Analysis of peptide length preference of the rat MHC class Ia molecule RT1-Au, by a modified random peptide library approach
James Stevens,
Karl-Heinz Wiesmüller1,
Geoffrey W. Butcher and
Etienne Joly
Laboratory of Functional Immunogenetics, The Babraham Institute, Cambridge CB2 4AT, UK
1 EMC Microcollections, Sindelfinger Strasse 3, 7020 Tübingen, Germany
Correspondence to:
J. Stevens
 |
Abstract
|
---|
Using random peptide libraries we have previously shown that both mouse and rat class I molecules can exhibit different peptide length preferences. Such studies required expression of the particular class I molecules in RMA-S, a cell line deficient in the transporter associated with antigen presentation (TAP). For another rat class I molecule called RT1-Au, however, we found that expression in RMA-S was poor and could not be increased sufficiently by incubation at 26°C. To circumvent this problem we performed our studies on C58, a rat cell line that expresses RT1-Au naturally in the presence of a functional TAP transporter. Using C58 cells, cell-surface-expressed class I molecules were `stripped' of peptides and ß2-microglobulin by washing the cells with an acidic citrate buffer (pH 3.3). Peptide stabilization assays, assessed by FACS analysis, were then performed using either specific peptides or synthetic random peptide libraries of different lengths (715 amino acids), supplemented with recombinant rat ß2-microglobulin. As a positive control an RT1-Au-specific nonamer peptide was designed using the previously determined peptide binding motif and this was found to bind to RT1-Au at nanomolar concentrations. Both length preference and importance of free N- and C-termini were tested using free base, formylated and acetylated peptide libraries. Results showed that RT1-Au was not able to accommodate N- or C-terminally blocked peptides but displayed a preference for peptides of 912 amino acids, similar to the preference observed for the RT1-A1c allotype, the other rat TAP-B-associated molecule tested thus far. These results suggest that length preference remains a consideration to explain the allelic class ITAP associations of the RT1-A region.
Keywords: class I MHC, FACS, MHC, peptide stabilization, random peptide library, RT1-A
 |
Introduction
|
---|
It is generally accepted that the majority of peptides bound to MHC class I molecules are generated in the cytoplasm, principally by the proteasome (1). For presentation, these peptides are transported into the endoplasmic reticulum (ER) by the heterodimeric transporter associated with antigen presentation (TAP). In the laboratory rat, two allelic variants of TAP have been described and found to differ by as many as 25 amino acids in the TAP2 chain of the transporter (2). These differences were shown to affect the supply of peptides to the MHC class I molecules. The TAP-A form of the transporter (TAP1/TAP2-A) is more permissive for peptide access, whereas the TAP-B form (TAP1/TAP2-B) preferentially transports peptides with aromatic or hydrophobic amino acid residues at the C-terminus (3,4).
Interestingly, it was shown that class I MHC alleles, encoded by the classical class Ia locus RT1-A, are genetically associated with only one form of the transporter (5). Of the 13 MHC class I molecules identified thus far, seven appear to associate with the TAP-A form of the transporter and six with the TAP-B form (see Table 1
for summary) (6). The reasons for such strong allelic class ITAP associations cannot be explained simply by the preference of the MHC class I molecule for the C-terminal amino acid residue, since by this reasoning RT1-Al, a TAP-A-associated class I molecule which has an aromatic/hydrophobic preference (see Table 1
) (7,8), should be associated with TAP-B. Other factors must therefore be operating and one possibility is that TAP-A-associated class I molecules have a different peptide length preference from those associated with the TAP-B transporter. Transport data from Heemels and Ploegh support this theory by suggesting that the TAP-B form of the transporter can transport longer peptides (9). Purified rat liver microsomes were used and TAP transporter activity assayed with specific peptides of differing length (of the type: GY...NATI), therefore not excluding the possible effects of sequence preference in their assays. In addition, each strain used has its own TAP1 subunit and although no functional polymorphisms have been described in the rat, there is evidence that splice variations may occur in humans which influences peptide selectivities (10). Conflicting data from Koopmann et al. (11) proposes the TAP-A transporter to be the more permissive for longer peptides. In this case, the study was performed using permeabilized TAP1/TAP2-A and TAP1/TAP2-B transfected cell lines, therefore removing possible influences of the TAP1 subunit variations. Both a specific peptide series (TNKT...Y) and a randomized peptide library [RY(X)nNKTL] were used, thus reducing the possible effects of sequence preference on transport. In this latter study both types of peptides showed the same length preference results.
