THE JOURNAL OF BIOLOGICAL CHEMISTRY Vol. 265, No. 19, Issue of July 5, pp. 11177-11184,lSSO 0 1990 by The American Society for Biochemistry and Molecular Biology, Inc. Printed in (I S. A.

The Sites in the I-Ak Histocompatibility Molecule Photoaffinity Labeled by an Immunogenic Lysozyme Peptide*

(Received for publication, September 22, 1989)

Immanuel F. Luescher, Dan L. CrimminsS, Benjamin D. Schwartz& and Emil R. Unanue From the Department of Pathology, the *Howard Hughes Medical Institute Core Protein-Peptide Facility, and the §Howard Hughes Medical Institute and Department of Mkdicine, Division of Rheumatology, Washington University School of Medicine, St. Louis, Missouri 63110

The class II histocompatibility molecule I-Ak was photoaffinity labeled by NH2- and COOH-terminal photoreactive conjugates of an immunogenic hen egg white lysozyme (HEL) peptide. The labeled (Y and B chains were digested with protease from Staphylococ- cus aureus strain V-S (protease V-8) and/or trypsin, and the proteolytic fragments were separated by high performance liquid chromatography (HPLC) (peptide mapping). Reproducible peptide maps containing a ma- jor labeled component were obtained from the three conjugates reported here whose photoreactive group was attached via short spacers of limited flexibility. The COOH-terminal conjugate N-acetyl HEL-(49-61)- iodo-4-azidosalicyloyl thioester (compound 1) labeled hydrophilic tryptic digest fragments on both chains of I-Ak. The labeled digest fragments were homogeneous in reverse-phase and anion-exchange HPLC, indicat- ing that the photoaffinity labeling was site-specific. Conversely, the NH&erminal conjugate iodo-4-azido- salicyloyl HEL-(46-61) (compound 2: IASA-(46-61)) labeled exceptionally hydrophobic sequences on both chains of I-Ak. The labeling was also site-specific be- cause reverse-phase HPLC of primary digests with protease V-S and secondary digests with trypsin showed single major labeled components. The labeling of I-Ak by IASA-(46-61) was fully inhibitible by HEL- (46-61). In contrast, IASA attached to the smallest immunogenic peptide 52-61 (compound 3) labeled a distinctly different hydrophilic tryptic fragment.

The site of the I-Ak molecule that was photoaffinity labeled by IASA-(46-61) (compound 2) was deter- mined. IASA-(46-61) labeled selectively at Pro- 118 of a primary cu chain fragment most likely encompassing residues 115-134. It labeled Thr-121 of a primary B chain fragment most likely encompassing residues 109-138. We also obtained evidence that IASA-(46- 61) occupied the antigen-specific site; the conjugate stimulated a T-cell hybridoma that recognizes the se- quence 52-6 1 and also competed for the binding of this smaller peptide to I-Ak. Thus, peptides that bind to the allele-specific binding site and are long enough to ex- tend beyond it can interact with a hydrophobic area of class II molecules. This area is formed by sequences of the first halves of the second domain of both a and fi chains.

* These investigations were supported by grants from the National Institutes of Health, the American Cancer Society, and the Mon- santo-Washington University agreement. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked “advertisement” in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

Class 11 major histocompatibility complex molecules (MHC)’ are intimately involved in the activation of CD4- positive T-lymphocytes (1). Their function is to bind specifi- cally protein antigen or antigen-derived fragments, in corre- lation with the underlying immune response pattern (2, 3). The three-dimensional structure of class II MHC molecules is not known. According to a model proposed by Brown et al. (4), the antigen binding site is formed by the first domains of the 01 and @ chain in a structure similar to the antigen binding site of class I MHC molecules (5, 6). In this model, the NH,- terminal halves of the first domains of the (Y and p chains form a ,&pleated sheet platform, and the COOH-terminal halves form two anti-parallel ol-helices. The binding site is an extended groove whose floor is formed by the P-pleated sheet platform and whose sides are formed by the anti-parallel LY- helices. This hypothetical antigen binding site therefore has a geometry similar to the class I binding site, which is ap- proximately 25 A long, 10 A wide, and 11 A deep (5, 6).

Two issues appear prominent, particularly with regard to a better understanding of the recognition of the MHC-antigen complex by the T-cell receptor. First, the hypothetical antigen binding site is large in comparison with immunogenic struc- tures, which can be as small as pentapeptides (7) or the heme molecule (8). This size difference suggests that topological variability may exist among the different immunogenic struc- tures that bind to a class II molecule. If this variability is small, different T-cell receptors restricted by the same class II molecule are likely to recognize identical or closely related determinants on the MHC molecules. Conversely, if the top- ological variability is large, different T-cell receptors are expected to recognize a more variable spectrum of MHC determinants.

The second issue is whether class II MHC molecules can bind immunogenic peptides in more than one configuration. If so, an expanded spectrum of T-cell reactivities could be elicited against single immunogenic structures (discussed in Ref. 9). Both of these issues can be addressed by determining antigen contact points on class II MHC molecules. In this study we explored the possibility of determining such antigen contact points via photoaffnity labeling.

The I-Ak class II MHC molecule specifically binds the immunogenic hen egg white lysozyme peptide (HEL)-46-61 (2, 10) and its NHZ-terminal truncated homologues (HEL)-

i The abbreviations used are: MHC, major histocompatibility com- plex; ABA, 4-azidobenzoyl; ASA, 4-azidosalicyloyl; HEL, hen egg white lysozyme; HPLC, high performance liquid chromatography; IASA, iodo-4-azidosalicyloyk MEGA 8, octanoyl-N-methylslucamide; MEGA 9, nanoyl-N-methyl glucamide; ONSuT the hydro&succinim- idyl ester of either ABA or ASA; PBS, phosphate-buffered saline: Protease V-8, protease from Staphyloc&us &reus strain V-8; AC, acetyl; HEPES, 4-(Z-hydroxyethyl)-l-piperazineethanesulfonic acid.

