Introduction

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Most MHC class I molecules are capable of binding a large array of individual peptides (1). In contrast, the murine class Ib molecule, Qa-1^b, predominantly binds a single species (2, 3). We refer to this peptide as Qdm (for Qa-1 determinant modifier; reference 2), and it is derived from amino acids 3-11 of class Ia D-region-encoded molecules. HLA-E, which differs from Qa-1^b in 55 of 181 residues in the 1 and 2 domains, binds leader peptides from human class Ia molecules that are very similar to the murine class Ia leader peptide bound by Qa-1^b (4). HLA-E and Qa-1^b, unlike other class Ia molecules, have serines rather than the conserved residues threonine and tryptophan at positions 143 and 147 in the "F" pocket, respectively. In the "B" pocket, HLA-E and Qa-1^b also share the key residues methionine and alanine at positions 45 and 67, respectively. The HLA-E crystal structure reveals that side chains of five of the nine amino acids of the bound peptide protrude into the pockets of the HLA-E groove (5). Based on this structure of HLA-E, it would be predicted that only a few substitutions in the native Qdm peptide would be tolerated for binding to Qa-1^b. This use of multiple anchors would also account for our previous finding that the Qdm peptide binds to Qa-1^b with a very high affinity (6). Here, we test this issue by examining the ability of class I leader- derived peptides from several mammalian species to bind Qa-1^b and define a minimum Qa-1^b binding peptide. Using surface plasmon resonance (SPR), we find that Qa-1^b binds class I leader peptides from almost all species tested. Unlike most class Ia molecules, the binding of peptide to Qa-1^b requires the retention of multiple amino acids from the native Qdm peptide sequence. The fact that this single peptide dominates the occupancy of Qa-1^b/HLA-E may also be related to the functional properties of these molecules, since recent data show that HLA-E interacts with CD94/NKG2 receptors on NK cells to deliver an inhibitory signal (7, 8).

	Materials and Methods

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Cells.

Cloning Soluble Qa-1^b for the Production of Soluble Molecules.

Production of Soluble Qa-1^b.

SPR.

View larger version (25K): [in this window] [in a new window]   View larger version (14K): [in this window] [in a new window]   View larger version (22K): [in this window] [in a new window]   View larger version (19K): [in this window] [in a new window]   Fig. 1.   Characteristics of sQa-1b/2m secreted from transfected D. melanogaster cells and its specific binding to immobilized QdmC5 peptide. (A) After concentration and purification, the supernatant from Qa-1b/2m-transfected Drosophila cells was resolved on a 15% SDS-PAGE, and stained using Coomassie brilliant blue. Arrows, Qa-1b heavy chain (Hc) and 2m. (B-D) SPR demonstrating binding of sQa-1b/2m to immobilized QdmC5. Sensorgrams were obtained using injection volumes of 20 µl at a rate of 1 µl/min. Mass increase due to macromolecular binding is measured in resonance units (RU). Arrowheads, Start ( ) and end ( ) of the injection. (B) Injection of 0.5 µM sQa-1b or sM3. (C) 0.5 µM sQa-1b was run over the chip alone, or in the presence of 20 µM Qdm (AMAPRTLLL), QdmC5 (AMAPCTLLL), or two control peptides, PMLTMCHAL and YPHFMPTNL. (D) 0.5 µM sQa-1b was run at 1 µl/min for 20 min over immobilized QdmC5 in the absence or presence of competitor peptides. Results are presented as relative binding of sQa-1b, where 0 represents binding in the presence of 20 µM Qdm (25 RU), and 1 is the binding in the absence of peptide (263 RU). *, +, and O represent binding in the presence of 20 µM of the entire 24-mer Dd leader, MGAMAPRTL and MAPRTLLLL, respectively.

Peptide Synthesis.

	Results

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We showed previously that Qa-1^b predominantly binds a single peptide species, Qdm (AMAPRTLLL; reference 3). This result precludes our ability to identify anchor residues by conventional techniques. Therefore, the approach used in this investigation was to generate sQa-1^b molecules and test their binding ability to a series of related ligands using SPR.

To study antigen binding to the Qa-1^b molecule, recombinant sQa-1^b/₂m dimers were generated in D. melanogaster (S2) cells following established protocols (11, 12). Truncated Qa-1^b molecules secreted by stably transfected cells were purified on Ni-coated beads followed by anion exchange. Both heavy chain and ₂m were visible on Coomassie-stained SDS-PAGE (Fig. 1 A).