View this table:
[in this window]
[in a new window]
|
Table 1. Summary of transporter associations and peptide preferences for rat class I molecules encoded by the classical class Ia locus RT1-A)
|
|
Certain residues surrounding the A and F pockets of the peptide binding groove, which are conserved across species, act to lock both ends of the peptide into the groove. In the rat these residues are frequently non-canonical, particularly in the TAP-B-associated MHC class I molecules (6). We previously tested a hypothesis that certain TAP-B-associated class I molecules might be able to bind longer peptides (12) by extension out of the A and/or F pockets. Two molecules, RT1-Aa and RT1-A1c were expressed in the TAP-deficient mouse cell line RMA-S, and tested for stabilization by random peptide libraries of different lengths. Surprisingly, results showed that it was the TAP-A-associated MHC class I molecule, RT1-Aa, which is orthodox in all of the canonical A and F pocket residues, which was more permissive to longer peptides. Extension of this study to several other known RT1-A-encoded molecules would help to determine if this peptide preference is truly TAP associated. We therefore decided to continue this study with the TAP-B-associated MHC class I molecule, RT1-Au, which is frequently present in important rat stocks/strains [i.e. Wistar (Furth, WAG, BB), Long Evans and Sprague-Dawley]. Previously published data revealed a binding motif with strong preferences for hydrophobic and uncharged residues, in particular with a C-terminal preference for tyrosine, thus agreeing with its TAP association (13). Unfortunately, when we expressed this molecule by transfection into RMA-S, we found that expression was poor and could not be increased sufficiently by incubation at 26°C.
We thus describe here a protocol for length preference studies capable of by-passing the requirement for a TAP-deficient cell line. It is a modification of a method described to isolate class I presented peptides from viable cells and reconstitute them with known peptides (14,15). Acid elution was performed on a cell line expressing RT1-Au naturally at high level. The `acid-stripped cells' were then allowed to re-stabilize their cell surface heavy chains in the presence of recombinant rat ß2-microglobulin with serial dilutions of random peptide libraries of different length and the ability of the libraries to stabilize RT1-Au was subsequently determined by FACS analysis.
 |
Methods
|
---|
Cloning RT1-Au into the RMA-S cell line
The rat MHC class Ia molecule RT1-Au was cloned into a retroviral expression system as previously described (4) and the resulting construct was used to infect mouse RMA-S cells. Additionally, RT1-Au was also cloned into the pcDNA3 vector (Invitrogen, Groningen, The Netherlands), which has the stronger cytomegalovirus enhancer promoter, and the linearized construct was transformed into RMA-S cells by electroporation and selected with G418 sulfate (Calbiochem, Nottingham, UK).
Preparation of peptides
Two specific peptides were tested prior to performing the length preference assays. For RT1-Aa, the 13mer MTA peptide (16) was synthesized by Alta Biosciences (University of Birmingham, UK). For RT1-Au, a synthetic binding peptide designed from the previously published binding motif (13), was synthesized at EMC Microcollections (Tübingen, Germany). Peptide stocks were dissolved in DMSO and their concentrations determined by the micro BCA assay (Pierce, Rockford, IL) using BSA as standard. Stocks were diluted with RPMI 1640 containing 0.5% (w/v) BSA (RPMI/0.5% BSA) to give 5-fold diluted concentrations starting from 200 µM (a minimum of six samples in total).
The synthetic random peptide libraries (715 amino acids in length) were prepared by solid-phase peptide synthesis using Fmoc/tBu chemistry for the introduction of random degenerate positions (x = 19 L-amino acids, cysteine excluded) as described previously (13). For each of the peptide libraries, 1 mg was dissolved in 50 µl DMSO (Pierce; packed under nitrogen), aliquoted (10 µl) to Eppendorf tubes, gassed with nitrogen and stored at 70°C. Peptide concentrations were determined using the micro BCA assay as above. Peptide stocks, sufficient for the assay, were diluted with RPMI/0.5% BSA to give starting concentrations of 2 mM, assuming an average mol. wt of 119.73 for amino acid residues in the random positions (19 amino acid residues, cysteine excluded). Serial 3-fold dilutions were made resulting in eight peptide concentrations for each different peptide pool.