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11178 Peptide Binding by Histocompatibility Molecules

49-61 (2, 10) and (HEL)-52-61 (11). The interaction of these peptides with the I-Ak molecule can also be studied by photo- affinity labeling (12), which creates a covalent bond between the photoreactive peptide conjugate and the class II molecule. In the present study we analyzed the photoaffinity labeling of I-Ak by different photoreactive 4-azidobenzoyl derivatives of (HEL)-46-61. The UV light-induced intermediate nitrenes have a very short life span (13) and are able to react with virtually any amino acid residue in proteins (14). The rapid reaction time of the photoreactive peptide conjugates coupled with the extremely slow dissociation of immunogenic peptides from class II MHC molecules (15) facilitates the identification of antigen contact sites via photoaffinity labeling.

MATERIALS AND METHODS

Syntheses of Photoreactive Peptide Conjugates-The peptides (HEL)-46-61, (HEL)-49-61-Cys(SH) and (HEL)-Ac-49-61-Cys(SH) were synthesized on a Applied Biosystem peptide synthesizer follow- ing standard procedures. The deprotected peptides were purified by reverse-phase HPLC using a C-18 Vydac column and analyzed by amino acid composition. They were used to prepare the photoreactive conjugates depicted in Fig. 1.

General reagents were obtained from Sigma in the highest purity available. The photoreactive hydroxysuccinimidyl esters of 4-azido- benzoic acid (ABA-ONSu) and 4-aiidosalicylic acid (ASA-ONSu) were from Pierce Chemical Co. Sodium 1Yliodide. 1YSlmethionine. and [%]cysteine having average specific akivity of 2iOO Ci/mmoi (lz51) and 1000 (35S) Ci/mmol were obtained from Du Pont-New England Nuclear.

The conjugates and their intermediates were purified by HPLC using an Altex C-18 reverse phase column (ultrasphere ODS-IP, 5 pm, 4.6 X 250 mm). The column was eluted by the following linear gradients of acetonitrile in 0.1% trifluoroacetic acid in water; the acetonitrile concentration increased from 3.75% (v/v) to 33.75% within the first 15 min, then to 56.25% within 45 min, and finally to 75% within 60 min. Maximally 300 pg of material were applied to the column per chromatography. Ultraviolet absorption spectra were recorded on a Beckman photospectrometer either in the HPLC sol- vent system or in PBS. All procedures involving photoreactive ma- terials were performed under dimmed light.

Compound 1: (HEL)-Ac-49-61-Cys [Iodo-4-azidosalicyloyl] Thio- ester-Compound 1 was obtained by acylation of Ac-49-61-Cys(SH) (Fig. 1) with IASA-ONSu (Fig. 1). Compound 1 was freshly reduced with 2-mercaptoethanol in Tris buffer, pH 8.0, and HPLC-purified prior to use. It was dissolved in dimethyl sulfoxide (10 pg in 6 ~1) and added to fresh radioiodinated compound 2b (2 mCi, see below). Two ~1 of methylmorpholine were added, and the solution was stirred under argon at room temperature for 3-4 h. Following addition of water (800 ~1) the mixture was subjected to HPLC. The major radioactive peak (0.9 mCi, 45%) eluted after 27.25 min. Ultraviolet absorption spectra of similarly prepared nonradioactive material showed absorption maxima at $19, 270, and 231 nm (see Fig. 1).

ComDound 2: Zodo-4-azidosalicvlovl fHEL)-46-61-Comnound 2 was prepared by reacting compound-2 (2 in Fig. 1) with (GEL)-46- 61 (Fig. 1). Compound 2 was obtained by radioiodination of N- hydroxysuccinimidyl4-azidobenzoate (ASA-ONSu). In brief, four ~1 of ASA-ONSu in dimethyl sulfoxide (5 mg/ml) were added to phos- phate-buffered (pH 6.0, 10 mM) sodium [‘*‘I]iodide (5 mCi in lo-15 ~1). Four ~1 of fresh chloramine-T solution (5 mg/ml in dimethyl sulfoxide) were immediately added. After 15 s 4 ~1 of sodium meta- bisulfite (5 mg/ml in water) were added followed by the addition of 300 ~1 of ethyl acetate and 300 ~1 of saline (lo%, w/v). After mixing, the organic phase was recovered, dried over Na2S04, and dried in uacuo. The recovered radioactivity was approximately 4 mCi (80%). Thirty ~1 of purified (HEL)-46-61 (6 mg/ml in anhydrous dimethyl sulfoxide) and 3 ~1 of N-methylmorpholine were added, and the reaction mixture was stirred under &y argon at room temperature overnight. The reaction mixture was dissolved in 800 ~1 of water and subjected to HPLC. The main product eluted at 30.5 min (600 &i, 12%) and a minor product at 31.6 min (170 &i, 3.4%). Ultraviolet absorption spectra-on similarly prepared n&radioactive materials showed absorption maxima at 320 and 280 nm in the case of the first component (30.5 min) and at 325 and 275 nm in the case of the second component. Both materials were indistinguishable in photo- affinity labeling and peptide mapping experiments (see “Results”).

As suggested, the main product probably is the 3-iodo-4-azidosalicy- loyl derivative and the minor product the 5-iodo-4-azidosalicyloyl isomer (24). We also prepared the homologous (IASA)-52-61, using peptide 52-61 of HEL. The main product eluted from the HPLC column at 29 min with a minor product at 30 min.

