak band appeared to be also present in {beta}2m-defective cells (lane 2), in the absence of detectable H chains (lane 2). Possibly, this reflects coimmunoprecipitation with a very small (or poorly radiolabeled) pool of W6/32 H chains synthesized in the absence of {beta}2m. H chains free of {beta}2m have been described to carry the W6/32 epitope under certain conditions (27). Alternatively, calnexin may associate nonspecifically with the immunoadsorbent in this lane.

A component comigrating with {beta}2m was visible within a smear of calnexin-bound proteins (lane 10). Because it was absent in the corresponding immunoprecipitate from {beta}2m-defective parental cells (lane 5), it is likely to coincide with {beta}2m. Its presence is not surprising, because significant levels of {beta}2m-associated H chains were associated with calnexin in FO-1-{beta}2m and other cell lines (see Fig. 2, lanes 39, 45, and 51). More surprisingly, a similar component may be present in L31 and HC10 immunoprecipitates (lanes 8 and 9). Assuming this extremely weak component coincides with {beta}2m, its presence is easier to explain in HC10 than L31 immunoprecipitates, because only the former Ab was shown to immunoprecipitate low levels of {beta}2m (16, 17, 33), whereas no {beta}2m was detected in L31 immunoprecipitates by sensitive coimmunoprecipitation/blotting experiments (Fig. 2, lane 26, and data not shown). Therefore, we conclude that the presence of trace amounts of {beta}2m in L31 immunoprecipitates cannot be excluded, although we have no direct confirmatory evidence.

Several additional bands were present in the basic area of the gel around and above {beta}2m. One of these even exceeded the intensity of the calnexin band directly immunoprecipitated by the polyclonal Ab to calnexin (lanes 5 and 10, arrows), and was presumably identical with a weak band detectable in some L31 and HC10 immunoprecipitates (lanes 3 and 4). No such band was seen by probing IEF blot filters with the same polyclonal to calnexin (data not shown), suggesting that an unknown protein, primarily associated with calnexin, was detected as a result of coimmunoprecipitation rather than Ab cross-reactivity. This component was not further characterized.

Finally, from the results depicted in Fig. 3A, it is evident that, as expected, L31 only reacted with HLA-B8 and HLA-Cw7 H chains, whereas HC10 reacted more widely (lanes 3, 4, 8, and 9). Accordingly, immunodepletion experiments, performed to reveal a possible overlapping in the molecular pools of {beta}2m-free H chains identified by the two Abs, resulted in the complete immunodepletion of all L31-reactive H chains (Fig. 3B, lane 16) by HC10, and in the partial depletion of HC10-reactive H chains by L31 (lane 18). The results of the immunodepletion experiment demonstrate that two nearly adjacent linear epitopes of the {alpha}1 domain {alpha} helix and binding groove become simultaneously accessible to, and hidden from, Ab binding on HLA-B8 and -Cw7 H chains, when these are {beta}2m associated and {beta}2m free, respectively.

In conclusion, the results shown in Figs. 2 and 3A are consistent with the existence of at least two class I intermediates in which the binding groove remains unfolded during successive chaperoning/quality control steps taking place on calnexin and on some members of the peptide-loading complex, respectively.

HLA-Cw1-free H chains do not bind naturally occurring peptides

To determine whether L31-reactive H chains naturally bind peptides in 221.Cw1 and LG2 cells (also expressing Cw1 as the only L31-reactive allele), low molecular mass peptides were eluted from W6/32-reactive and L31-reactive HLA class I molecules. Mass spectrometry identified several (20–40) prominent and distinct peptide peaks with estimated molecular masses ~1 kDa in the W6/32 eluates of 221.Cw1 transfectants. One dominant peptide peak was identified as NXPERIITL, in agreement with a previous report (34). Database searches identified heterogeneous nuclear ribonucleoprotein X (aa 53–61) as the putative peptide donor, and NCPERIITL as the putative ligand. The corresponding synthetic peptide was shown to be a specific HLA-Cw1 ligand in in vitro assembly assays (see below). In contrast, no peptide species, in a mass range compatible with the classical class I (9–11 mer) ligands, were detected in the L31 eluates from the two cell lines, indicating that L31 conformers are not only free of {beta}2m, but also free of peptides.