Peptide stabilization assays
C58 (17) or C58-3.3/1 cells (2) were grown at 37°C in RPMI/10% FCS. For the assay, cells were spun and resuspended in 1 ml of pre-chilled citrate buffer (131 mM citric acid, 66 mM Na2HPO4, pH 3.3 and 1.0% BSA) (15) and incubated for 5 min at room temperature. RPMI/0.5% BSA (25 ml; pre-chilled to 4°C) was added and the cells were then centrifuged at 240 g. The medium was removed and the cell pellet was resuspended and washed twice more with fresh pre-chilled RPMI/0.5% BSA. After the final wash, cells were resuspended in a volume of RPMI/0.5% BSA containing 200 nM recombinant rat ß2-microglobulin (13), to give ~3x106 cells/ml. Cells (25 µl) were aliquoted to a 96-well plate and 25 µl peptide solution was added to the appropriate well (highest final concentration of peptide was 1 mM). The plate was incubated at 37°C for 4 h after which the cells were washed once with PBS containing 0.5% BSA, and then re-suspended in 150 µl PBS, 2% (v/v) FCS and 0.05% (w/v) sodium azide (PFN). The cells were subjected to FACS analysis with first-stage anti-rat mAb: U9F4 (anti-RT1-Au) (18) or MAC 30 (anti-RT1-Aa) (19). For the second-stage antibodies, FITC-conjugated rabbit anti-mouse Ig (Dako, Denmark) was used at a 1/100 dilution for U9F4 whilst for MAC 30, a FITC-conjugated rabbit anti-rat antibody was used (Dako). All samples were subjected to FACS analysis using a FACSCalibur (Becton Dickinson, Mountain View, CA) in the presence of propidium iodide (2.5 µg/ml).
 |
Results
|
---|
We wished to study the peptide binding requirements of RT1-Au as was done previously for both RT1-A1c and RT1-Aa (12), so attempts were made to express RT1-Au in the RMA-S cell line using both a retroviral vector (4) and the pcDNA3 expression system. Both of these approaches were, however, unsuccessful in that incubation of the transfected cell line at 26°C resulted in minimal induction of the surface expression of the class I molecule, as judged by FACS staining with the antibody U9F4 (18) (results not shown). Western blot analysis of cell lysates of RMA-S transfected with RT1-Au and RT1-A1c using a rabbit anti-rat class I antibody, F88/5 (S. J. Powis and G. W. Butcher; unpublished) revealed the expression of both heavy chains at similar levels and no staining with untransfected cells (results not shown), suggesting that RT1-Au is retained in the ER, but this has yet to be investigated further.
For peptide stabilization studies using TAP-defective cell lines to succeed, sufficient induction by incubation at a lower temperature is an essential requirement for comparing each peptide library and to distinguish weak peptide binding from peptide-independent background fluctuations. Cloning MHC class I molecules into the RMA-S cell line is both time consuming and expensive, and is not guaranteed to produce cell lines with sufficient cell surface expression for use in such assays (as shown for RT1-Au in this present study). We therefore developed a new method inspired by the results of Storkus et al. (14) as a quick and inexpensive alternative to using the RMA-S cell line. The method is simple: cells are washed briefly at pH 3.3 with a citrate-phosphate buffer to remove ß2-microglobulin and bound peptides. This can be confirmed by the loss of conformational class I epitopes on the cell surface, determined by FACS analysis. The simplicity of the system is that theoretically any cell line can be used provided a specific antibody to the class I molecule under study is available. For this study, the cell line C58 (RT1-Au/TAP-B) (17) was used, as well as its derivative, C58-3.3/1 (2) that also expresses RT1-Aa through transfection. Initially, C58-3.3/1 was the cell line of choice because the known length preferences for RT1-Aa could be compared directly with RT1-Au, thus providing a convenient internal control.
