From the Department of Immunology, The Babraham
Institute, Cambridge CB2 4AT, United Kingdom, the
¶ Naturwissenschaftliches und Medizinisches Institut,
Universität Tübingen, D-72762 Reutlingen, Germany, the
Microchemical Facility, The Babraham Institute, Cambridge
CB2 4AT, United Kingdom, and the ** Dermatologische Klinik,
Charité, Humboldt-Universität, Schumannstrasse 20/21,
D-10117 Berlin, Germany
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ABSTRACT |
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The use of synthetic random peptide libraries is
a powerful technology for the study of many aspects of antigen
presentation and peptide selection by major histocompatibility complex
(MHC) molecules. Here we have used them in conjunction with a
recombinant system to determine the peptide binding motifs of three
classical class I MHC molecules of the laboratory rat:
RT1-Aa, RT1-Au, and RT1-A1c.
Described is a method for producing large amounts of soluble class I
heavy and light chains in bacteria. Refolding RT1-Aa heavy
chain (HC) with rat 2-microglobulin (
2m)
in the presence of a specific peptide and the subsequent purification
of the complex yielded conformationally correct material. This was
assessed by gel chromatography, SDS-polyacrylamide gel electrophoresis,
isoelectric focussing gel electrophoresis, enzyme-linked immunosorbent
assay, and fluorescence-activated cell sorter analysis employing a
previously unreported method utilizing a His-Tag affinity silica. By
refolding RT1-Aa HC and rat
2m around a
random nonapeptide library and subjecting the resulting complex to acid
elution of the bound peptides and pool sequencing, the peptide binding
motif for this MHC class I molecule was determined. Results
corresponded well with those previously determined from naturally bound
peptides and in addition gave a clear and unambiguous signal for the
C-terminal anchor residue. This method was then applied to determine
the previously undescribed binding motifs for RT1-Au and
RT1-A1c. For both molecules, the whole motif was confirmed
from naturally bound peptides. We propose this method as an alternative
way to obtain the whole class I MHC peptide motif, particularly when a
specific antibody is unavailable and/or natural expression of the class
I molecule of interest is low.
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INTRODUCTION |
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Antigenic peptides generated principally by the proteasome are transported into the endoplasmic reticulum (ER)1 via the heterodimeric transporter associated with antigen presentation (TAP) (1, 2) where they associate with newly synthesized major histocompatibility complex (MHC) class I molecules. In the rat, the TAP2 subunit of the transporter exists as two main allotypes (TAP2-A and TAP2-B) (3, 4), and it appears that class I MHC alleles, encoded by the classical class Ia locus RT1-A, are genetically associated with only one form of the transporter (4). In recent years, it has been shown that the two forms of the transporter display different peptide selectivities, such that TAP-A binds and transports peptides with many different amino acids present at the C terminus while TAP-B generally allows only hydrophobic and aromatic residues in that position (5, 6). In addition, the TAP-B form of the transporter has been reported to be able to accept longer peptides than the usual 8-11 amino acid lengths (7) although this is disputed (8). Despite extensive studies on TAP peptide selectivities, few peptide binding motifs for rat class Ia molecules (RT1-A) have been reported. Thus far, the motifs of only two TAP-A associated molecules, RT1-Al (9, 10) and RT1-Aa (11), have been determined.
The main method used to determine a peptide binding motif for an MHC class I molecule, including those rat alleles already reported, is the pool sequencing method of Falk et al. (12), that consists of acid eluting peptides from a natural source of class I, separating the heavy and light chains from the peptides by reverse phase HPLC, pooling the predetermined peptide containing fractions, and subjecting the pool to automated sequence analysis. This method has proven extremely useful for obtaining peptide binding data for class I from different species. However, for its success, the method relies on a specific antibody for the purification of the class I under study, but this is not always available. Antibodies can often have cross-reactivities with other known or unknown class Ia or class Ib molecules (13). If expression of the molecule under study is low, then cloning and expression in a suitable cell line is needed. In addition, the C-terminal anchor residue is not always clearly defined by this sequencing method since class I alleles can bind peptides of varying lengths, thus spreading the C-terminal signal over several sequencing cycles (14-16).
