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Jun 18, 1996 - USA. Vol. 93, pp. 9724-9729, September 1996. Immunology. Kinetic analysis of peptide loading onto HLA-DR molecules mediated by HLA-DM.
Proc. Natl. Acad. Sci. USA Vol. 93, pp. 9724-9729, September 1996

Immunology

Kinetic analysis of peptide loading onto HLA-DR molecules mediated by HLA-DM (antigen presentation/Michaelis-Menten kinetics/biosensor/real time analysis)

ANNE B. VOGTt, HARALD KROPSHOFERt, GERHARD MOLDENHAUER, AND GUNTER J. HAMMERLINGt Department of Molecular Immunology, German Cancer Research Center, Im Neuenheimer Feld 280, D-69120 Heidelberg, Germany

Communicated by Harden M. McConnell, Stanford University, Stanford, CA, June 18, 1996 (received for review April 5, 1996)

ABSTRACT The nonclassical major histocompatibility complex class II molecule HLA-DM (DM) has recently been shown to play a central role in the class 11-associated antigen presentation pathway: DM releases invariant chain-derived CLIP peptides (class II-associated invariant chain protein peptide) from HLA-DR (DR) molecules and thereby facilitates loading with antigenic peptides. Some observations have led to the suggestion that DM acts in a catalytic manner, but so far direct proof is missing. Here, we investigated in vitro the kinetics of exchange of endogenously bound CLIP for various peptides on DR1 and DR2a molecules: we found that in the presence of DM the peptide loading process follows MichaelisMenten kinetics with turnover numbers of 3-12 DR molecules per minute per DM molecule, and with KM values of 500-1000 nM. In addition, surface plasmon resonance measurements showed that DM interacts efficiently with DR-CLIP complexes but only weakly with DR-peptide complexes isolated from DM-positive cells. Taken together, our data provide evidence that DM functions as an enzyme-like catalyst of peptide exchange and favors the generation of long-lived DR-peptide complexes that are no longer substrates for DM.

In the present study we demonstrate that DM catalyzes the peptide exchange processes in an enzyme-like fashion following Michaelis-Menten kinetics. The peptide exchange is terminated once a kinetically stable peptide has been loaded onto the class II molecule, indicating that the catalytic action of DM favors the generation of long-lived class II-peptide complexes that will then accumulate at the cell surface.

MATERIALS AND METHODS Cells. The Epstein-Barr virus-transformed homozygous B cell lines WT-100 (DRlDwl), LD2B (DR15Dw2), and COX (DR17Dw3) as well as the T x B hybrid cell line T2 transfected with the cDNA for DR1 (DRA*0101/ DRB1*0101), DR2a (DRA*01O1/DRB5*0101), or DR3 (DRA*0101/DRB1*0301) were used for the isolation of the respective DR molecules. Cell lines were maintained in roller bottles at 37°C and cultured in RPMI 1640 medium with 20 mM Hepes (GIBCO) containing 10% heat-inactivated fetal calf serum (Konco Laboratories, Wiesbaden, Germany). Peptides. The following peptides were synthesized and labeled with the fluorophor 7-amino-4-methylcoumarin-3acetic acid (AMCA; Lambda Fluorescence Technology, Graz, Germany) as described (20): CLIP(81-105), LPKPPKPVSKMRMATPLLMQALPMG [a synthetic peptide containing residues 81-105 of human Ii (p33 form)]; DQw6(43-58), DVGVYRAVTPQGRPDA (a synthetic peptide containing residues 43-58 of HLA-DQw6); HA(307-319), PKYVKQNTLKLAT (synthetic peptide containing residues 307-319 of influenza virus hemagglutinin); and MBP(87-99), VHFFKNIVTPRTP (synthetic peptide containing residues 87-99 of human myelin basic protein). Purification of DR Molecules. HLA-DR molecules were isolated from B-cell lines or T2 transfectants by affinitychromatography using anti-DR mAb L243, as described (7). Purification of HLA-DM. HLA-DM was affinity-purified using the protocol described, for DR molecules, with the following modifications: 200 g of human spleen (containing -1011 cells) from a patient with B-chronic lymphocytic leukemia were homogenized and extracted for 2 h on ice in 1.0% Nonidet P-40. The lysate was applied to a Sepharose CL-4B column to which the monoclonal anti-DM antibody DM.K8 (directed against the cytoplasmic tail of the HLA-DM,B chain)