Initial studies showed that for both class I molecules on C58-3.3/1, cells needed to be incubated in the low pH buffer for a minimum of 5 min in order to achieve >80% loss of conformational class I epitopes, determined by FACS analysis (results not shown). This study used the same acidic buffer (pH 3.3) as described by Storkus et al. (14), who found >95% loss of class I expression after only 1560 s for the human cell line Mel 624 (HLA-A2+), indicating that both of the rat class I molecules tested here may be more stable at this pH. Having determined the time required to lose cell surface class I expression, specific peptides were tested for re-stabilization. For RT1-Aa, the 13mer mitochondrial minor histocompatibility antigen (ILFPSSERLISNR) (16) was used, whilst for RT1-Au, an `ideal peptide' (EIPDIFITY) was designed based on our previously published motif analysis (12) using the most abundant residues in the eluted peptide pool at each position of the peptide. Figure 1
shows that using this stabilization system, both RT1-Au and RT1-Aa re-stabilize specifically in the presence of the appropriate peptide. The stabilization curve for RT1-Aa is in agreement with previously published data (16), obtained using RMA-S transfected with RT1-Aa. As a measure of affinity, peptide binding is given in C50 values (the concentration of peptide required to achieve 50% of the level of cell surface expression of cells with no acid treatment and without any exogenously added peptide). The `ideal peptide' for RT1-Au, designed to have optimum residues in all positions, resulted in a peptide that was almost 10-fold better at binding to RT1-Au (C50 = 0.015 µM) than the natural 13mer peptide binding to RT1-Aa (C50 = 0.105 µM). This very high affinity can be explained by accommodation of more preferred residues in secondary anchor pockets within the groove. For the RT1-Au `ideal peptide', C58 and C58-3.3/1 produced identical binding curves (see Fig. 1
). However, we found that the expression level of RT1-Au on C58-3.3/1 was only 2030% of that on C58. This explains the much more erratic profile of the binding curve (see Fig. 1
), thus making any stabilization with weak binding peptide libraries difficult to detect or quantify accurately. Such reduced expression may be explained by the competitive inhibition of RT1-Au's association with TAP by accumulating RT1-Aa, within the ER (20). The C58 parent line was therefore used for subsequent length preference studies. Also worthy of note is the ability of the RT1-Au `ideal peptide' to bind weakly to RT1-Aa (C50 = 12 µM; Fig. 1
). The peptide EIPDIFITY has none of the primary anchors in the RT1-Aa binding motif previously reported (4), although those results did show minor preferences for Pro and Asp at P3 and P4 respectively. This suggests that when RT1-Aa is expressed in a cell line where the TAP transporter is unable to provide optimum peptides, it might be able to bind peptides in which only the secondary anchors are contributing to MHC interaction. This may be the case in the C58-3.3/1 cell line where we see good expression of RT1-Aa in the presence of TAP-B (staining was 4-fold higher than for RT1-Au by FACS analysis; results not shown) and in vivo, in the PVG.R1 rat strain.

View larger version (25K):
[in this window]
[in a new window]
|
Fig. 1. Comparison of stabilization of specific peptides on the C58 cell line and C58-3.3/1 (transfected with RT1-Aa). After acid-stripping of the peptides naturally bound to class I molecules, synthetic peptides were added at varying concentrations together with recombinant rat ß2-microglobulin (see Methods). After 4 h at 37°C, the level of restabilization was determined by FACS analysis using class I-specific antibodies. Results shown are the average of three independent experiments.
|
|
Length preference studies of RT1-Au on the C58 cell line (Fig. 2
and Table 2
), using random peptide libraries of variable length, revealed a similar length preference profile to RT1-A1c, the other TAP-B-associated class I tested. The optimum length appeared to be peptides of only 912 amino acids in length, with markedly weaker binding with the 8mer and 13mer libraries (C50 data in Table 2
). In addition, we also tested the ability of RT1-Au to bind peptides with modified N- or C-terminal residues. We found that acetylation or formylation of the N-terminal amine resulted in a dramatic drop in the levels of stabilization attained for the optimum length nonapeptide library (Table 2
). Acetylated nonapeptide amide libraries produced no detectable re-stabilization of RT1-Au. Altogether, these results indicate that this class I molecule is unable to accommodate modified residues such as is seen for the mitochondrion-derived N-terminal formylated peptides bound by H2-M3 (21).