An alternative method called positional scanning (17) has been shown to be extremely useful within the field of immunology (18-20). Synthetic random peptide libraries coupled to single defined amino acids at set positions within the peptide have been used successfully to probe the effect of the defined amino acid on peptide-MHC interactions, induction of T-cell responses, specificity of proteasomes (21), and TAP (22, 23).
Here, we have combined the above methods to determine peptide binding
motifs for class I MHC molecules. We report the use of a bacterial
system to produce recombinant protein of three truncated rat class Ia
MHC molecules and rat 2-microglobulin (
2m). We have successfully refolded these rat class I
heavy chains in the presence of rat
2m and a random
nonapeptide library and have successfully determined the binding motifs
for all three molecules under study.
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EXPERIMENTAL PROCEDURES |
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Expression and Purification--
The regions coding for amino
acids 1-276 of the RT1-Aa, RT1-Au, and
RT1-A1c heavy chains were amplified from cloned cDNA by
polymerase chain reaction (PCR) using the oligonucleotide
primers: forward
5-CGGGATCCCCATATGGGCTCACACTCGCTGCGGTATT-3
and
reverse
5
-GAAGATCTCGAGAGGCTCCCATCTCTGGGAAAGTGGC-3
(bold are coding nucleotides and underlined are restriction sites used). Full-length rat
2m was amplified from
PVG-RT1u(AO) poly T-primed reverse strand
concanavalin A blasts, with primers designed using the nucleotide
sequence from the EMBL data base: accession number Y00441, forward
5
-CGGGATCCCCATATGATTCAGAAAACTCCCCAAATTCAA-3
and reverse 5
-CGCGGATCCAGATGATTCAGAGCTCC-3
(anneals
11 base pairs downstream of the stop codon). The resulting fragments
were digested with NdeI/XhoI (heavy chain) and
NdeI/BamHI (
2m), ligated separately into the T7 expression plasmid pET-22b(+) (Novagen Inc.,
WI), and transformed/selected in XL2-Blue (Stratagene). DNA sequences
from these constructs were checked, and plasmids were retransformed
into the Escherichia coli strain BL21(DE3) (Novagen Inc.).
Gel Electrophoresis--
Sodium dodecyl sulfate-polyacrylamide
gel electrophoresis (SDS-PAGE) was performed as described (24). The
concentration of acrylamide used was 15%, and gels were stained with
Coomassie Brilliant Blue R-250. Native isoelectric focusing was
performed using pH 3-10 mini gels. Gels were blotted onto
polyvinylidene difluoride membranes and probed with either anti-heavy
chain (MRC-OX18, Ref. 25) or anti-2m antibodies (C9,
Ref. 26). Membranes were developed by ECLTM Western
blotting system (Amersham Corp.).
DNA/Protein Sequencing-- Samples for DNA sequencing were submitted to the sequencing service at the Microchemical Facility, The Babraham Institute. Purified peptides were applied to polybrene-coated glass fiber filters and sequenced by Edman degradation using an ABI model 492 sequencer with 610A data analysis software.
Peptide Synthesis--
The specific 13-mer peptide,
ILFPSSERLISNR (27), was synthesized by Alta Biosciences, The University
of Birmingham, UK. The synthetic nonapeptide library was prepared by
fully automated solid phase peptide synthesis using
Fmoc/tert-butyl chemistry. For coupling of amino acids in
randomized sequence positions (X), double couplings were
performed with diisopropylcarbodiimide/butanol and an equimolar mixture
of Fmoc-L-amino acids and Fmoc-Gly (cysteine was excluded),
used in an equimolar ratio with respect to the coupling sites of the
resins. The peptide mixture was cleaved from the resins and side chain
deprotected with trifluoroacetic acid:phenol:EDT:thioanisole (96:2:1:2,
v/w/v/v) and filtered from the resin. The products were precipitated at
20 °C by the addition of cold n-heptane:diethylether
(1:1, v/v), washed twice by sonification, and lyophilized from acetic
acid:water:t-butyl alcohol (1:10:50, v/v/v), yielding the
completely randomized nonapeptide library. The amino acid composition
of the peptide library was determined by pool sequencing (28),
electrospray mass spectrometry (29), and amino acid analysis.