HLA-Cw1-free H chains do not bind exogenous peptides or {beta}2m in in vitro assembly assays

The absence of natural class I ligands in L31 eluates raises the possibility that peptides with low affinity and high dissociation rates might be lost during the purification procedures. In this case, however, it should be possible to force HLA-C ligands to bind back to L31 conformers, by in vitro incubation with excess amounts of synthetic peptides. Therefore, we examined the binding of NCPERIITL to the L31 conformers present in cell lysates. To this aim, the TAP-deficient T2 cell line was initially selected, because it synthesizes H chain:{beta}2m complexes largely devoid of high affinity, endogenous peptides, and naturally expresses HLA-Cw*0102 molecules. The in vitro assembly assay (13) was conducted at 4°C to enhance low affinity interactions, either in the absence or in the presence of exogenously added {beta}2m, in the event that this might further stabilize transient H chain:peptide assemblies. As expected, the addition of {beta}2m enhanced the recovery of W6/32-reactive HLA-A2, -B51, as well as -Cw1 H chains (Fig. 4A, compare lane 2 with lanes 5, 11, 14, and 17), while the addition of NCPERIITL selectively enhanced the W6/32 reactivity of the HLA-Cw1 allele only (lane 8). The addition of both {beta}2m and peptide promoted W6/32 reactivity in a roughly additive fashion (compare Cw1 bands in lanes 2, 5, 8, and 17, and also see HLA-A2 in lanes 2, 5, and 14, the latter supplemented with an HLA-A2-specific ligand). In contrast, {beta}2m had a marginal effect, and the NCPERIITL peptide had no effect, on the amounts of L31-reactive HLA-Cw1 conformers (lane 1 compared with lanes 4, 7, 10, 13, and 16). A densitometric analysis of these results, reported in the right panel of Fig. 4, confirmed that L31-reactive H chains do not appreciably assemble with peptide. Other HLA-Cw1-free H chain conformers, barely detectable in T2 soluble extracts, became more reactive with an Ab to the {alpha}3 domain (Q1/28) following addition of the Cw1 peptide (Fig. 4, A, lane 9, and B, histograms to the right end). Q1/28 conformers were not further investigated. Thus, L31 appears to identify a subset of free H chain conformers only marginally affected by the addition of exogenous {beta}2m, and refractory to peptide-mediated stabilization, even at 4°C.



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  		FIGURE 4. In vitro assembly of HLA-Cw1 molecules with exogenous peptide and {beta}2m in TAP-deficient T2 cells. A, Nonidet P-40 extracts from metabolically labeled (30-min) T2 cells were incubated at 4°C for 4 h with {beta}2m (10 µg/ml) and/or peptide ligands (20 µg/ml; see Materials and Methods) of HLA-Cw1 and HLA-A2 molecules, or in the absence of ligands, as indicated. Class I H chains were immunoprecipitated and resolved on an IEF slab gel. B, Densitometric scans of HLA-A2 and HLA-Cw1 bands in the different lanes shown in A.

 
An additional experiment, shown in Fig. 5, was conducted to test peptide receptivity and thermal stability of free H chains in the presence of TAP and tapasin. Soluble extracts of 221.Cw1 transfectants were incubated at 4°C (to enhance binding) with specific Cw1 peptides, and then the temperature was increased to 37°C (to enhance the detection of minor subpopulations of L31 conformers through the removal of the bulk of thermally unstable molecules). Thermal stability was assessed at 37°C following two different chase periods (15 min and 120 min). The stability of W6/32 and F4/326 conformers was assessed in parallel. As reported (12), L31 did not bind nonclassical class I H chains expressed (35) by 221 cells and their transfectants (Fig. 5A, compare lanes 8 and 21 with lanes 2 and 15). In contrast, W6/32 reacted with both nonclassical (lower band, thin line) and HLA-Cw1 (upper band, thick line) H chains (lanes 3, 9, 16, and 22), and the two bands were poorly resolved when simultaneously present (i.e., lanes 9 and 22). As expected, W6/32-reactive Cw1 H chains were detected at low levels at the early (15-min) chase point, and completely melted at 37°C (Fig. 5, A and B, lane 11, compared with lane 9), unless incubated with the Cw1-specific peptide (lane 13), suggesting that they were largely free of, but receptive to, peptide at this biosynthetic stage. In addition, they significantly accumulated, and became thermally stable, at the end of the long (120-min) chase period (lanes 22 and 24). Peptide addition enhanced stability even after a long chase (lane 26), suggesting suboptimal loading by natural peptide ligands in mature HLA-Cw1 molecules. To detect conformed Cw1 H chains without the interference of closely migrating nonclassical H chain bands, the same experiment was repeated using F4/326, an Ab that does not bind nonclassical class I H chains, and similar results were obtained (lanes 27–32).