Compound 3: Iodo-4-azidosalicvll (HEL)-52-61-It was nrevared the same as compound 2 but using the shorter peptide (HEi)-g2-61.

Purification and Labeling of I-Ak-The class II molecules I-Ak, I- Ad, and I-Ek plus I-Ed were purified from TA3 cell membranes by affinity chromatography using Sepharose coupled with 10.3.6.2 (anti- I-A’), MKD6 (anti-I-Ad), and 14.4.4 (anti-I-E’ and I-Ed) monoclonal antibodies, respectively. The photoreactive conjugates (5 X lo-l4 mol) were incubated with purified class II molecules (20 rg) in PBS (60 ~1) containing 20 mM MEGA 8 and 20 mM MEGA 9 detergents, in the absence or presence of the indicated molar-fold excesses of (HEL)- 46-61, at room temperature for 2 days. The samples were irradiated with UV light (8-watt low pressure mercury lamp with parabolic reflector for 7 min at 4 “C) in Pyrex glass tubes (which strongly absorb light below 270 nm) and then subjected to sodium dodecyl sulfate-polyacrylamide gel electrophoresis (12.5%) under reducing conditions. In some experiments I-Ak on cell membranes was photo- affinity labeled as described nreviouslv (12). The dried gels were exposed with an amplifying slreen to -Kodak XAR-5 x-rai film at -80 ‘C. The labeled bands were cut out from the gels, rehydrated in 250 mM Tris buffer, pH 8.0, containing 8.3% glycerol and 0.3% SDS for 3 h at 4 “C. The labeled a and j3 chains were eluted by electropho- resis using a disc electrophoresis apparatus with a constant current of 3 mA/disc overnight at 4 “C. The anodic end of the discs was plugged with glass wool and connected to a closed dialysis membrane tubing (25-kDa cut-off). The eluted proteins were concentrated in a Centricon concentrator (lo-kDa cut-off, Amicon) to approximately 100 ~1. Two ml of water were added, and the samples were concen- trated again. This procedure was repeated three times.

Labeling of I-A” with Amino Acids-CH-27 B-lymphoma cells (1 x 107), in logarithmic growth phase, were washed three times and resuspended in 2 ml of RPM1 1640 medium deficient in the amino acid used for labeling, and complemented with 7% dialyzed fetal calf serum. 10 mM sodium pvruvate (Difco), 10 mM HEPES (Difco), 10m5 M 2-mercaptoethanol,-and 5 mCi of either [35S]cysteine (Du Pont- New England Nuclear, 900-1000 Ci/mmol) or 10 mCi each of [3H] tryptophan or proline (Amersham Corp., 40-70 Ci/mmol). Cells were incubated at 37 “C in a humidified atmosphere containing 4% COs for 6 h, washed three times. and then solubilized at 0 “C in 20 ml of PBS cdntaining 0.7% Triton X-100, and 20 mM iodoacetamide, 1 mM phenylmethanesulfonyl fluoride, and 10 @g/ml leupeptin. After 30 min incubation, the detergent-insoluble components were removed by centrifugation (100,000 x g, 1 h), and the supernatant was incu- bated with 1 to 2 ml of Sepharose 4B under agitation at 4 “C for approximately 6 h. The Sepharose was removed by centrifugation, and the supeinatant was filtered through 0.2-pm disposable mem- brane filters (Millinore, Waters). All further nurification procedures of I-Ak were ‘as described using antibody-afknity chromatography. Approximately 1.2 X 10’ dpm [3SS]cysteine, 8 x lo6 dpm [3H]trypto- phan, or 1.5 x lo7 dpm 13Hlproline were incorporated in the purified i-Ak,.which was coice&rated to a final volime of 60-80 il. The biosvntheticallv labeled I-Ak was incubated with 700 &i of 1”‘Il (IASA)-46-61 and 1 x 10m7 mol of nonradioactive (iASA)-4&6i (approximately 300-fold molar excess) in 20 ~1 of PBS containing the same detergents.

Digestions of the Labeled Chains and Peptide Mapping-Aliquots (lo-20 ~1) of the above solutions were added to 180 ~1 of the following digestion buffers: 100 mM ammonium bicarbonate, pH 8.25, for trypsin; 100 mM phosphate buffer, pH 7.8, for protease V-8; 100 mM ammonium carbonate, pH 8.65, for protease Lys-C; 100 mM phos- phate buffer, pH 7.8, in 150 mM saline for clostripain; and 100 mM phosphate buffer, pH 8.25, for mouse submaxillary endoprotease Arg- C. Digestions were performed at 37 “C in the presence of 100 pg of human y-globulin. Complete digestion with trypsin was achieved by adding 3 aliquots of 20 rg of L-l-tosylamido-2-phenylethyl chloro- methyl ketone-trypsin (Worthington) at 12-h intervals. Digestion with protease V-8 was performed over 2 days by adding 4 aliquots (20 pg) of enzyme (Boehringer Mannheim) in 12-h intervals.