Deviations from equimolar representation of the amino acids were found
to be within the error limits of the analytical methods (30).
Refolding Experiments--
Refolding was performed by the
dilution method (31, 32). Briefly, 360 µg of 2m and
150 µg of peptide were added to 15 ml of 50 mM Tris-HCl,
pH 8.0, 400 mM arginine, 0.1 mM EDTA, 0.1 mM PMSF. Denatured heavy chain (465 µg) was added to give
a final molar ratio of 1:2:10 (heavy chain:
2m:peptide).
For RT1-Au and RT1-A1c, the peptide
concentration was increased to give a final ratio of 1:2:100. (For
RT1-A1c, the results presented here were from an experiment
using three times the amounts shown above for all three components).
After 24-48 h at 20 °C, the refolding mixture was concentrated down to 100 µl using a Centriprep-10 and Centricon-10 (Amicon). Refolded class I complex was purified by gel filtration on an FPLC Superdex 75 column (Pharmacia) equilibrated in 20 mM Tris-HCl, pH 8.0, 100 mM NaCl.
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Conformational Antibody Experiments--
Successful refolding
was determined by ELISA using a conformational RT1-Aa
specific antibody (MAC 30, Ref. 34), a general class I reactive antibody (MRC-OX18, Ref. 25), and a 2m specific antibody
(C9, Ref. 26). The complex to be tested was coated onto plates
overnight at 4 °C before addition of antibodies. A horseradish
peroxidase-secondary antibody was used, and the plates were developed
with TMB and read at 450 nm.
Immunoprecipitation Experiments-- Monoclonal antibodies U9F4 (anti-RT1-Au, Ref. 35) and YR5/310 (anti-RT1-A1c, Ref. 13) were purified and coupled to cyanogen bromide-activated Sepharose (Pharmacia) according to the manufacturer instructions. C58 cells (RT1-Au/TAP2-B) (3) were cultured to approximately one million cells per ml (1.5 liters total volume). Splenocyte cell suspensions were made from 15 spleens of PVG.R20 (RT1-A1c/TAP2-B) rats.
For both cell types, the cell suspension was centrifuged and the pellets were washed with buffer A (10 mM Tris, pH 7.7, 150 mM NaCl). The cell pellets were resuspended in 100 ml of buffer A before addition of 100 ml of buffer B (10 mM Tris-HCl, pH 7.7, 150 mM NaCl, 2% Nonidet P-40, 1 mM PMSF). The suspension was mixed at room temperature for 30 min and then spun at 20,000 × g for 30 min at 4 °C. The supernatants were decanted into fresh bottles, and 3 ml of non-immune rat serum-Sepharose was added. This was mixed for 1 h at 4 °C before the matrices were removed by centrifugation/filtration. U9F4-Sepharose and YR5/310-Sepharose (1.5 ml for both) were then added to their respective lysates and mixed at 4 °C for a further 90 min. The Sepharose was collected and washed thoroughly with buffer B followed by buffer A. Peptides were eluted by incubating the antibody-coupled Sepharose with 15 ml of 10% acetic acid for 5 min at room temperature. After filtration, the solutions were then passed through Centriprep-3 units and concentrated, and the peptides were separated by reverse phase chromatography using the same method described above for the peptides released from the refolded complex. The cut-off values used to determine binding motifs were a 300% increase over the previous cycle for cycles 1-6 and 150% for cycles 7-10. ![]() |
RESULTS |
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Expression and Purification--
Using PCR, we have engineered
expression plasmids to produce three different soluble rat class I
RT1-A molecules in E. coli, RT1-Aa,
RT1-Au, and RT1-A1c. Under
isopropyl--D-thiogalactopyranoside induction, both
2m and the heavy chains were expressed as inclusion
bodies and therefore required urea solubilization before further
purification could be performed. Induction of the heavy chain
constructs produced material consistent in size, by SDS-PAGE, with that
expected for the truncated, 1-276-amino acid heavy chains coupled at
their C-termini to an 8-amino acid stretch containing a His-Tag motif (LEHHHHHH) coded by the pET-22b(+) plasmid. The resulting class I heavy
chains were further purified on an Ni-NTA-agarose column to >95%
purity (Fig. 4). The yield of heavy chain
was 10-20 mg/liter of bacterial culture. This eluted off the Ni-NTA
column as two species, one in pH 5.9 and the other in pH 4.5 buffer
(Fig. 4; last two lanes). SDS-PAGE in the presence/absence
of reducing agent confirmed the mixture eluting in the pH 5.9 buffer to
be mainly composed of monomers, while protein eluting in the pH 4.5 buffer comprised large molecular weight multimers (results not shown).