View this table:
[in this window]
[in a new window]
|
Table 2. Specificity of RT1-Au for random peptide libraries of different lengths or for N- and C-terminally modified nonapeptide libraries
|
|
 |
Discussion
|
---|
The results presented here are part of an ongoing study attempting to explain the reasons for the observed genetic association of rat class I MHC alleles with only one form of the TAP transporter (5). Such associations cannot be explained simply by the preference of the MHC class I molecule for the peptide's C-terminal residue since, at first examination, the permissiveness of the TAP-A rat transporter allele for almost any C-terminal residue should make it an adequate supplier of peptides for all rat class I alleles. Other factors are likely to be operating and one possibility is that class I molecules associated with a certain transporter have a different peptide length preference compared to those associated with the other transporter. Such differences in the length preferences were observed between two rat MHC class I molecules previously studied, i.e. RT1-Aa (915mer) and RT1-A1c (912mer) (12). In this report we show that the TAP-B-associated molecule RT1-Au has similar restrictive characteristics to RT1-A1c.
Peptides generally bind to MHC class I molecules in an extended conformation with three residues at the N-terminus and two residues at the C-terminus adopting an essentially invariant conformation in the MHC class I binding site (22). Consequently, central residues of the peptide can be more flexibly accommodated within the peptide binding site allowing for longer peptides to bind by `bulging' out of the groove (23). Although this is the generally accepted model for the binding of long peptides, there are exceptions such as extension of the C-terminal residue out of the groove (24). In the rat, data for binding longer peptides to RT1-Aa suggest that the general `bulging' model is occurring and this has been specifically confirmed by recent crystal structure data for RT1-Aa complexed to the 13mer MTA peptide used in this study (Speir et al., manuscript in preparation).
Both of the TAP-B-associated class I molecules studied (RT1-A1c and RT1-Au) only appear to accommodate peptides of up to 12 amino acids in length into their grooves, suggesting that they have a reduced ability to accommodate `bulging' peptides compared to RT1-Aa, which can bind 15mer random peptide libraries almost as well as the 9mer library (12). One possible explanation is that the TAP-B-associated molecules depend more upon additional internal anchor pockets for stable peptide binding than their TAP-A counterparts. If this were the case, the distance from one anchor to the next would be less, therefore reducing the gap from which a stable `bulge' could occur. Evidence consistent with this proposal comes from the recombinant random peptide refolding system described for obtaining binding motif data (13): a molecule with more anchors along the length of the groove ought to bind to a reduced population within the random peptide library (i.e. be more selective). Consequently more random peptide would be needed to obtain complex formation. Indeed this was the observation for both RT1-Au and RT1-A1c where a concentration of 100 µM nonamer random peptide library was required for a comparable level of complex formation seen with RT1-Aa at a concentration of 10 µM of the peptide library (13). Subsequent binding motif data also revealed an increased number of amino acid preferences among the middle residues (P5P8) of nonamer peptides eluted from TAP-B-associated MHC class I molecules (RT1-Au and RT1-A1c) compared to the TAP-A counterpart studied (RT1-Aa).
Sequence analysis and structural modeling also suggests the possibility of increased importance of secondary anchors with TAP-B-associated molecules. On the floor of the peptide binding groove of TAP-A associated molecules, and also seen with human class I molecules (25), bulky aromatic residues at positions 79 of the heavy chain protrude up, providing a `ridge' which acts to force a kink in the peptide and direct it to the top of, and possibly out of the groove. For the TAP-A associated molecules, five out of seven retain Tyr at position 9, whereas in five out of the six known class I molecules associated with TAP-B, position 9 is mutated from a Tyr to an amino acid with a smaller side chain residue (see Table 1
). Thus, the majority of TAP-B-associated molecules might prefer shorter peptides which, with a substantial reduction of this `ridge', can establish secondary anchor binding along the middle part of the binding groove (Fig. 3
). This possibility is being actively studied.