Digestions with protease Lvs-C (Boehringer Mannheim) were per- formed within 36 h by adding 3 aliquots of-1 unit of enzyme. Diges- tions with clostripain were performed following preactivation of the enzyme as recommended by the supplier (Sigma) for 24 h by adding 3 aliquots (40 pg) in 8-h intervals. Digestion with endoprotease Arg-C was performed, likewise, using 5-unit aliquots. All digests

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Peptide Binding by Histocompatibility Molecules 11179

were kept at -20 “C until analysis by HPLC. Reverse-phase HPLC was performed on C-18 or C-4 Vydac col-

umns (5 pm, 4.6 x 250 mm). Fractions were eluted from the columns with a linear gradient of acetonitrile (O-90%) in 0.1% trifluoroacetic acid in water over 60 min at 1 ml/min. Anion-exchange HPLC was performed on a DEAE-Protpak column (8 X 100 mm) (Waters). Fractions were eluted from the columns with a linear sodium chloride gradient (O-500 mM) in 10 mM Tris/HCl in water, pH 8.0, over 60 min at 1 ml/min. One-ml fractions were collected and counted in a gamma counter. Primary peptide maps were made with at least 20,000 cpm of photoaffinity labeled protein and secondary maps with at least 10,000 cpm of labeled peptide fragment. The chromatographic hardware and data acquisition procedures were described in Refs. 16 and 17.

Sequence Analysis-NH2-terminal sequencing was performed on an Applied Biosystems gas-phase sequenator using an on-line 120 A phenylthiohydantoin analyzer essentially as described (16, 17). The ‘251-radiolabeled fractions from the C-4 HPLC chromatogram were pooled and lyophilized. The samples, reconstituted in 50 ~1 of triflu- oroacetic acid, were applied to Polybrene-coated and trifluoroacetic acid-treated glass filters. The sample was then washed with 50 ~1 of 88% formic acid, and the solution was applied to the glass filter. This procedure resulted in 90-95% transfer of the labeled material to the filter. The iodine content of each cycle was assessed by gamma counting. In some of the sequencing studies, 200 pmol of fl-lactoglob- ulin was added, and the resulting phenylthiohydantoin derivatives were analyzed to validate the performance. For 3H,1251-double-labeled samples, the iodine content was first determined by gamma counting. The cycle fractions were then mixed with scintillation fluid and the 3H radioactivity was determined by double label counting as described above.

Antigen Presentation and Binding-The basic system was as de- scribed in Ref. 11. A T-cell hybridoma (3A9) that recognizes (HEL)- 52-61 was cultured for 24 h with B-lymphoma cells (CH27) that bear I-A!+ molecules and the indicated peptides. The CH27 was highly fixed in paraformaldehyde just prior to the culture. After 24 h the culture fluid was tested for the presence of interleukin-2. 3A9 cells release interleukin-2 in amounts proportional to their recognition of (HEL)- 52-61-I-Ak complex (1, 11).

I-Ak molecules purified from CH27 cells were incubated with either (HEL)-52-61 (11) or (HEL)-34-45 (9), as described (18), in the presence of unlabeled (HEL)-46-61 or compound 2 ((IASA)-46-61). The incubation mixtures usually consisted of 15-20 ~1 that contained about 100 pmol of I-A’, 5 pmol of peptides, in 20 mM each of Mega 8 and Mega 9 and 5 mM lysophosphatidylserine. After 48 h the amounts of bound peptide were determined by Sephadex G-50 filtration (3, 18).

RESULTS

Peptide Mapping-We labeled the I-A’ molecule with dif- ferent photoreactive conjugates of (HEL)-46-61 and (HEL)- 49-61-Cys and analyzed the labeled peptides after digestion of the separated (Y and /3 chains with trypsin or protease V-8. In this paper we will examine our initial results with the three compounds shown in Fig. 1 and in particular with compound 2 ((IASA)-46-61). The photoreactive IASA group was directly conjugated at the COOH-terminal cysteine of Ac-49-61-Cys (compound 1) or at the NH2 terminus of either (HEL)-46-61 (compound 2) or (HEL)-52-61 (compound 3). With these three compounds, the tryptic peptide maps of the labeled (Y and /3 chain revealed resolved single major labeled components (Figs. 1 and 2). The fragment from the @ chain labeled by compound 2 eluted considerably later (46 min) from the reverse-phase column than the one labeled by compound 1 (21 min) (Fig. l), indicating that it was more hydrophobic. Indeed, peptide maps of unlabeled I-Ak showed very few fragments eluting in the last third of tryptic peptide maps (see Refs. 19, 20). Furthermore, we failed to obtain a tryptic peptide map of the (Y chain labeled by compound 2, suggesting that an even more hydrophobic digest fragment was labeled which was no longer amenable to reverse-phase chromatog- raphy. In contrast to compound 2, a similar conjugate, but shorter, of the minimal immunogenic peptide from residues

A COMPOUND FORMVLA SYNTHESIS

1) Ac-~BB?-CYS(IASA)

I on0

2) IA%-48-6, N,+kVSu +4641 s N

I on

FmcII(II (na, Fnclbm (nh)

FIG. 1. Structures of the compounds 1,2, and 3 (A). Reverse-phase HPLC (C-4) of the tryptic digests of I-Ak p labeled by compound 1 (B) and compound 2 (C). The digest fragment labeled by the COOH- terminal conjugate eluted 24.7 min earlier than the one labeled by the NH2-terminal conjugate of (HEL)-46-61. The peptide fragment labeled by compound 3 ((IASA)-52-61) eluted at 30 min (C, dotted line).

A 6

1 P -L 54

z 2

6 16 224 32 40 48 56

Fractions (min)

FIG. 2. Reverse-phase HPLC and DEAE HPLC of a and fi chains labeled by compound 1. The tryptic digest of the I-Ak OL chain labeled by compound 1 and analyzed by reverse-phase HPLC (C-4) exhibited one major labeled component eluting at 22 min (A). This labeled species rechromatographed on DEAE HPLC eluted again as a major labeled component at 21 min (B). The labeled tryptic digest fragment of I-Ak fl chain that eluted at 21 min (shown in Fig. 1B) was likewise rechromatographed and shown to elute as a major labeled component at 27 min (C).

