©1995 by The American Society for Biochemistry and Molecular Biology, Inc.
Requirements for Peptide Binding to the Human Transporter Associated with Antigen Processing Revealed by Peptide Scans and Complex Peptide Libraries (*)

(Received for publication, February 10, 1995; and in revised form, April 10, 1995)

Stephan Uebel (1) Thomas H. Meyer (1) Wolfgang Kraas (2) Stefan Kienle (2) (3) Gnther Jung (3) Karl-Heinz Wiesmller (3) Robert Tamp (1) (4)(§)

From the  (1)Max-Planck-Institut fr Biochemie, Am Klopferspitz 18a, D-82152 Martinsried, the (2)Institut fr Organische Chemie der Universitt Tbingen, Auf der Morgenstelle 18, D-72076 Tbingen, the (3)Naturwissenschaftliches und Medizinisches Institut, Eberhardstrasse 29, D-72762 Reutlingen, and the (4)Lehrstuhl fr Biophysik E22, Technische Universitt Mnchen, D-85747 Garching, Germany

ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
FOOTNOTES
REFERENCES

ABSTRACT

Antigenic peptides are translocated into the lumen of the endoplasmic reticulum by the action of the transporter associated with antigen processing (TAP), where they are subsequently needed for the correct assembly of major histocompatability complex molecules. The transport function was reconstituted in insect cells by expression of both TAP genes. On the basis of this overexpression system, substrate selection was analyzed in detail by a direct bimolecular peptide binding assay. Competition assays with peptide variants, including substitutions of residues with alanine or structurally related amino acids, underline the broad peptide specificity of the human TAP complex. Steric requirements of the substrate-binding pocket were mapped using elongated peptides and scans with bulky, hydrophobic amino acids. Complex nonapeptide libraries were used to determine the contribution of each residue to stabilize peptide-TAP complexes. For the first time, this approach lets us directly evaluate the importance of peptide selection for the overall process of antigen presentation on the level of the peptide transporter.


INTRODUCTION

Cytotoxic T lymphocytes recognize peptides derived from endogenous proteins in association with major histocompatibility complex (MHC) (^1)class I molecules. These peptides are believed to be translocated into the lumen of the endoplasmic reticulum (ER) by the action of the transporter associated with antigen processing (TAP) for binding to assembling MHC class I molecules (Neefjes et al., 1993; Shepherd et al., 1993; Androlewicz et al., 1993). Formation of MHC-peptide complexes induces a conformational change that is required to overcome retention in the ER and to allow subsequent transport of these complexes to the cell surface (Suh et al., 1994; Ortmann et al., 1994). The transport complex is a heteromer (Spies et al., 1992; Kelly et al., 1992; Meyer et al., 1994) composed of two MHC-encoded subunits, TAP1 and TAP2, with homology to the ATP-binding cassette superfamily of transport proteins (Monaco, et al., 1990). The stoichiometry of the solubilized and purified TAP complex was revealed to be 1:1 (TAP1/TAP2) (Meyer et al., 1994).