Immediately after biosynthesis in the endoplasmic reticulum, major histocompatibility complex (MHC) class II molecules associate with the invariant chain (Ii) (1), which prevents the MHC class II peptide binding groove from association with peptides (2) or polypeptides (3). A sorting signal in the cytoplasmic tail of Ii targets MHC class II molecules to endosomal compartments (4), where stepwise proteolytical degradation of Ii takes place (5). The final fragment of Ii that stays associated with class II molecules is designated CLIP (class II-associated invariant chain peptide) and encompasses residues 81-105 of the p33 form of human Ii (6). Since CLIP occupies the peptide binding groove (7, 8), its removal is a prerequisite for loading of MHC class II molecules. From some class II alleles, CLIP can rapidly dissociate by itself at endosomal pH (7, 9, 10). However, analysis of mutant antigen presenting cell lines that mainly displayed HLA-DR (DR)-CLIP complexes instead of DR-self-peptide complexes (6, 11) led to the identification of the nonclassical MHC class II molecule HLA-DM (DM) (12) as a key player involved in the peptide loading process (13, 14). Subcellular fractionation and immunoprecipitation studies have revealed that DM can physically associate with DR molecules (15), and in vitro experiments have shown that DM enhances CLIP release from DR (16, 17), but the mechanism is still unknown. It has been speculated that DM may act as a CLIP acceptor or a peptide shuttle, but recent studies have suggested that DM functions substoichiometrically and, therefore, may act as a catalyst (18, 19). However, so far there is no formal evidence for the catalytic nature of DM.

Abbreviations: AMCA, 7-amino-4-methyl-coumarin-3-acetic acid; CLIP, class II associated invariant chain peptides; CLIP(81-105), synthetic peptide containing residues 81-105 of human Ii (p33 form); DM, HLA-DM; DR, HLA-DR; DQw6(43-58), synthetic peptide containing residues 43-58 of HLA-DQw6; HA(307-319), synthetic peptide containing residues 307-319 of influenza virus hemagglutinin; HLA, human leucocyte antigen; HIPSEC, high-performance size exclusion chromatography; Ii, invariant chain; MALDI-MS, matrix-assisted laser desorption ionization-mass spectrometry; MBP (87-99), synthetic peptide containing residues 87-99 of human myelin basic protein; MHC, major histocompatibility complex. tA.B.V. and H.K. contributed equally to this work. *To whom reprint requests should be addressed.

The publication costs of this article were defrayed in part by page charge payment. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. §1734 solely to indicate this fact.

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was coupled. HLA-DM was eluted in 0.1% Zwittergent-12 (Calbiochem), 100 mM sodium phosphate, 50 mM sodium acetate (pH 5.0), yielding -250 ,ug HLA-DM. Purity of the isolated material was higher than 90%, as assessed by SDS/

PAGE. Mass Spectrometry. Self-peptides bound to purified DR molecules were analyzed by matrix-assisted laser desorption ionization-mass spectrometry (MALDI-MS) as described (10). Peptide Binding Assay. Purified DR molecules and the respective AMCA-labeled peptide were coincubated in the absence or presence of purified DM for the indicated times at 37°C in 50 mM sodium phosphate, 50 mM sodium citrate, 0.1% Zwittergent-12 (pH 5.0). Binding was quantitated by high performance size-exclusion chromatography (HPSEC) as described (7). Soluble DM. HLA-DM molecules lacking the transmembrane and the cytoplasmic region were produced in Spodoptera frugiperda (Sf9) cells and isolated as decribed (16). BlAcore Analysis. Real time analysis of DM-DR interaction was performed in a BIAcore2000 biosensor (Pharmacia Biosensor, Upsala). Soluble DM molecules were immobilized in a BIAcore2000 flow cell by standard amine-targeted chemistry by activating the carboxy-detran layer as described (21). DM was coupled via the active esters by injection into the flow cell in 10 mM acetate (pH 4.5) until 4000-5000 resonance units were bound. The surface was inactivated with ethanolamine. Measurements were performed by injection of different amounts of DR molecules in 50 mM sodium phosphate, 50 mM citrate, 1% octyl glucoside (pH 5.1) at a flow rate of 5 gl/min over the DM surface and simultaneously over a control surface. Background binding was subtracted from the data presented in Fig. 4. The DM surface was regenerated by injecting binding buffer (pH 7.4).