Only the monomer fraction was used for refolding work (yield 5-10
mg/liter). Protein sequencing of the monomer fraction revealed that the
bacteria had successfully cleaved the initiation methionine to give the
expected N-termini (results not shown).
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Specific Peptide Refolding--
Refolding tests were initially
performed using the RT1-Aa heavy chain because, thus far,
this is the only rat class Ia molecule for which a specific peptide has
been identified and reported to bind (27). The peptide used for
refolding was ILFPSSERLISNR, a 13-mer peptide from the rat
mitochondrial A chain of ATP synthase (SwissProt accession number
P05504). Results from a typical refolding experiment, using a 10-fold
excess of peptide over the heavy chain, are shown in Fig.
5A. Gel filtration of the
refolding mixture through a Superdex 75 column gave three peaks eluting at >100 kDa (Peak A), ~40 kDa (Peak B), and 12 kDa (Peak C). These corresponded to aggregates, refolded
complex, and 2m, respectively. Peak B increased in the
presence of peptide, and SDS-PAGE analysis showed that there were both
heavy chain and
2m bands present (Fig. 5B).
When refolding is performed without peptide, the observed peak at ~40
kDa could be an empty heavy chain/
2m complex, but this has
yet to be characterized. Isoelectric focussing gel electrophoresis and
a subsequent Western blot of the peptide/MHC complex revealed bands
with an approximate pI of 6.7 for free
2m (C9 positive) and with pI 5.5 for the heavy chain/
2m complex (MRC-OX18
and C9 positive), and at pI 5.1, two almost indistinguishable bands (MRC-OX18 positive) were revealed that could be heavy chain
with/without peptide (results not shown).
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Random Peptide Refolding Experiments-- Direct pool sequencing of the random nonapeptide library showed no significant enrichments for any amino acids at any position (results not shown). Therefore, any enrichment seen in the material eluted from the refolded RT1-A molecules was considered specific.
For RT1-Aa, a 10-fold molar excess of random peptide:heavy chain was sufficient to give equivalent successful refolding compared with the specific 13-mer peptide (results not shown). Acid elution and subsequent pool sequencing of the released peptides yielded the results and motif presented in Fig. 1A. Dominant residues (boxed values) as determined previously (12, 33) were seen at positions 2, 3, 4, 8, and 9. These results are comparable with the RT1-Aa motif results already published by Powis et al. (11) and reproduced in Fig. 1B. It can be noted that, in their results obtained from natural isolates, the residues which increased at positions 3 and 4 (Phe and Pro, respectively) were not seen as large enough increases to be considered anchor residues although both are present in the natural mitochondrial peptide ligand.RT1-Au Peptide Motif from Recombinant and Natural Sources-- Having obtained successful results for RT1-Aa, this method was used to refold around the TAP2-B-associated class I molecules, RT1-Au and RT1-A1c (see below). Initial experiments with a 10-fold excess of random peptide:heavy chain failed to yield sufficient refolded complex. Only when the peptide ratio was increased to 100-fold did the complex refold sufficiently (see "Discussion"), with the peak area being 41% of the total area of the aggregate and complex (Fig. 7). With the higher ratio of peptide to heavy chain, the peptide motif for RT1-Au was determined (Fig. 2A). RT1-Au revealed a motif with strong preferences for hydrophobic and uncharged residues. Dominant residues appeared at positions 2, 3, 7, and 8 and, in particular, showed a C-terminal preference for Tyr at position 9.