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  		FIGURE 5. In vitro assembly and thermal stability of HLA-Cw1 molecules in 221.Cw1 cells. A, Parental 221 cells and 221.Cw1 transfectants were metabolically labeled for 10 min, and chased for either 15 min or 120 min. At the end of chase, Nonidet P-40 extracts were prepared, divided in three parts for incubation (4 h) at 4°C, 37°C, or 37°C in the presence of the Cw1 peptide ligand NCPERIITL (20 µg/ml). Class I HLA H chains were then immunoprecipitated with or without (–) specific Abs, and run on SDS-PAGE slabs. A nonspecific (n.s.) immunoprecipitate is indicated. B, Densitometric analysis of the Cw1 band in the indicated lanes of an underexposed autoradiogram.

 
In contrast, nonclassical H chains reactive with W6/32, and Cw1 H chains reactive with L31, both melted at 37°C, irrespective of the presence of the HLA-Cw1 peptide and the length of chase (compare lanes 3 and 16 with lanes 7 and 20; lanes 9 and 22 with lanes 10, 23, 12, and 25). Thus, neither peptides nor {beta}2m (naturally occurring in biosynthetically active cells or exogenously added) could detectably influence the fate of L31-reactive conformers.


   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
Disclosures
 References
 
In this study, we have shown that L31-reactive free H chains bind calnexin, calreticulin, and TAP, although part of these free H chains may also exist free of all class I chaperones. A natural, dominant Cw1 peptide ligand and/or {beta}2m, even provided in excess amounts, could not significantly stabilize L31 conformers in experiments of in vitro assembly performed in various experimental conditions.

A spectrum of H chain intermediates with different degrees of folding

Among murine and human H chain intermediates with different degrees of folding, the peptide-free murine Ld alt molecules identified by the 64-3-7 Ab have been shown to be partially folded, because they: 1) are weakly associated with {beta}2m (16, 18); 2) bind all members of the peptide-loading complex (19); and 3) can be readily converted into fully conformed H chains displaying stable association with {beta}2m by peptide addition (7). This combination of features has not been described, to our knowledge, in any of the human H chain intermediates described to date, although HC10-reacting H chains were reported to weakly bind {beta}2m (16, 17, 33), and to be coimmunoprecipitated with calnexin, calreticulin, and TAP (15, 16, 36). Likewise, another Ab (MARB-4) was shown to recognize H chains free of {beta}2m and associated with peptides (37), but no information is available about its ability to coimmunoprecipitate class I chaperones.

In this study, L31-reactive H chains have been systematically characterized. An association with {beta}2m could not be demonstrated conclusively, using a sensitive immunoprecipitation/blotting method in lysates prepared with the mild nonionic detergent CHAPS. Like HC10, L31-reactive H chains were found to interact with calnexin and at least two members of the peptide-loading complex (TAP and calreticulin). Unlike 64-3-7-reactive H chains, L31-reactive H chains were free of peptides, and unreceptive to a major peptide ligand, as shown by mass spectrometry and in vitro assembly (Figs. 4 and 5). These results complement the observation that HC10-reactive conformers were not spontaneously chased into assembled class I molecules (20). Thus, human class I molecules appear to be more sensitive to conformational melting in cell lysates as compared with mouse class I molecules. Possibly, this folding impediment of human {beta}2m-free H chains reflects a more stringent quality control of class I assembly in humans than in mice (reviewed in Ref.17).

In this respect, it is of interest that the 64-3-7 epitope is on a protruding loop connecting the {beta}-sheet floor of the binding groove with the beginning of the {alpha}1 domain {alpha} helix, just outside the peptide binding site, whereas the H chain epitopes recognized on human free H chains by several Abs such as HC10 (residues 57–62), LA45 (residues 62–63), L31 (epitope centered on residues 67–69), and HCA2 (residues 77–84) are clustered on the {alpha}1 domain {alpha} helix (9, 10, 38), i.e., they line up on one of the sidewalls of the peptide binding site. The availability of linear epitopes outside and inside the peptide-binding groove (in murine and human free H chains, respectively) may correlate with a different plasticity of the groove, and a distinct ability to achieve a peptide-receptive state.