Members of this ATP-binding cassette superfamily, including the multidrug resistance P-glycoprotein and the oligopeptide transporter of Salmonella typhimurium, are involved in the transport of substrates across membranes (Higgins, 1992). Hydrophobicity profiles of these proteins suggest two domains of six to eight membrane-spanning regions and two hydrophilic domains that contain the ATP binding motifs Walker A and Walker B. Indeed, it was shown by ATP cross-linking that the individually expressed hydrophilic C-terminal domains of either TAP1 or TAP2 bind ATP (Mller et al., 1994; Wang et al., 1994). Several assay systems have been developed so far to confirm the function of the TAP complex as peptide translocator. These experiments are based on the restoration of cytotoxic T cell recognition (i.e. correct antigen presentation in mutant cell lines by transfection with tap genes (Spies & DeMars, 1991)) or accumulation of peptides in the ER of permeabilized cells or TAP1bulletTAP2-containing microsomes measured by quantitation of radiolabeled peptide. The latter can be recovered as peptide glycosylated in the ER (Neefjes et al., 1993), or microsomal lumen (Meyer et al., 1994), or as peptide bound to MHC class I molecules (Shepherd et al., 1993; Androlewicz et al., 1993) or simply associated with microsomes (Shepherd et al., 1993; Heemels et al., 1993; van Endert et al., 1994). Translocation was demonstrated to be ATP-dependent. Peptide specificity (e.g. C-terminal preference and length selectivity) was examined in these and various other studies (Schumacher et al., 1994a, 1994b; Momburg et al., 1994; Androlewicz & Cresswell, 1994). Nevertheless, all of these previous assay systems are based on a retention system (i.e. glycosylation or MHC class I binding), because transported peptides without it were hard to detect, possibly because of another active export system for peptides (Schumacher et al., 1994a). It remains to be shown that an assumed substrate specificity of the retention system, a proteolytic activity in the cytoplasm and ER, or the export machinery do not alter the experimental results.

In order to circumvent the problems associated with these indirect assays of peptide translocation, a direct bimolecular binding and competition assay was established, because an isolated and reconstituted in vitro translocation system is not available so far. Substrate specificity and selection of the TAP complex were examined in detail by competition studies using reporter and various modified competitor peptides. Although peptide binding was discussed in studies relying on microsome-associated radioactivity, peptide binding to TAP1bulletTAP2 has recently been shown by photoaffinity cross-linking of peptides (Androlewicz & Cresswell, 1994; Androlewicz et al., 1994) and binding assays involving an overexpression system for human TAP1bulletTAP2 in insect cells, which has been used to screen various peptides derived from MHC class I epitopes (Meyer et al., 1994; van Endert et al., 1994). In this article, we report a systematic approach to determine the requirements for peptide-TAP complex formation, using variants of the histone H3-derived peptide (RRYNASTEL) that have been elongated or modified by alanine, structurally related, or bulky amino acids. In addition, complex nonapeptide libraries, including all possible sequence variations, are used to determine the stabilization or destabilization effect for each peptide residue individually in terms of Gibbs free enthalpy (DeltaDeltaG).


MATERIALS AND METHODS

Overexpression of TAP1bulletTAP2 with the Baculovirus Expression System

The generation of recombinant baculoviruses carrying genes of human tap1 and tap2 has been described previously (Meyer, et al., 1994). Sf9 (Spodoptera frugiperda) cells were grown as a monolayer according to standard procedures in TC100 insect cell medium with 10% fetal calf serum, and infection was routinely performed with a multiplicity of infection of 3-5.

Preparation of Microsomes

Sf9 cells were harvested 60-72 h postinfection by centrifugation and washed once with phosphate-buffered saline. The cells were lysed after resuspension in cavitation buffer (250 mM sucrose, 25 mM KOAc, 5 mM MgOAc, 0.5 mM CaOAc, 50 mM Tris-Cl, pH 7.4) with proteinase inhibitor mix at 10^8 cells/ml by repeated drawing through a 26 gauge needle. Nuclei and nonlysed cells were removed by centrifugation (300 g for 5 min at 4 °C), and the supernatant was diluted 6.4-fold with 2.5 M sucrose in gradient buffer (150 mM KOAc, 5 mM MgOAc, 50 mM Tris-Cl, pH 7.4) and stepwise overlaid with 2.0 and 1.3 M sucrose in gradient buffer and with cavitation buffer. After centrifugation overnight (104,000 g at 4 °C), the turbid fraction at the interface of the 2.0 and 1.3 M sucrose solution was collected, diluted 2-fold with phosphate-buffered saline and 1 mM 1,4-dithio-DL-threitol and centrifuged (227,000 g for 1 h at 4 °C). The vesicles were resuspended in phosphate-buffered saline and 1 mM 1,4-dithio-DL-threitol, snap frozen in liquid nitrogen, and stored at -80 °C.