52-61 (11) labeled a different, more hydrophilic, tryptic pep- tide fragment (Fig. lC, dotted line).

The tryptic peptide map of the (Y chain labeled by compound 1 showed a major labeled component eluting at 22.0 min (Fig. 2A). To assess the homogeneity of this fragment, it was rechromatographed on anion-exchange (DEAE) HPLC. Again a single major labeled fraction was observed at 21 min (Fig. 2B). Likewise, rechromatography on DEAE HPLC of the major labeled p chain fragment (Fig. 1B) revealed a single major component eluting at 27 min (Fig. 2C). This later elution indicated that the labeled p chain fragment was more acidic than the labeled (Y chain fragment. The observation that the major labeled fragments of both chains were homo-

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11180 Peptide Binding by Histocompatibility Molecules

8 16 24 32 40 48 56 6 16 24 32 40 fbl 56

Fractions (min] 52 Fractions (min) 47

FIG. 3. Reverse-phase HPLC of the a and /3 chains labeled by compound 2. The protease V-8 digest of the I-Ak LY chain labeled by compound 2 and analyzed by reverse-phase HPLC (C-4) exhibited one major labeled component eluting at 48 min (A). The same experiment for the I-Ak 0 chain showed one major component eluting at 41 min (B). These major components were digested with trypsin and the peptides chromatographed by reverse-phase HPLC. In the case of the (Y chain a derivative secondary labeled peptide product eluted at 52 min (C) and in the case of the p chain a derivative secondary peptide eked at 47 min (D).

geneous in two different HPLC systems strongly suggests that compound 1 labeled I-Ak in a site-specific manner.

In the remainder of this study we will focus on compound 2. The site of labeling of I-Ak was entirely inhibited by about a lOOO-fold molar excess of unlabeled compound 2 and by about a 5000-fold molar excess of (HEL)-46-61. The photo- affinity labeling of I-Ak by compound 2 was further studied with protease V-8 peptide maps. In the case of the LY chain a single major labeled component eluted at 48 min and in the case of the p chain at 41 min (Fig. 3, A and B). The homoge- neity of these labeled peptides was assessed by secondary tryptic peptide maps. (The high degree of hydrophobicity of these labeled fragments excluded their analysis by ion-ex- change chromatography.) The secondary fragment of the LY chain eluted again as a major single component at 52 min and that of the p chain at 47 min (Fig. 3, C and D). These results indicate that both primary labeled digest fragments are ho- mogeneous and contain tryptic cleavage site(s).

We assessed whether compound 2 labeled the membrane spanning domains of the I-Ak molecule. I-Ak was photoaffinity labeled on cells, and the separated chains were subjected to primary protease V-8 and secondary tryptic digestion. In contrast to purified I-Ak in detergent, in which both chains were labeled, I-Ak on cells was selectively labeled on the LY chain (12).* The peptide map of the I-Ak (Y chain labeled on the membrane was identical to that of I-Ak labeled in deter- gent (not shown). This result suggests that the I-Ak (Y chains on the membrane and in detergent were labeled at the same site, therefore excluding the transmembrane domain as the labeled site.

Identification of the Site Labeled by Compound 2 ((IASA)- 46-61)--We examined whether the tryptic cleavage sites of the a and @ chains labeled by compound 2 were argininets) and/or lysine(s). The labeled fragments from protease V-8 digestion were treated with endoprotease Arg-C or Lys-C, which selectively cleaves COOH-terminal to arginine or ly- sine, respectively, and were rechromatographed under iden-

‘1. F. Luescher and E. R. Unanue, manuscript submitted for publication.

tical conditions (C-4 reverse-phase HPLC). In the case of the (Y chain, the secondary endoprotease Lys-C peptide map showed a new labeled fragment eluting at 51 min (Fig. 4A). Based on the high specificity of this enzyme (21), this result indicated the presence of a lysine residue in the primary fragment. Similarly, treatment with clostripain or endopro- tease Arg-C from mouse submaxillary glands also resulted in a secondary digest fragment (Fig. 4.4). Clostripain treatment gave similar results (data not shown). Since both enzymes are specific for arginine bonds (22, 23), these results indicate the presence of an arginine residue in the primary protease V-8- labeled peptide.

In the case of the @ chain, peptide maps of the labeled primary protease V-8 digest fragment secondarily digested with trypsin suggested the presence of multiple basic amino

FIG. 4. Secondary peptide mapping of the [‘251](IASA)-46- 61 (compound 2) labeled protease V-8 digest products of the I-A“ a and @ chains. A shows the proteolytic digestions of the LY chain; top, primary protease V-8 product; bottom, secondary proteo- lytic digests with Lys-C, trypsin, or Arg-C (left to right). The arrow in the chromatograms of these secondary digests denotes the elution position of the primary protease V-8 product (fraction 48). B shows the proteolytic digestions of the p chain; top, primary protease V-8 product; bottom, secondary proteolytic digests with Lys-C, trypsin, or Arg-C (left to right). The arrow in these secondary digests denotes the elution position of the primary protease V-8 product (fraction 41). For secondary tryptic digestion of both the (Y and p chains, a short digestion time (12 h) was used to produce incomplete proteol- ysis. All peptide maps were analyzed under identical conditions by reverse-phase (C-4) HPLC.

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Peptide Binding by Histocompatibility Molecules

acid residues (Fig. 4B). Secondary peptide maps with protease Lys-C and Arg-C showed two new labeled peptides, suggesting the presence of 2 lysines and arginines in the primary digest fragment (Fig. 4B). The relative amount of the secondary digest products in these peptide maps varied in different experiments. The earlier eluting fragment in the protease Arg-C peptide map (44 min) was the major peptide when clostripain rather than protease Arg-C from mouse submax- illary gland was used.