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RT1-A1c Peptide Motif from Recombinant and Natural Sources-- As for RT1-Au, successful refolding with recombinant RT1-A1c was achieved only with a 100-fold excess of peptide over the heavy chain. The motif obtained from pool sequencing for recombinant RT1-A1c is shown in Fig. 3A. Dominant residues appeared at positions 2, 3, and 8 and with an almost singular preference for proline at position 2. Interestingly, there was an observed increase in tyrosine at position 8 while no obvious C-terminal anchor residue was observed at position 9, unlike for the two other class I heavy chains that we have used in this study. The motif derived from the rat splenocytes (Fig. 3B), revealed similar anchor residues to the recombinant system. The strong proline at position 2 is confirmed as is the preference for positive residues, particularly His and Arg at position 3. An increase for tyrosine at position 8 was observed, and in addition, a C-terminal (P9) anchor, leucine, was indicated.
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DISCUSSION |
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In this study we have successfully used a random nonamer peptide
library to determine the peptide binding motifs for RT1-Aa,
RT1-Au, and RT1-A1c using a recombinant system.
We describe a method to produce large amounts of rat MHC heavy and
light chains. We have cloned, expressed, and purified truncated
versions of three rat RT1-A class I heavy chains containing only the
1,
2, and
3 domains. Engineering a His-Tag at the C-terminal
end of the heavy chains enabled a quick and simple purification
strategy while leaving the protein denatured in 8 M urea
and ready for refolding. Purification in the absence of reducing agents
yielded both monomers and aggregates with interdisulfide bonds. The use
of the Qiagen Ni-NTA matrix allowed for the enrichment of monomers from
aggregates. Attempts to denature aggregates in the presence of reducing
agents and to refold them subsequently were unsuccessful (results not
shown). The
2m was purified by urea solubilization,
followed by renaturation by dialysis. Ion exchange chromatography with
a Q-Sepharose column successfully purified monomers from
aggregates.
In recent years, large scale protein expression of MHC molecules in bacteria has mainly been directed toward crystal production for structure determination, when a binding peptide is already known (31, 32, 38-42). Here we present results showing that the same method can be adapted successfully to elucidate the binding motifs of rat RT1-A MHC molecules by using a random peptide library. Refolding with a fixed length random peptide library has a clear advantage over immunoprecipitation of MHC class I molecules expressed in mammalian cells. Unlike class II MHC molecules, peptides are usually locked into the groove of class I molecules at their N and C termini. In a natural system, peptides of different lengths can still bind to class I molecules (7, 14-16), and when eluted and subjected to pool sequencing, the C-terminal residues are often "diluted" away because of the different lengths of the population. This should not often occur with a fixed length random peptide library although it is possible for a nonamer library to bind with an end extending out of the groove. In addition, immunoprecipitation requires a rich source of class I and a specific antibody to extract a homogeneous population of the class I under study. This is often unavailable, especially with the poorly expressed non-classical (class Ib) molecules. The cloning scheme described here, coupled to refolding around random peptides, could also produce conformationally correct material for raising anti-class I antibodies. Raising antibodies against a complex with a specific peptide bound within the groove of a specific class I is also possible with this system. Injection of a single peptide·MHC complex, rather than a cell presenting the MHC with the peptide as was done by Porgador et al. (43), should produce more specific antibodies.