Synthesis of Peptides

The peptides in this study were synthesized on a multiple peptide synthesizer (MultiSynTech, Bochum, Germany) by conventional Fmoc chemistry. Identity of the peptides was checked by mass spectrometry, and the purity was determined by reversed phase HPLC. Purity was at least 93% for each peptide of the alanine scan, 89% for the beta-(1-naphthyl)-L-alanine scan, and 80% for the other peptides. The reporter peptide (RRYNASTEL) was HPLC-purified using a C-18 column and a gradient of acetonitrile in water (purity, >99%). Peptides containing -dansyl-lysine were synthesized by removal of the Boc group from N-alpha-Z-N--Boc-Lys-OH with 40% trifluoroacetic acid in water, reaction of the product with dansylchloride in acetone/NaOH, removal of the Z group by catalytic hydrogenation, reaction with Fmoc-N-hydroxysuccinimide, and subsequent use of the product after crystallization in peptide synthesis.

Synthetic nonapeptide libraries were generated on a robot system using Fmoc amino acids, solid phase chemistry, and Fmoc leucine-loaded Wang resin or an equimolar mixture of 19 Fmoc amino acid resins loaded with all proteinogenic amino acids except cysteine. Equimolar mixtures of Fmoc amino acids equimolar to the coupling sites on the resins were used for the X positions, and 5-fold molar excess was used for coupling of single amino acids in defined positions, using the diisopropylcarbodiimide/1-hydroxybenzotriazole method for coupling. Details of the procedure are described elsewhere (Udaka et al., 1995). To confirm the sequences and the amino acid frequency in the X positions, the peptide library was subjected to amino acid analysis, pool sequence analysis (Stevanovic & Jung, 1993), and electrospray mass spectrometry (Metzger et al., 1994). Close to equimolar distribution of every amino acid was found to be within the error limits of the analytical methods (Kienle et al., 1994). Peptide concentrations were determined using an orthophthalic aldehyde assay (Pierce).

Iodination of Peptides

Peptides were iodinated as described previously (Hunter & Greenwood, 1962). In brief, the reaction was routinely performed with 15 nmol of peptide and 3.7 10^7 Bq (1 mCi) NaI using the chloramine-T method. Free iodine was removed by gel filtration through a Sephadex G-10 column (Pharmacia). The specific activities were 0.8-1.3 10 Bq/mol for *RRYNASTEL* and 0.2-0.3 10 Bq/mol for -dansyl-lysine-containing peptides.

Peptide Binding and Competition Assays

Microsomes were diluted with assay buffer (phosphate-buffered saline with 1 mg/ml dialyzed bovine serum albumin, 1 mM 1,4-dithio-DL-threitol, 2 mM MgCl(2)) to a final protein concentration of 75 µg/ml in binding assays and 25-50 µg/ml in competition assays. The protein concentration was determined using the bicinchoninic acid assay (Pierce). This suspension was homogenized by drawing through a 23 gauge needle. For the determination of the binding affinity of TAP1bulletTAP2, various amounts of radiolabeled reporter peptide *RRYNASTEL* were added as indicated at 4 °C. In the case of competition assays, 300 nM of the reporter peptide and the appropriate amount of unlabeled competitor were added at 4 °C. After incubation for 5 min on ice, 350 µl of ice-cold assay buffer were added, and the microsomes were pelleted by centrifugation (12,000 g for 10 min at 4 °C). Vesicle-associated radioactivity was quantified by counting after one washing step with 500 µl of ice-cold assay buffer. The amount of bound peptide was corrected by unspecific binding of baculovirus wild type microsomes. The data set was fitted by the competition function: the percentage of inhibition = 100 ([competitor]/IC)/(([competitor]/IC) + 1)). The concentration needed for 50% inhibition was determined from three to four competition assays.