We next examined whether the labeled protease V-8 digest fragments contained cysteine or tryptophan, infrequent amino acids in I-Ak (Fig. 9). Cysteine labeled I-Ak was incu- bated with compound 2 and digested with protease V-8. Dou- ble counting of the peptide maps showed that the photo- affinity labeled fragment of the (Y chain did not contain [35S] cysteine (Fig. 5, A and B). Interestingly, the same peptide maps were obtained whether the labeled (Y chain was reduced and alkylated prior to digestion or whether the digest products were reduced and incubated with 5,5’-dithiobis(2-nitroben- zoic) acid which gives typical shifts of thiol-containing pep- tides in reverse-phase HPLC (24) (not shown). It thus appears that the thiol function in this sequence became irreversibly “blocked,” during the UV irradiation which is known to destroy cysteine structures (25). The same experiment on the p chain was inconclusive since multiple ‘S-containing frag- ments eluted in the region of the lz51-labeled component (not shown). The latter lz51-labeled component was isolated and then treated with trypsin. The secondary tryptic peptide map on this ‘Y-labeled component revealed that the fragment contained [35S]cysteine (Fig. 5, C and D).

Protease V-8 peptide maps showed that the photoaffinity labeled products of both chains contained 13H]tryptophan (Fig. 6). Secondary tryptic peptide maps further indicated that the products of the (Y but not the /3 chain contained tryptophan (Fig. 7). The photoaffinity labeled site and tryp- tophan had therefore to be located on the same side of the tryptic cleavage site(s) in the case of the (Y but not p chain.

I-Ak was then intrinsically labeled with [3H]proline and photoaffinity labeled with (IASA)-46-61. Primary peptide maps with protease V-8 showed proline to be present in the labeled digest fragments of both chains (not shown), which were subjected to amino acid sequencing. In the case of the (Y

FIG. 5. Reverse-phase (C-4) HPLC of [‘261](IASA)-46-61 (compound 2)-labeled [36S]Cys-I-Ak (I and fl chain fragments. A and B are chromatograms of the cy chain and C and D are chromatograms of the fl chain. For the (Y chain, the fractions of the primary protease V-8 digest were counted for 9 in A and for %l in B; the arrow in B denotes elution position of the “‘1 component. For the p chain, a primary V-8 fragment was digested with trypsin; peptides were fractionated in HPLC and counted for “9 in C and %3 in D.

Fractions [min] Fractions [min]

FIG. 6. Reverse-phase (C-4) HPLC of [‘251](IASA)-46-61- labeled [3H]Trp-I-Ak (I and fl chains. I-Ak was intrinsically labeled with [sH]tryptophan and photoaffinity labeled with [iz51](IASA)-46- 61. The isolated chains were subjected to protease V-8 peptide maps. The fractions of the 01 chain map (A and B) and p chain map (C and D) were counted for 9 (A and C) and 3H (B and D). Peptide maps were performed on C-4 reverse-phase HPLC.

47

FIG. 7. Secondary tryptic peptide maps of the primary pro- tease V-8-derived peptides of [‘261](IASA)-46-61-labeled [3H] Trp-1-A’ a and fl chains. Fraction 48 (Fig. 4A) of the primary protease V-8 digest of the cy chain was digested with trypsin and analyzed by reverse-phase (C-4) HPLC. A shows the “‘1 and B the 3H elution patterns. Fraction 41 (Fig. 4B) of the primary protease V- 8 digest of the p chain was digested with trypsin and the digest analyzed by reverse-phase (C-4) HPLC. C shows the “‘1 and D the 3H elution patterns. Note that in D the sH radioactivity did not co- elute with iz51 radioactivity.

chain, [3H]proline eluted in the 4th and 5th cycle and the 1251- photoaffinity-labeled residue in the 4th cycle (Fig. 8, A and B). In the case of the fi chain, 3H-proline eluted at the 17th cycle and the ‘Y-labeled residue at the 13th cycle (Fig. 8, C and D). The findings are compatible with the photoaffinity labeled sites being located in the primary (Y chain fragment at residues 115-134 and in the primary /I chain fragments at residue 109-138. The (Y chain fragment contains proline at positions 4 and 5 (Pro-118 and -119) and the /3 chain fragment at position 17 (Pro-125) (Fig. 9). (Assuming that the trypsin digestion was complete, the secondary peptides likely include residues 115-127 for the (Y chain and 109-127 for the /3 chain.)

Interactions of Compound 2 with Z-Ah-Dose-response curves showed that compound 2 ((IASA)-46-61) was slightly less stimulatory than (HEL)-46-61 (Fig. 10). Since the acti- vation of this T-cell hybridoma depends on the recognition of the I-Ak. (HEL)-46-61 complex, these results indicate that the photoreactive groups did not substantially alter this in-

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Peptide Binding by Histocompatibility Molecules 11182

B f 6 x E4 0

5?