View larger version (20K):
[in this window]
[in a new window]
|
Fig. 3. Proposed mechanism for peptide binding to TAP-A- and TAP-B-associated MHC class I molecules. (A) The majority of TAP-A-associated molecules possess a bulky tyrosine at position 9 of the heavy chain. In conjunction with the conserved Tyr at position 7, a bulky `ridge' is formed in the bottom of the peptide binding groove. This may result in the bound peptide being forced up and out of the groove. Thus, by-passing interactions in the middle of the groove allows the binding of longer peptides. (B) At the same position in the majority of TAP-B-associated molecules is a smaller residue resulting in a reduced `ridge'. This might allow preferred binding of shorter peptides which can establish secondary anchor binding along the middle part of the binding groove.
|
|
In conclusion, our results indicate that in the rat system, the observed genetic association of the RT1-A-encoded class I MHC molecules with only one form of the TAP transporter might be, in part, due to differences in peptide length preference. Results presented here and elsewhere point to secondary anchors playing prominent roles in peptide binding by TAP-B-associated class I molecules which could be responsible for the poor ability of these molecules to bind longer peptides. The assay described here will allow this hypothesis to be tested by simplifying the study of the length preference of additional rat MHC class I molecules, subject to the availability of a specific antibody.
 |
Acknowledgments
|
---|
This work was supported by a BBSRC Project Grant and BBSRC Senior Postdoctoral Fellowship to E. J., BBSRC CSG funding to the Laboratory of Immunogenetics, Babraham, and funding by the Deutsche Forschungsgemeinschaft (SFB 510/C3) to K.-H. W. The authors would like to thank R. J. M. Stet for the kind gift of the U9F4 hybridoma and N. G. A. Miller for maintenance of the FACS machine.
 |
Abbreviations
|
---|
ER endoplasmic reticulum |
TAP transporter associated with antigen processing |
 |
Notes
|
---|
Transmitting editor: A. Cooke
Received 24 May 1999,
accepted 5 October 1999.
 |
References
|
---|
-
Rock, K. L., Gramm, C., Rothstein, L., Clark, K., Stein, R., Dick, L., Hwang, D. and Goldberg, A. L. 1994. Inhibitors of the proteasome block the degradation of most cell-proteins and the generation of peptides presented on MHC class I molecules. Cell 78:761.[ISI][Medline]
-
Powis, S. J., Deverson, E. V., Coadwell, W. J., Ciruela, A., Huskisson, N. S., Smith, H., Butcher, G. W. and Howard, J. C. 1992. Effect of polymorphism of an MHC-linked transporter on the peptides assembled in a class I molecule. Nature 357:211.[ISI][Medline]
-
Momburg, F., Roelse, J., Howard, J. C., Butcher, G. W., Hämmerling, G. J. and Neefjes, J. J. 1994. Selectivity of MHC-encoded peptide transporters from human, mouse and rat. Nature 367:648.[ISI][Medline]
-
Powis, S. J., Young, L. L., Joly, E., Barker, P. J., Richardson, L., Brandt, R. P., Melief, C. J., Howard, J. C. and Butcher, G. W. 1996. The rat cim effect: TAP allele-dependent changes in a class I MHC anchor motif and evidence against C-terminal trimming of peptides in the ER. Immunity 4:159.[ISI][Medline]
-
Joly, E., Deverson, E. V., Coadwell, J. W., Günther, E., Howard, J. C. and Butcher, G. W. 1994. The distribution of TAP2 alleles among laboratory rat RT1 haplotypes. Immunogenetics 40:45.[ISI][Medline]
-
Joly, E., Le Rolle, A.-F., González, A. L., Mehling, B., Stevens, J., Coadwell, W. J., Hünig, T., Howard, J. C. and Butcher, G. W. 1998. Co-evolution of rat TAP transporters and MHC class I RT1-A molecules. Curr. Biol. 8:169.[ISI][Medline]
-
Powis, S. J., Young, L. L., Barker, P. J., Richardson, L., Howard, J. C. and Butcher, G. W. 1993. Major histocompatibility complex-encoded ABC transporters and rat class I peptide motifs. Transplant. Proc. 25:2752.[ISI][Medline]