Peptide Translocation Assays

Peptide translocation assays were performed as described previously (Meyer et al., 1994). In brief, 150 µl of microsome suspension (20 µg of total amount of protein) were incubated with 50 ng of radioactive peptide in the presence of 0.3 unit of apyrase, 10 mM ATP, 100-fold excess of competitor peptide, or nonhydrolyzable ATP analogue (10 mM AMP-PNP). After 10 min of incubation at 37 °C, microsomes were lysed by adding detergent. Glycosylated and therefore translocated peptides are quantified by counting after binding to concanavalin A-Sepharose (Sigma) and elution with alpha-methylmannoside.


RESULTS

Peptide Binding to the TAP Complex

The affinity constant of the radiolabeled peptide, *RRYNASTEL*, for the TAP complex was determined in a saturation binding experiment to be 310 ± 30 nM at 4 °C (Fig. 1). No specific binding to microsomes containing either only TAP1 or TAP2 was observed (data not shown). The linear Scatchard plot indicates a noncooperative ligand-receptor interaction with identical, independent binding sites. Saturation was reached at 0.175 nmol/mg microsomal protein. Assuming one binding site and a molecular mass of about 150 kDa of the transport complex, 2.4% of the total microsomal protein represents functional TAP complex expressed in insect cells. The affinity constant and the saturation value are not influenced by ATP (data not shown). Peptide binding to a high affinity substrate binding site generated by TAP1 and TAP2 therefore represents an ATP-independent process, whereas peptide translocation was shown before to be strictly ATP-dependent (Meyer et al., 1994).


Figure 1: Peptide binding affinity of the reporter peptide *RRYNASTEL* used in the experiments. Each value () represents specific binding to TAP1bulletTAP2-containing microsomes background-corrected for binding to the equivalent amount of microsomes from baculovirus wild type-infected insect cells (). The total protein amount for each assay (150 µl) was 11.5 µg. The inset shows the data of a Scatchard plot leading to K = 310 ± 30 nM. Saturation is reached at 0.175 nmol/mg microsomal protein.



Amino Acid Substitutions

To analyze the affinity of various peptides for the TAP complex, competition assays were carried out using *RRYNASTEL* (300 nM) as reporter peptide. The percentage of inhibition of peptide binding at various competitor concentrations was plotted against the molar excess of competitor. The concentration needed for 50% inhibition (IC) was calculated by fitting a competition function to the data set. In order to compare various peptides, the IC of each competitor was expressed in relation to the IC of the reference peptide (RRYNASTEL). To reveal possible anchoring positions for peptide binding, each amino acid at an individual position of the reporter peptide (RRYNASTEL) was sequentially exchanged against an alanine. The results of this alanine scan and of the substitutions by related amino acids are summarized in Fig. 2. With the exception of phenylalanine at position 9 (IC/IC = 0.27), nearly all peptides show a decreased affinity for TAP1bulletTAP2, ranging from an up to 10-fold lower affinity of peptides with a changed N or C terminus to nearly unchanged affinity by exchange of glutamic acid to alanine in position 8 (IC/IC = 0.88). The destabilization effect of each lysine in position 1 and 2 is additive with relation to the free enthalpy. The slightly increased affinity of peptide substitution, asparagine to glutamine at position 4, reflects the 2-fold higher affinity of TAP for the original histone H3-derived epitope RRYQKSTEL compared with the reporter peptide RRYNASTEL that is modified with a glycosylation sequence (Fig. 3).


Figure 2: Summary of competition assays using peptides with alanine and homologous substitutions of RRYNASTEL. In relation to RRYNASTEL (IC), the IC/IC values of these competitor peptides are given. Varied positions in the peptides are underlined.




Figure 3: Summary of competition assays using elongated peptides of the modified epitope of histone H3 (RRYNASTEL). The IC/IC ratios of various competitor peptides are given. For comparison, the data of the original epitope (RRYQKSTEL) and the reference peptide (RRYNASTEL) of human histone H3 are given on top. Varied positions in the peptides are underlined.