A2 L 2 ts 8 12 14 18 20 45

10 18 Cycle.9

FIG. 8. NH2-terminal sequence analysis of [3H]Pro-I-Ak a and @ chain fragments labeled with [1261](IASA)-46-61. The ‘251-labeled (Y and p chains were obtained from a primary protease V- 8 digestion after chromatography (as in Fig. 4, A and B). A and B show the results for the a chain. A indicates the lz51 cycle profile showing the label at position 4 of the peptide. B indicates the 3H cycle showing proline at positions 4 and 5 of the peptide. C and D show the results for the fl chain. C indicates the “‘1 cycle profile showing the label at position 13 of the peptide. D indicates the 3H cycle profile showing proline at position 17 of the peptide.

teraction (11). We also examined whether unlabeled com- pound 2 competed effectively for the binding of ‘251-labeled (HEL)-52-61 and rz51-labeled (HEL)-34-45 to I-Ak (9). Both peptides bind to the single peptide binding site of I-Ak. Ad- dition of unlabeled compound 2 inhibited the binding of the radioactive peptides. (For example, addition of labeled (HEL)- 52-61 to I-Ak resulted in specific binding of 23,500 cpm; addition of lo-fold excess of compound 2 reduced the binding to 4,120 cpm; addition of peptide 34-45 resulted in 8,680 cpm

FIG. 9. Amino acid sequence of the (I (A) and j3 chain (31). The acidic amino acids residues D and E (protease V-8 cleavage sites) are circled, and the basic residues K and R (tryptic cleavage sites) are marked by boxes. The hydro- phobic amino acid residues (A, F, I, L, M, V, W, and Y) are marked by a dot above the letter. The first two lines are

the sequences of the first domains (cul, 81) and the following two lines of the second domain (a2, @2). The bottom lines are the sequences of the transmembrane and cytoplasmic domain (01 TM-CY, p TM-CY). The rare cysteine (C) and tryp- tophan (w) residues are underlined by a solid line or a broken line.

bound; and this binding was entirely abolished by lo-fold excess of compound 2.)

DISCUSSION

We are in the process of determining the contact points of the HEL peptides 46-61 and 49-61 for the I-Ak MHC molecule by photoaffinity labeling. The identification of photoaffinity labeled sites requires that the photoaffinity labeling be site- specific and that the labeled proteins can be fragmented in a defined and reproducible manner, preserving the photocross- linked bond. The findings of this first study indicate that photoaffinity labeled sites on the I-Ak molecule can, indeed, be determined. Site-specific labeling that leads to resolved peptide maps, however, requires that the photoreactive groups be conjugated to the peptide via short spacers of limited flexibility. Our other studies3 using photoconjugates contain- ing cysteines resulted in unresolved peptide maps perhaps as a result of a longer and more flexible molecule. In this study we have also been able to identify the site of labeling of the (Y and p chains by an NH*-terminal conjugate of HEL 46-61 (compound 2 in the text).

The photoreactive IASA group conjugated directly at the NH*-terminal amino group of (HEL)-46-61 (compound 2), and the COOH-terminal conjugate, compound 1, yielded re- solved, labeled peptide maps (Figs. l-3). However, compound 2 labeled exceptionally hydrophobic protease V-8 as well as tryptic fragments. The hydrophobicity of the labeled digest fragments was not accounted for by the photoconjugation. (HEL)-46-61 itself is a tryptic digest fragment that contains two protease V-8 cleavage sites (Fig. 1); the protease V-8 digest fragment will therefore be labeled by a derivative of 4- amino-[‘251]iodosalicyloyl Asn-Thr-Asp. This relatively hy-

3 I. F. Luescher, D. L. Crimmins, B. D. Schwartz, and E. R. Unanue, unpublished data.

Sequence of A!

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Antigen [,A]

FIG. 10. Dose-response curves of HEL 46-61(O) and IASA- 46-61 (A) on 3A9 T-cell hsbridomas. The indicated concentra- tions of p&tide or conjugates were added to a constant number of fixed CH-27 cells and 3A9 cells. The supernatants were assayed after 24 h for interleukin-2 by [3H]thymidine incorporation by the inter- leukin-2-dependent CTLL cells. The bars indicate standard deviation of the triplicate culture.

drophilic “tag” is expected to give only a small delay in the elution of the labeled fragments.

Reverse-phase peptide maps of I-Ak labeled by (IASA)-46- 61 indicated that the labeled primary protease V-8 digest fragments of both chains contained tryptic cleavage sites (Fig. 4). In the case of the cy chain, digestion of the V-8 fragment with endoprotease Arg-C and Lys-C showed new labeled fragments, indicating the presence of an arginine and a lysine residue (Fig. 4A). In the case of the p chain, the same exper- iment showed two new digest fragments in both peptide maps. Because the 125I label was found at only one position during sequencing, these results suggest the presence of 2 lysine and 2 arginine residues in the primary protease V-8 digest frag- ment (Fig. 4B). Assuming that protease V-8 cleaved at all acidic residues, the following generated peptides are compat- ible with these findings: I-Ak a-76-89 and -115-134 and I-Ak p-123-138 and -195-238 (Fig. 9). In the case of the (Y chain the former peptide contains three and the latter eight hydro- phobic amino acids. In view of the late elution of the labeled protease V-8 peptide (Fig. 4A), the latter sequence seems to be more likely to contain the labeled site(s). This hypothesis was further supported by the failure to obtain a primary tryptic peptide map of the labeled cy chain. The corresponding tryptic peptide that encompasses residues 99-127 contains 15 hydrophobic amino acids and may indeed not be amenable to reverse-phase HPLC (Fig. 9).

Peptide maps on [35S]cysteine intrinsically labeled I-Ak showed that only the labeled peptide derived from the /3 chain contained cysteine (Fig. 5). In contradiction to these findings, however, the predicted labeled /3 chain peptide, including residues 123-138, did not contain cysteine (Fig. 9). It is, however, conceivable that protease V-8 failed to cleave at Asp-122, resulting in the primary labeled protease V-8 peptide being 109-138. Protease V-8 has been reported to cleave sluggishly when aspartic acid residues are followed by bulky hydrophobic residues, such as is found in the present sequence (Asp-Phe-Tyr) (26). Furthermore, a primary V-8 peptide spanning residues 109-138 better accounts for the secondary tryptic peptide maps. The secondary digest /? chain peptide derived from this larger peptide spans residues 109-127 and contains eight hydrophobic amino acids. It, rather than the alternative hydrophilic tryptic peptide spanning residues 123- 127, would be expected to elute at 47 min (Figs. 4B and 7C).