Only the monomer fractions for both subunits were used during the refolding experiments presented here. This enrichment produced reproducible refolding of between 35 and 45% of the starting material, when using both the specific 13-mer peptide and the random peptide library. This value is higher than any previously reported for refolding with bacterial protein; 20% was the maximum yield reported by Reid et al. (32), who "pulsed" the refolding mixture with aliquots of heavy chain. We have yet to determine whether such pulsing could improve yields in our system. The purified complex was shown to contain both heavy and light chains, by SDS-PAGE and isoelectric focussing gel electrophoresis, and to have conformationally correct material, by ELISA and FACS. In this report, we have used Ni-NTA silica (Qiagen Inc.) for FACS analysis. The product, sold as a solid support for FPLC, HPLC, and microspin miniprep applications, has a bead size of 16-24 µm, which was small enough to be unhindered in the tubing of the FACSCaliburTM (Becton Dickinson). Binding via the His-Tag allows complete access of the antibodies to the native complex and produced an increase in fluorescence with the RT1-Aa specific antibody, MAC 30. Such material could provide a replacement for TAP-deficient cell lines such as RMA-S and T2, which are routinely used for providing "empty" class I molecules.
Pool sequencing results for acid-eluted random nonamer peptides from
RT1-Aa, when compared with the motif reported by Powis
et al. (11), show a comparable motif with identical amino
acid preferences at positions 2, 8, and 9. In addition to these
positions, our results suggest a strong preference for serine at
position 1 and for phenylalanine and proline at positions 3 and 4, respectively. The latter two were both observed in the previous report
but not as anchor residues. This discrepancy may be explained by
reduced availability of such peptides in a natural system compared with the random library. Similarly, results for the TAP2-B-associated RT1-Au from both random peptide refolding and
immunoprecipitation were comparable and revealed a general preference
for aliphatic and aromatic residues at positions 2, 3, 6, and 7 and a
strong preference for tyrosine at position 9 (even to the exclusion of
Phe). As for RT1-Aa, there was also a preference for serine
at position 1 in addition to threonine, alanine, and glutamic acid. The
major differences between the two systems occur at positions 3 and 4, which once again may be due to a more stringent peptide availability in
the natural system. Fig. 8 shows a
comparison of the hydrophobic residues in the theoretical structures of
RT1-Aa, RT1-Au, and RT1-A1c. The
circled region clearly shows an increase in hydrophobic residues for
RT1-Au in the -sheets at the bottom of the groove. This
provides a likely explanation for the hydrophobic residues observed at
positions 6-8 of the immunoprecipitation motif (Fig. 2), which would
fit into this area if placed in the groove.
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In both the motifs for RT1-A1c, we find a strong preference for proline at position 2. This anchor residue has already been reported in other class I molecules such as H-2Ld (12), HLA-B7 (44), HLA-B*3501 (45), and HLA-B53 (45). It is of interest to note that the TAP/peptide translocation work of Neefjes et al. (46) found that a proline at position 2 or 3 of the peptide negatively influenced transport in both the rat TAP-A and TAP-B transporters (also observed with human TAP, Ref. 22). Our data do not necessarily contradict the TAP data since longer, Pro-containing peptides could be transported and amino-terminal trimming in the ER would make them suitable for binding to RT1-A1c.
Other interesting observations from the recombinant RT1-A1c are the significant increase in tyrosine at position 8 and surprisingly, no observed specific increase at the ninth position, whereas from the natural system (Fig. 3B), a leucine was observed at position 9. The C-terminal anchor residues for the other motifs reported, RT1-Aa and RT1-Au, were determined accurately and unequivocally to be an arginine and tyrosine, respectively. Previous attempts to determine MHC peptide binding motifs using recombinant protein and a random peptide library, with mouse H-2Kb from insect cells (47) and human HLA-A2, B8 and B53 from bacteria (48), were only partially successful as both were unable to report any of the C-terminal anchor residues. Interestingly, a C-terminal leucine appears in all of the motifs for the above examples (except HLA-B53 whose motif is yet to be completed). It may be possible that a C-terminal leucine is lost to a "washout" effect during the Edman degradation. Closer examination of Fig. 3A does show a hint of a leucine at cycle 9 since it shows only a small drop in signal compared with the other residues from the previous cycle. A larger scale prep may circumvent this problem.