-
Reizis, B., Schild, H., Stevanovi
, S., Mor, F., Rammensee, H. G. and Cohen, I. R. 1997. Peptide binding motifs of the MHC class I molecules (RT1.A) of the Lewis rat. Immunogenetics 45:278.[ISI][Medline]
-
Heemels, M. T. and Ploegh, H. L. 1994. Substrate-specificity of allelic variants of the TAP peptide transporter. Immunity 1:775.[ISI][Medline]
-
Yan, G., Shi, L. J. and Faustman, D. 1999. Novel splicing of the human MHC-encoded peptide transporter confers unique properties. J. Immunol. 162:852.[Abstract/Free Full Text]
-
Koopmann, J. O., Post, M., Neefjes, J. J., Hämmerling, G. J. and Momburg, F. 1996. Translocation of long peptides by transporters associated with antigen-processing (TAP). Eur. J. Immunol. 26:1720.[ISI][Medline]
-
Stevens, J., Wiesmüller, K.-H., Walden, P. and Joly, E. 1998. Peptide length preferences for rat and mouse MHC class I molecules using random peptide libraries. Eur. J. Immunol. 28:1272.[ISI][Medline]
-
Stevens, J., Wiesmüller, K.-H., Barker, P. J., Walden, P., Butcher, G. W. and Joly, E. 1998. Efficient generation of major histocompatibility complex class I-peptide complexes using synthetic peptide libraries. J. Biol. Chem. 273:2874.[Abstract/Free Full Text]
-
Storkus, W. J., Zeh, H. J., Salter, R. D. and Lotze, M. T. 1993. Identification of T-cell epitopes: rapid isolation of class I-presented peptides from viable cells by mild acid elution. J. Immunother. 14:94.[ISI][Medline]
-
Zeh, H. J., Leder, G. H., Lotze, M. T., Salter, R. D., Tector, M., Stuber, G., Modrow, S. and Storkus, W. J. 1994. Flow-cytometric determination of peptideclass I complex formation: identification of p53 peptides that bind to HLA-A2. Hum. Immunol. 39:79.[ISI][Medline]
-
Bhuyan, P. K., Young, L. L., Fischer Lindahl, K. and Butcher, G. W. 1997. Identification of the rat maternally transmitted minor histocompatibility antigen. J. Immunol. 158:3753.[Abstract]
-
Silva, A., Macdonald, H. R., Conzelmann, A., Corthesy, P. and Nabholz, M. 1983. Ratxmouse T-cell hybrids with inducible specific cytolytic activity. Immunol. Rev. 76:105.[ISI][Medline]
-
Stet, R. J. M., Zantema, A., Vanlaar, T., Dewaal, R. M. W., Vaessen, L. M. B. and Rozing, J. 1987. U9F4a monoclonal antibody recognizing a rat polymorphic class I determinant. Trans. Proc. 19:3004.[ISI]
-
Stephenson, S. P., Morley, R. C. and Butcher, G. W. 1985. Genetics of the rat CT system: its apparent complexity is a consequence of cross-reactivity between the distinct MHC class I antigens RT1.C and RT1.A. J. Immunogenet. 12:101.[ISI][Medline]
-
Knittler, M. R., Gulow, K., Seelig, A. and Howard, J. C. 1998. MHC class I molecules compete in the endoplasmic reticulum for access to transporter associated with antigen processing. J. Immunol. 161:5967.[Abstract/Free Full Text]
-
Shawar, S. M., Cook, R. G., Rodgers, J. R. and Rich, R. R. 1990. Specialized functions of MHC class I molecules. 1. An N-formyl peptide receptor is required for construction of the class I antigen MTA. J. Exp. Med. 171:897.[Abstract]
-
Madden, D. R., Gorga, J. C., Strominger, J. L. and Wiley, D. C. 1992. The three-dimensional structure of HLA-B27 at 2.1 angstrom resolution suggests a general mechanism for tight peptide binding to MHC. Cell 70:1035.[ISI][Medline]
-
Guo, H. C., Jardetzky, T. S., Garrett, T. P. J., Lane, W. S., Strominger, J. L. and Wiley, D. C. 1992. Different length peptides bind to HLA-Aw68 similarly at their ends but bulge out In the middle. Nature 360:364.[ISI][Medline]
-
Collins, E. J., Garboczi, D. N. and Wiley, D. C. 1994. Three-dimensional structure of a peptide extending from one end of a class I MHC binding site. Nature 371:626.[ISI][Medline]
-
Saper, M. A., Bjorkman, P. J. and Wiley, D. C. 1991. Refined structure of the human histocompatibility antigen HLA-A2 at 2.6Å resolution. J. Mol. Biol. 219:277.[ISI][Medline]