Thus, our results are consistent with an unfolding of at least some of the H chains identified through the {alpha}1 domain {alpha} helix epitopes, with the L31 conformers most likely lying at the unfolded end of the H chain spectrum.

Clues to the structure of class I HLA intermediates

As noted above, the HC10 and L31 epitopes are adjacent. Both epitopes are accessible on free H chains, but hidden in H chain:{beta}2m complexes, and spatially distant from {beta}2m. This rules out direct epitope masking by the L chain subunit in assembled class I molecules, but remains compatible with masking by: 1) molecules other than the class I structural subunits, namely TAP or other members of the peptide-loading complex; 2) peptides in the binding groove; 3) H chain residues upon a conformational rearrangement; or 4) a combination of some or all of the above.

Mechanism 1 (see above paragraph) is not supported by the available experimental evidence, because association with TAP and calreticulin is compatible with L31 binding (Fig. 2) and, reciprocally, W6/32-reactive molecules are free of all members of the peptide-loading complex (39) (Fig. 1), and yet they do not react with L31. In contrast, mechanism 2 is plausible, because the HC10 epitope is located at the N-terminal end of the {alpha}1 domain {alpha} helix, right above the N terminus of bound peptides. These could mask the HC10 epitope without any significant contribution of a conformational rearrangement in H chain residues. Although this also applies to the L31 epitope, it may be noted that residue 67, i.e., the single most important residue for L31 binding (12), is buried deep in the B pocket of conformed H chains, in a region involved in anchoring antigenic peptides (5). This makes epitope unmasking unlikely, unless peptide loss is accompanied by an extensive loss of conformation in the peptide-binding groove (e.g., mechanism 3).

In support of this mechanism, we present three observations: 1) free H chains are simultaneously reactive with L31 and HC10 in the absence of {beta}2m (Fig. 2), demonstrating linkage in the acquisition and loss of two linear epitopes on a long amino acid stretch (residues 57–69) spanning the entire N-terminal end of the {alpha}1 domain {alpha} helix; 2) L31-reactive H chains are not only peptide free, but also peptide unreceptive (Figs. 3 and 4), indicating a degree of unfolding that precludes not only peptide binding, but also the formation of a potentially peptide-receptive interface; 3) conversely, thermally unstable HLA-Cw1:{beta}2m complexes in both TAP-deficient and TAP-expressing cells are free of peptides, but receptive to class I ligands, and yet they are not reactive with L31 (Figs. 4 and 5), demonstrating that the absence of peptides per se is insufficient to elicit L31 reactivity.

Thus, in agreement with mechanism 3, L31 identifies an extensive local unfolding incompatible with peptide binding, possibly an extended conformation of an otherwise helical region surrounding the binding groove. The absence or presence of peptides either plays a marginal role, or has no effect, on epitope masking/unmasking.

Class I HLA intermediates

In this study, we have identified two {beta}2m-free H chain intermediates, the former associated with calnexin, and the latter associated with at least two members of the peptide-loading complex (calreticulin and TAP). Both intermediates carry an unfolded binding groove. Their relative amounts depend on the availability of {beta}2m (Figs. 2 and 3), suggesting an equilibrium between the two intermediates. In {beta}2m-defective cells, the association of L31 conformers with calnexin stoichiometrically exceeds their overall accumulation. Two interpretations are possible to explain this feature: 1) with no {beta}2m available, H chains have no alternative but passive calnexin association; 2) calnexin actively retains its substrates because it is capable of sensing an early qualitative impairment in the H chain fold consequent to the absence of {beta}2m. In support of the latter possibility, previous phase partitioning studies with the nonionic detergent TX-114 demonstrated that {beta}2m transfection of KJ-29 cells partially restores, in free HLA-C H chains, a detergent-accessible interface typical of folded proteins, somewhat similar to that of {beta}2m-associated H chains (24).

The interpretation that calnexin-mediated sorting differs in the absence and presence of {beta}2m, and the finding that L31-reacting H chains better associate with calreticulin and TAP in the presence of {beta}2m, are in agreement with studies by Degen et al. (14), and the original view by Sugita and Brenner (15), further elaborated by Solheim et al. (36). These authors suggested that calnexin retains misfolded class I H chains in the ER exclusively in cells defective in peptide loading or {beta}2m expression, while in physiological conditions H chains are released from calnexin, and additional quality control steps may take place on the peptide-loading complex.