Primary protease V-8 peptide maps of I-Ak labeled intrin- sically with tryptophan showed that the labeled digest prod- ucts of both chains contained tryptophan (Fig. 6). This finding is compatible with the primary labeled protease V-8 fragments being I-Ak a-115-134 and I-Ak p-109-138. More importantly,

the secondary tryptic digest product of the LY chain, but not the /I chain, contained tryptophan (Fig. 7). Tryptophan in the predicted primary B chain fragment is located between tryptic cleavage sites and is therefore expected to be absent in the secondary peptide. Conversely, in the case of the (Y chain, tryptophan is located in the hydrophobic sequence NH*- terminal of the tryptic cleavage sites and is therefore expected to be present in the secondary peptide. In fact, there are no other protease V-8 peptides that contain tryptophan and tryptic cleavage sites in a manner compatible with these peptide maps.

The predicted labeled protease V-8 cy chain peptide 115- I34 contains proline at positions 4 and 5 (Pro-118 and -119) and the p chain fragment 109-138 at position 17 (Pro-125). Sequencing of V-8 protease peptides prepared from [3H]pro- line intrinsically labeled I-Ak molecules localized proline at the predicted sites (Fig. 8). These sequencing experiments further showed lZ51 at the 4th residue of the LY chain and at the 13th residue of the /3 chain, indicating that the photo- affinity labeled residues were I-A” Pro+118 and I-Ak Thr- p121.

Our interpretation is that (IASA)-46-61 folds into the allele-specific peptide-binding site at the critical stretch from residues 52 to 61 and that the peptide from residues 46 to 51 extends beyond the peptide-binding site to a nearby area rich in hydrophobic residues in the (Y* and /3* domains. Several observations support this interpretation. First, the finding that (IASA)-46-61 stimulated a T-cell hybridoma similarly to the unmodified peptide (Fig. 1) argues that the 52-61 residues of the peptide must be specifically bound to the allele-com- bining site. This was also confirmed by showing that it com- peted for the binding of labeled 52-61 or an unrelated lyso- zyme peptide. We had previously established that (HEL)-46- 61 can be NH*-terminally truncated to the minimal immu- nogenic structure 52-61 (27). The residues that contact I-Ak are Asp-52, Ile-58, and Arg-61. The photoreactive group is spaced by 7 residues from Asp-52. Second, a shorter compound such as compound 3, (IASA)-52-61, failed to label these hydrophobic sites on I-Ak, indicating that their labeling re- quires a spacer. Third, a less hydrophobic conjugate ABA- Met-46-61 only partially labeled these hydrophobic sequences but also labeled more hydrophilic ones (data not reported). Fourth, the ability of (IASA)-46-61 or (IASA)-52-61 to label other alleles of class II molecules correlated with the labeling of this hydrophobic site, suggesting allele nonspecific hydro- phobic interactions with the photoreactive groups (12). The potential of the conjugated photoreactive groups to undergo hydrophobic interactions was indeed observed by reverse- phase HPLC; ABA-Met-46-61 eluted 4 min later from a C-18 column than (HEL)-46-61, and (IASA)-46-61 was even 10 min later.

The presence of a nonpolymorphic, hydrophobic domain located in the vicinity of the allele-specific antigen binding site of class II molecules constitutes a major difference from class I molecules. In class I molecules, the beginning of the constant (third) domain forms a “loop” at the outer side of the molecule, connecting the helical part of the second domain with P-pleated sheet structures in the constant domain (5). This class I constant domain is hydrophilic, containing mainly charged amino acids. In contrast, the sequences of the corre- sponding constant (second) domains of class II molecules show a considerably different hydrophobicity pattern, con- taining mainly uncharged, hydrophobic amino acids. Further- more, in contrast to class I, class II molecules have two such sequences which, according to the model, exit the antigen binding site on opposite sites (4). It is tempting to speculate

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that these hydrophobic areas would associate to minimize their contact with the hydrophilic medium, rather than as- sume conformations at the surface of the molecule, as their counterparts do in class I molecules (5).

The configuration in which these hydrophobic areas inter- act may be expected to depend on the symmetry of class II molecules, in particular on the angle between the floor of the antigen binding site and the center axis of the constant domain. If this angle is close to 90”, these sequences would “coil together” under the antigen binding site, approximately in the center of the molecule. In this case, the photoreactive groups conjugated NH&erminally to (HEL)-46-61 would need to access the labeled sites through a sufficiently wide intramolecular opening. Conversely, if this angle is consider- ably less than 90”, the labeled sites would be located closer to an end of the antigen binding site. In this case, this hydro- phobic domain could be accessible for macromolecules from the aqueous milieu. It is possible that allele nonspecific inter- actions of class II molecules involve hydrophobic interactions with this site. Examples include the interaction of class II molecules with the CD4 structure (28), the invariant chain (29), and “super antigens” such as staphylococcal enterotoxin B or Mls molecules (30).

Acknowledgments-We thank R. S. Thoma and D. W. McCourt for their technical assistance.

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I F Luescher, D L Crimmins, B D Schwartz and E R Unanueimmunogenic lysozyme peptide.

The sites in the I-Ak histocompatibility molecule photoaffinity labeled by an

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