Due to the SPR limitations in detecting the binding of small molecular weight peptides to immobilized class I molecules, we decided to attach the peptide to the chip. In the following experiments, we used QdmC5 (arginine cysteine substitution at position 5), which readily bound to the biosensor chip and in turn was bound by sQa-1^b (Fig. 1, B and C). This binding is specific, since sM3 (Fig. 1 B) and sCD1 (not shown) failed to bind. Binding of Qa-1^b to immobilized QdmC5 was blocked by adding QdmC5 or Qdm in solution, but not irrelevant control peptides (YPHFMPTNL) or (PMLTMCHAL), the latter of which contains the putative Qa-1^b peptide anchors methionine at P2 and leucine at P9 (Fig. 1 C).

Since peptides in solution can compete with immobilized QdmC5 for binding to soluble Qa-1^b, we used this approach to further analyze the peptide binding characteristics of this molecule. The Qdm nonamer peptide completely blocked binding at concentrations between 200 nM and 20 µM (Fig. 1 D). Extending the Qdm peptide by adding a leucine at position 10 (10-mer) results in decreased binding relative to the nonamer at 20 and 2 µM concentrations, and almost no binding at 200 nM. Trimming the Qdm peptide at the COOH end to an 8-mer gives a similar result. A 7-mer lost virtually all of its binding ability. We also tested the entire 24 amino acid leader of D^d from which the Qdm peptide is derived, and found that it failed to block the binding of Qa-1^b to immobilized peptides. Finally, we generated two more nonamers from the leader or D^d. Instead of spanning from residues 3 to 11, these peptides span amino acids 1-9 (MGAMAPRTL) and 4-12 (MAPRTLLLL). They both failed to bind to Qa-1^b.

Since HLA-E and Qa-1^b share unique features in their peptide binding grooves, we tested whether Qa-1^b can bind the same human class I-derived peptides that bind to HLA-E. We found that all of the tested peptides except for the one originating from the leader of HLA-A3 bound to Qa-1^b (Fig. 2). All of the peptides that bound to both Qa-1^b and HLA-E have very similar sequences that are derived from positions 3-11 of the leader. Peptides with a threonine methionine change at P2 (HLA-B27, -35) bound less well, and this was more evident in experiments where the inhibiting peptides were titrated at lower concentrations (data not shown).

View larger version (24K): [in this window] [in a new window]   Fig. 2.   Blocking of the binding of sQa-1b to immobilized QdmC5 by peptides derived from leader sequences of human class I molecules. Results are presented as relative binding, where 0 is the binding of sQa-1b in the presence of 20 µM Qdm (32 RU), and 1 is the binding in the absence of peptides (345 RU). 0.5 µM sQa-1b was run over immobilized QdmC5 for 20 min at the rate of 1 µl/min in the presence of 20 µM competitor peptides. For HLA-E binding, + indicates strong binding,  indicates no binding, and +/ indicates weak binding. Data taken from * Table 1 in reference 18,  reference 8, and § reference 4.

The unusual finding that HLA-E and Qa-1^b bind the same set of peptides, all derived from leader sequences of MHC class I molecules, raises the possibility that there is a conservation of similar epitopes in other mammals. In fact, inspection of representative class Ia sequences from a variety of mammalian species reveals a conserved "Qdm-like" epitope (Table 1). We tested the ability of these putative peptides to bind to Qa-1^b. Most of the tested peptides, except those from dog or cow class I molecules, bound well to Qa-1^b (Fig. 3 A). It is likely that the presence of the positively charged arginine at P3 of the peptide results in weaker binding.

View larger version (20K): [in this window] [in a new window]   View larger version (14K): [in this window] [in a new window]   Fig. 3.   Putative peptides from leaders of various mammalian class I molecules bind to Qa-1b. Results are presented at relative binding, where 0 is the binding of sQa-1b in the presence of 20 µM Qdm (25 RU in A, 26 RU in B), and 1 is the binding in the absence of blockers (276 RU in A, 230 RU in B). Running buffer was Hepes-buffered saline (HBS) in A and 2% DMSO in HBS in B.

Some of these leader peptides are extremely hydrophobic and not soluble in aqueous solvents. One such peptide is VMSPTVLLL, a Qdm-like epitope derived from the cat class I leader. To circumvent this problem, we diluted the peptide in 2% DMSO, where it remained soluble, and then used 2% DMSO as a running buffer. Under these conditions, we demonstrate that both the murine Qdm peptide and the peptide derived from the cat sequence bind Qa-1^b (Fig. 3 B).

We next determined the minimum requirement for ligand binding to Qa-1^b by synthesizing a number of peptides in which glycines were introduced in different positions (Table 2). We used glycine instead of alanine because the native Qdm sequence contains two alanines. Of the minimal peptides we tested, those with two or three nonglycine residues showed no (GMGGGGGGL, GMGGRGGGL) or very little binding (GMGGGGLGL, GMGGGGGLL) (Table 2). However, a peptide with four of nine native residues GMGGGGLLL blocked >50% of binding of sQa-1^b to immobilized QdmC5 peptide at the highest concentration tested (20 µM). However, when this peptide was titrated, we noted relatively little blocking activity at 2 µM and none at 200 nM, in marked contrast to the titration seen when more homologous peptides were tested (Fig. 3 A). This indicates that methionine at P2 and the three COOH-terminal leucines are sufficient for detectable although relatively very weak binding to Qa-1^b. Side chains of other amino acids in Qdm also play a role in the overall peptide binding. There is apparently a fine balance in their contribution which is dependent on the neighboring residues, since a peptide with five native residues (GMGGRGLLL) binds better to Qa-1^b than a peptide with six native residues (GMGPRGLLL).