Elongated Peptide Epitopes

Within the scope of antigen processing, protein fragments variable in size are generated in the cytosol or nucleus. In order to determine the length selectivity of the TAP complex, elongated modifications derived from the histone H3 epitope were used (Fig. 3). No length selectivity was observed in the range of 9-15-mers, because the longest peptide tested (REIRRYNASTELLIR) showed only a slightly decreased binding affinity (IC/IC = 1.25). Interestingly, peptides containing an N-terminal arginine bound very efficiently, whereas peptides with an altered amino acid at position 1 have a decreased binding affinity.

Bulky, Hydrophobic Substitutions

In experiments originally designed to introduce cross-linker, biotin, or fluorescence labels and to map the steric requirements of peptide binding to the TAP complex, peptides that contain substitutions of (1-naphthyl)alanine or -dansyl-lysine at different positions were screened for binding to the TAP complex (Fig. 4). Most strikingly, almost every substitution causes an increase in peptide affinity independent of the relative position of the bulky, aromatic side chain in comparison with the reference peptide and reached values of 12-fold increased affinity for peptides dansylated at positions 6-8 (IC/IC = 0.081). Only the peptide with an N-terminal (1-naphthyl)alanine exhibits a decreased affinity (IC/IC = 11.3).


Figure 4: Summary of competition assays using peptide scans containing bulky, hydrophobic amino acids of (1-naphthyl)alanine and -dansyl-lysine. The IC values (top) of these modified peptides are given in reference to the IC of RRYNASTEL along with the structures (bottom) of the modifications. Varied positions in the peptides are underlined.



In an inverse competition experiment, a 14-fold molar excess of unlabeled RRYNASTEL as competitor was needed to achieve 50% inhibition of binding of the dansylated, radiolabeled peptide, *RRYUASTEL* (data not shown). An IC/IC of >200 was observed for the amino acid -dansyl-lysine in an experiment to rule out the possibility of an unspecific adsorption of these peptides to the peptide binding pocket via the aromatic side chain. The addition of an excess of lipid did not affect the IC value of RRYUASTEL, demonstrating that dansylated peptides do not accumulate in or at the membrane, thereby leading to an increase of the peptide concentration and the apparent binding constant. In the absence and the presence of a 20-fold excess of microsomal membranes (Sf9 baculovirus wild type) the IC values of the dansylated peptide (RRYUASTEL) are found to be identical within the range of error (data not shown). We can further demonstrate that the dansylated, radiolabeled peptide (*RRYNASTUL*) is directly translocated in an ATP- and TAP-dependent fashion (Fig. 5).


Figure 5: ATP- and TAP-dependent translocation of the -dansyl-lysine-modified peptide *RRYNASTUL* at 37 °C. Microsomes (20 µg of total amount of protein) were incubated with 0.3 unit of apyrase (- ATP), 10 mM ATP (+ ATP), 100-fold excess of unlabeled competitor peptide (RRYNASTUL) and 10 mM ATP (+ competitor), or 10 mM AMP-PNP (+ AMP-PNP). Glycosylated and therefore transported peptide was isolated by binding to concanavalin A-Sepharose and elution with alpha-methylmannoside. ConA, concanavalin A; BVwt, baculovirus wild type.



Peptide Libraries

We investigated the stabilizing or destabilizing effect of each individual peptide residue of the epitope RRYNASTEL bound to TAP, using peptide libraries representing up to 19^9 = 322,687,697,779 nonapeptides that are present at almost the same frequency. Therefore, we synthesized a nonapeptide library (X(9)) that contains every proteinogenic amino acid except cysteine at every position at the same frequency (X(9)) and sublibraries (X(8)O) containing one conserved residue (O) from each position of the reference epitope (RRYNASTEL). The IC of each sublibrary was determined in a competition assay using RRYNASTEL as reporter peptide. Binding was titrated to background level (100% inhibition), indicating that a large fraction out of the nonapeptide library can compete for TAP binding. Interestingly, only an 11-fold excess of totally randomized nonapeptides (X(9)) is needed for 50% inhibition, reflecting the broad substrate specificity of the human TAP complex.