RT1-Au and RT1-A1c, therefore, display anchor motifs with strong preferences for aromatic and hydrophobic residues, respectively. The restrictive TAP-B transporter to which RT1-Au and RT1-A1c are normally genetically linked is known to be able to transport these types of peptides (5, 6), so our results are consistent with the possibility that genetic associations of alleles at RT1-A and TAP2 may reflect the related specificities of transporter and presenting molecule (4). However, since the permissive TAP-A transporter can also transport such peptides, the allelic associations may be based on other unknown factors.
It is also interesting to note that, whereas only a 10-fold ratio of peptide:heavy chain was required for refolding with RT1-Aa, a 100-fold ratio was required for RT1-Au and RT1-A1c. Since pool sequencing of the untreated random peptide library showed no significant enrichments for any amino acids at any position, indicating a random population, there is an implication that both of these molecules have a more stringent requirement for particular peptides, possibly as the groove requires specific types of residues at more positions than does RT1-Aa. Alternatively, the increased preference for hydrophobic and aromatic residues in RT1-Au raises the possibility of reduced solubility for the preferred peptides and therefore reduced availability for refolding. But RT1-A1c also requires a 100-fold ratio for efficient refolding, and this has charged residue preferences at position 3. In vivo, before peptides can bind to class I molecules for presentation, at least two points have been identified where they are processed: the proteasome where the peptides are produced and the TAP transporter which transports peptides into the endoplasmic reticulum lumen. Therefore, peptide presentation in natural systems is subject not only to what peptides can bind to the class I but also to how proteins are processed and what peptides are able to be transported into the ER lumen. The method we have used here is free from the cellular processing and transport and as such, measures only the specificity of the groove of the class I molecule itself. Comparisons of the motifs from natural and recombinant sources for both RT1-Aa and RT1-Au do indeed show some differences, suggesting that the proteolytic and TAP transporter machineries may be imposing significant restrictions on the availability of peptides including positions other than the C-terminal anchor. If this were the case, then the method we describe here could prove extremely valuable for identifying such restrictions.
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ACKNOWLEDGEMENTS |
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The authors thank R. J. M. Stet for the kind gift of the U9F4 hybridoma, P. J. Bjorkman for the C9 hybridoma, and S. Jobson and P. Whiting for the unending supply of LB.
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FOOTNOTES |
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* This work was supported by a Biotechnology and Biological Sciences Research Council Project grant (to E. J.), Biotechnology and Biological Sciences Research Council Competitive Strategic Grant funding (to G. W. B.), and funding by the Deutsche Forschungsgemeinschaft (SFB 323) (to K. H. W.).The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
§ To whom correspondence should be addressed. Tel.: 44-1223-832912; Fax: 44-1223-837952; E-mail: james.stevens{at}bbsrc.ac.uk.
1
The abbreviations used are: ER, endoplasmic
reticulum; TAP, transporter associated with antigen processing; MHC,
major histocompatibility complex; 2m,
2-microglobulin; PCR, polymerase chain reaction; PMSF,
phenylmethylsulfonyl fluoride; Ni-NTA, nickel-nitrilotriacetic acid;
TMB, 3,3
,5,5
-tetramethylbenzidine; PAGE, polyacrylamide gel
electrophoresis; ELISA, enzyme-linked immunosorbent assay; FACS,
fluorescence-activated cell sorter; FPLC, fast protein liquid chromatography; HPLC, high performance liquid
chromatography; Fmoc,
N-(9-fluorenyl)methoxycarbonyl; PBS, phosphate-buffered saline.
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REFERENCES |
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