	Discussion

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Both Qa-1^b (3) and HLA-E (4) bind a similar peptide derived from the leader of class Ia molecules. It appears that these are the major peptides that both of these class I molecules bind. Although Qa-1^b and HLA-E share unique residues in their F pocket that are not found in other class I molecules, it is surprising that they bind similar peptides, since they differ considerably in their primary structure. Data presented in this paper show that Qa-1^b not only binds peptides derived from the leader of murine MHC molecules, but also binds all of the human class I-derived peptides that were reported to interact with HLA-E, as well as putative class I leader peptides from several other mammalian species. An examination of leader sequences from representative class I alleles from several mammalian species shows a conservation of the Qa-1^b/HLA-E binding epitope between positions 3 and 11 of the segment. In fact, among the class I-derived peptides we tested, there is relatively little variability in most of the amino acids. For example, P4 (proline) and P9 (leucine) had no variability, whereas P1, P2, P5, and P7 had a single predominant residue although an alternative amino acid was seen in some peptides. The binding of this array of xenogeneic leader peptides was almost as efficient as the binding of the murine leader itself. It is important to note that leaders of Qa-1^b, HLA-E, and their other mammalian homologues are unique and do not contain the conserved Qdm-like epitope. Consequently, the peptide binding grooves of these molecules are not occupied by peptides derived from their own leaders.

We have attempted to determine the minimum motif required for peptide binding to Qa-1^b. Since only one peptide has been eluted from the groove of this molecule, it is not possible to assign anchor residues in the conventional manner. In addition to embedding principal anchors, methionine at P2 and leucine at P9, we needed to introduce two more wild-type residues in the polyglycine chain, leucines at P7 and P8, to observe detectable binding. However, the binding of this pentaglycine analogue was still considerably weaker than that of native Qdm, suggesting that side chains of other residues also contribute to the overall interaction. Although it is possible that binding of the minimal peptide with fewer anchor residues could have been found had we used a backbone other than glycine (13), several other minimal peptides with glycine backbones have been used successfully to identify anchors that participate in binding to class I molecules (14, 15).

Thus, the finding that Qdm requires multiple anchors would explain the dominance of a single peptide in its groove. The data presented here, together with the recent crystal structure of HLA-E bound to its peptide (5), suggest that Qa-1^b and HLA-E can only bind Qdm-like peptides with high efficiency. However, it cannot be ruled out that their occupancy by these peptides is a result of a restrictive peptide antigen processing and/or presentation pathway. It is interesting to note that the common ligand that Qa-1^b and HLA-E bind is derived from a conserved part of class I leader segments that are expendable in the mature protein and thus would not affect selection for polymorphic peptide binding residues.

Boyson et al. (16) pointed out that a comparison of the rates of synonymous and nonsynonymous nucleotide substitutions in the peptide binding region versus the remainder of the molecule indicates that the peptide binding groove of HLA-E and its homologues in macaques has been conserved for over 36 million years, when the two last shared a common ancestor. Yeager et al. have communicated that, although not orthologous, Qa-1^b and HLA-E might have evolved similar functions through convergent evolution at the amino acid sequence level of the peptide binding region (17). Regardless of whether molecular-level convergence or evolutionary conservation of the peptide binding region accounts for the specificity of these grooves, this conservation of specificity suggests a crucial immunological function for these molecules. In this regard, it has recently been shown that HLA-E is a ligand for CD94/ NKG2 receptors on NK cells; interaction of HLA-E with this receptor protects target cells from NK-mediated lysis (7, 8). Although this has not yet been demonstrated for Qa-1^b, it is likely that it interacts with its murine CD94/ NKG2 counterpart in a similar manner. Class I molecules in mice could, through Qa-1^b, control the activity of NK cells which would be signaled upon interaction with cell surface-expressed Qa-1^b. Decreased expression and/or processing of class I molecules would decrease the expression of Qa-1^b, which would in turn result in a changed activity level of NK cells.

It is conceivable that occasionally Qa-1^b-bound class I-derived peptides could be replaced, or that some of the peptide binding grooves might be initially occupied by other self- or pathogen-derived peptides which would be presented to T cells. Future studies should show whether Qa-1^b is recognized by NK cell receptors, and what role peptides play in the response.