In Fig. 6, the IC values of each sublibrary (X(8)O) in relation to the IC of the totally randomized library (X(9)) were expressed in values of DeltaDeltaG, representing the stabilizing and destabilizing effect of each individual amino acid residue. Three groups of residues can be identified: (i) residues that promote peptide binding (DeltaDeltaG < -1.7 kJ/mol), (ii) residues with little effect on the binding affinity, and (iii) residues that do not favor peptide binding to TAP (DeltaDeltaG > +1.7 kJ/mol). In agreement to the alanine and homologous peptide substitutions, the first three residues of RRYNASTEL were found to be optimized for binding to the TAP complex. The strongest stabilization effect was observed with N-terminal arginine with a DeltaDeltaG = -4.6 kJ/mol.


Figure 6: Competition assays using complex peptide libraries. To analyze the effect of each amino acid residue on peptide selection, one position of the peptide epitope RRYNSATEL was kept constant and the rest was randomized by 19 amino acids (with the exception of cysteine). The IC = 11.0 of this X(9) peptide library was taken as reference. The IC/IC of each sublibrary (X(8)O) are expressed in terms of DeltaDeltaG, representing a stabilizing (- DeltaDeltaG) or destabilizing effect (+ DeltaDeltaG) of an individual amino acid residue.




DISCUSSION

Peptide binding to microsomes at 4 °C was found to be highly TAP1bulletTAP2-specific and ATP-independent. The dissociation constant was determined to be 310 ± 30 nM. This is consistent with recent findings of peptide binding to TAP at low temperatures (van Endert et al., 1994). Although there is a strict requirement of ATP hydrolysis for peptide translocation mediated by TAP (Neefjes et al., 1993; Shepherd et al., 1993, Meyer et al., 1994), we assume a mechanism of translocation that consists of at least two steps. If this mechanism involves transition from a high peptide affinity state to a low affinity state through binding of ATP (van Endert et al., 1994) or a conformational change after ATP hydrolysis remains to be elucidated, then our data indicate that addition of ATP does not alter the peptide binding affinity nor the accessible binding sites of the TAP complex at these temperatures.

Peptide binding studies allow us to determine substrate specificity of the TAP complex without the limitations of the translocation assays due to the absence of a retention system, peptide degradation, or an export machinery that may represent a bias for certain peptides. The fit of our data to the theoretical saturation binding function in the standard and Scatchard plot implies that TAP binding obeys a typical ligand-receptor interaction with identical, independent binding sites. In consequence, this allows us to use the competition assay for the determination of the IC values for various peptides. Unlike MHC class I molecules, no anchoring positions that are strictly required for peptide binding and that drastically decrease binding affinity, if altered, could be identified so far. We observe the strongest contribution to the binding affinity from residues at or close to the N or C terminus but with the effect being far from dramatic.

In detail, the experiments with elongated peptides point to a stabilizing effect of peptide binding by a N-terminal arginine. This is strongly supported by the evaluation of the stabilization effect determined individually for each residue with the use of random peptide libraries, which shows that the strongest contribution of DeltaDeltaG comes from this residue. Although acetylation (Schumacher et al., 1994a) and methylation (Momburg et al., 1994) of peptides were reported to decrease peptide translocation by TAP, no marked influence was reported so far with respect to the N-terminal residue (Momburg et al., 1994). With a C-terminal substitution to phenylalanine (IC/IC = 0.27, Fig. 3) we have identified a peptide consisting of proteinogenic amino acids that has an increased affinity compared with the reporter peptide. Taking into account the stabilizing effect of a C-terminal leucine, as determined from the random peptide library, we find that the hydrophobic residues leucine and phenylalanine are preferred to alanine, a pattern that correlates with the substrate specificity reported for the mouse TAP1bulletTAP2 but not with that found for human TAP (Momburg et al., 1994). The missing length selectivity up to a 15-mer is in line with recent findings that peptides of 8-16 amino acids are equally efficient in peptide competition (van Endert et al., 1994) and even a 24-mer efficiently competes for peptide translocation (Androlewicz & Cresswell, 1994).