In conclusion, we propose that at least two (and not mutually exclusive) pathways determine the accumulation of free H chains in human cells (see the diagram in Fig. 6). In one pathway, H chains might remain free of {beta}2m even following their emergence from calnexin, and in this conformation they would gain limited access to some members of the peptide-loading complex, with no chance of productive assembly. This pathway might function in {beta}2m-defective cells. In the other pathway, dominant in {beta}2m-expressing cells, H chains might bind {beta}2m, and be returned to the pool of unfolded H chains bound to TAP/calreticulin and calnexin, when they do not meet the requirements necessary for productive assembly. This may occur frequently in the case of HLA-C, a class I molecule characterized by restricted peptide binding, impaired assembly, and stable TAP association (39).



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  		FIGURE 6. HLA-C intermediates. In the hypothetical diagram, largely based on models of class I assembly proposed by others (2 15 16 17 36 ), the two HLA-C intermediates identified by L31 (Figs. 2 and 3A) are shown in association with calnexin (a), and either or both calreticulin and TAP (b). Their association with tapasin could be detected only in 221.Cw1 cells (lane 92 of Fig. 2). An association with a fully assembled peptide-loading complex cannot be excluded. The complete peptide-loading complex containing peptide-receptive/{beta}2m-associated H chains is depicted in c. The conformed, peptide-bound class I molecules released from the peptide-loading complex, as detected by W6/32 (Figs. 1, 4, and 5), are shown in d. Intermediate a is more abundant in the absence than in the presence of {beta}2m, whereas intermediate b exhibits a reciprocal trend (Fig. 2), suggesting an equilibrium between a and b. We propose that in cells expressing {beta}2m, b and a, as well as free H chains truly free of all class I chaperones (data not shown), result from the stepwise disassembly of the peptide-loading complex consequent to a failure of H chain:{beta}2m heterodimers (c) to be loaded with specific peptides. In contrast, in the absence of {beta}2m, a would accumulate most abundantly, and b would result from the escape of unfolded, free H chains from calnexin. The absence of a b to c rescue pathway is consistent with L31 conformers being largely refractory to peptide and {beta}2m binding (Figs. 4 and 5). However, it remains possible that class I H chains returned to calnexin (a) might regain a plastic conformation compatible with {beta}2m and peptide binding. Such a {beta}2m-associated/calnexin-bound intermediate (e), detected in Figs. 2 and 3A, is similar to a hypothetical backup intermediate described by others (16 ).

 
Whatever the exact mechanism, our study provides evidence that H chains free of {beta}2m are the substrates of different class I chaperones, presumably to prevent their premature melting. Despite this, large amounts of these H chains are not the immediate precursors of folding and folded intermediates, but dead ends of unsuccessful folding, i.e., they are intermediates dismissed from the proper class I assembly pathway at the calnexin and/or peptide-loading checkpoints.


   Acknowledgments
 
We are particularly grateful to Dr. Ettore Appella for many useful discussions and invaluable criticism; to Drs. H. Ploegh (Harvard University Medical School, Boston, MA), Soo Young Yang (New York Medical College, New York, NY), and A. Siccardi (Dibit S. Raffaele, Milano, Italy) for the HC10, F4/326, and L31 Abs, respectively; and to Dr. P. Cresswell for the 220 cell line. Maria Vincenza Sarcone and Paula Franke are gratefully acknowledged for secretarial support and for revising the English text, respectively.


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


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

1 This work was supported by Associazione Italiana per la Ricerca sul Cancro (to P.G. and D.F.) and National Institutes of Health GM 37537 and AI33993 (to D.F.H.) Grants. Back

2 Current address: Department of Animal and Human Biology, University "La Sapienza," Rome, V. le dell’Università 32, 00185 Rome, Italy. Back

3 Address correspondence and reprint requests to Dr. Patrizio Giacomini, Laboratory of Immunology, Regina Elena Cancer Institute Centro della Ricerca Sperimentale, Via delle Messi d’Oro 156, 00158 Rome, Italy. E-mail address: giacomini{at}ifo.it Back

4 Abbreviations used in this paper: {beta}2m, {beta}2-microglobulin; ER, endoplasmic reticulum; IEF, isoelectric focusing. Back

Received for publication December 29, 2004. Accepted for publication September 1, 2005.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 Disclosures
 References
 

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