Most strikingly, the binding affinity of peptides with bulky, hydrophobic amino acids, such as (1-naphthyl)alanine or -dansyl-lysine is significantly increased. This gives us the prospect of incorporating other amino acids with bulky side chains, for example photoaffinity cross-linkers, fluorescence, or biotin labels, to further investigate the molecular architecture and function of the TAP complex. The fact that these hydrophobic amino acids are preferred to naturally occurring amino acids may point to an amphipathic character of the peptide binding pocket formed by the N-terminal transmembrane domains of TAP1 and TAP2 at the lipid-protein interface or at amphipathic transmembrane helices.

The use of complex peptide libraries provides further clues on the function of the TAP complex. The relatively broad peptide specificity of human TAP (Androlewicz et al., 1993; Momburg et al., 1994; Androlewicz & Cresswell, 1994), which is reflected in the variety of MHC class I bound peptides, was confirmed by the fact that the affinity of the reporter peptide RRYNASTEL is only 11 times higher than the affinity of the totally random nonapeptide library (X(9)). The lack of a defined anchoring residue, identifiable by comparison of the effects of individual residues with the random library X(9), as well as the toleration of bulky amino acids at nearly every position, leads us to the speculation that the strongest contribution to peptide binding comes from the backbone and not through fixation of side chains.

For the first time, the restrictions of TAP on the repertoire of peptides available for presentation on MHC class I molecules can be directly quantified. The IC/IC of 11 for the nonapeptide library X(9) compared with an 180-fold increased binding affinity of the H-2K^b-restricted epitope SIINFEKL over an octapeptide library (X(8)) measured through the ability to stabilize the conformation of peptide-depleted MHC class I molecules (Udaka et al., 1995), points clearly to a restrictive role for TAP. With regard to the different combinatorial frequencies of each peptide in a 8- and 9-mer peptide library and the already optimized MHC-binding of the epitope SIINFEKL, this difference in the IC values clearly represents an upper limit. In conclusion, this means that the TAP1bulletTAP2 complex has a small but significant effect on the overall process of selection of peptides for antigen presentation.


FOOTNOTES

*
This work was supported by Grants SFB-266 (project D11-Tamp), SFB-323 (project C2-Jung), and Ta157/2 (to R. T.) from the Deutsche Forschungsgemeinschaft. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore by hereby marked ``advertisement'' in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

§
To whom correspondence should be addressed: Max-Planck-Institut fr Biochemie, Am Klopferspitz 18a, D-82152 Martinsried and Lehrstuhl fr Biophysik E22, Technische Universitt Mnchen, D-85478 Garching, Germany. Tel.: 49-89-8578-2646; Fax: 49-89-8578-2641; tampe{at}vms.biochem.mpg.de.

^1
The abbreviations used are: MHC, major histocompatibility complex; AMP-PNP, 5`-adenylyl imidodiphosphate; Boc, butyloxycarbonyl; ER, endoplasmic reticulum; Fmoc, fluorenylmethoxycarbonyl; HPLC, high performance liquid chromatography; TAP, transporter associated with antigen processing; Z, benzyloxycarbonyl; dansyl, 5-dimethylaminonaphthalene-1-sulfonyl.

The single letter code is used for peptide sequences. In addition, B means (1-naphthyl)alanine and U -dansyl-lysine. X stands for a mixed position in peptide libraries, where all proteinogenic amino acids (except cysteine) are randomly incorporated and present at the same frequency. O means one defined residue within the peptide sublibraries.


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