RESULTS AND DISCUSSION DR-CLIP Complexes Are Substrates for DM. The removal of CLIP from HLA-DR molecules seems to be a pivotal step in the peptide loading process which involves the action of DM. Therefore, for the in vitro investigation of the kinetics of DM-mediated peptide exchange purified DR-CLIP complexes were employed. DR2a molecules isolated from DM-negative T2 cells transfected with DR2a (DRA*0101/DRB5*0101) are exclusively occupied by CLIP variants (Fig. 1 Upper): analysis of the self-peptide fraction by MALDI-MS revealed that all six mass species correspond to CLIP truncation variants. Very similar CLIP variants have been described for DR3 molecules from T2 transfectants (6, 11). For comparison, the mass profile of DR2-associated self-peptides isolated from the DM-positive Epstein-Barr virus-transformed B-cell line LD2B is given in Fig. 1 Lower: a typical distribution of peptides with mass-tocharge ratios of m/z = 1400 to 2800, corresponding to 12-mers to 25-mers, is found, with a broad maximum around m/z = 1800, consistent with other reports (22). DR2a-DQw6(43-58) Complexes Are Stable in the Presence of DM. Using an in vitro peptide binding assay based on

HPSEC, we analyzed the time of half-maximal dissociation tl/2,off of different peptides bound to DR2a at endosomal pH 5.0 in the absence and presence of DM. Dissociation of CLIP(81105) from DR2a was found to be very fast (til/2,off 10 min), but even faster in the presence of DM (ti/2,off 3 min) (Fig. 2). The experiment was performed in the presence of the detergent Zwittergent, which has been described to increase the off-rate of CLIP (10). Nevertheless, also under these conditions, the dissociation of CLIP is enhanced by DM, but not the dissociation of the DQw6(43-58) peptide. The DM-sensitivity of the DR2a-CLIP complex is consistent with the findings described for other DR-CLIP complexes (16, 17, 19), although the CLIP dissocation rates may vary depending on the detergent used (10, 23). In contrast, DM did not affect the disso-

2150 11

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FIG. 1. Mass profile of DR2-associated self-peptides from DMpositive and DM-negative cells. (Upper) Self-peptides released by acid treatment of 5 lag affinity-purified DR2 molecules from T2.DR2a transfectants were analyzed by MALDI-MS. The indicated masses correspond to CLIP(83-100) (m/z = 2053), CLIP(82-100) (m/z = 2150), CLIP(82-101) (m/z = 2221), CLIP(82-102) (m/z = 2334), CLIP(82-103) (m/z = 2431), CLIP(82-104) (m/z = 2562), and CLIP(81-104) (m/z = 2675) (6, 11). (Lower) MALDI-MS analysis of DR2-associated self-peptides eluted from 5 Ag purified DR2 molecules from the DM-positive Epstein-Barr virus-transformed B cell line LD2B.

ciation rate of DQw6(43-58), abbreviated as DQw6 peptide, a DR2-associated self-peptide (22, 24), which bound to DR2a with a slow off-rate (tl/2,off 2400 min; Fig. 2). Thus, the intrinsically stable DR2a-DQw6 peptide complex is DMinsensitive, which is reminiscent of the previously described DR1-HA(307-319) complex (16). The finding that the kinetically stable DQw6 self-peptide DQw6(43-58) was resistant to DM-mediated removal, whereas the low-stability CLIP peptide was not is consistent with the hypothesis that DM may function as a peptide editor in the class II loading compartment by skewing the self-peptide repertoire toward stably binding peptides. The Michaelis-Menten Model. Recent data suggested that substoichiometric amounts of natural DM were sufficient to enhance formation of SDS-stable MHC class II a13-peptide complexes in vitro (19), but in another study an excess of recombinant DM was necessary to demonstrate enhanced loading (16). To address the question of whether DM acts as a catalyst, we tested, by using HPSEC, whether DM-mediated peptide loading of DR molecules would follow classical Michaelis-Menten kinetics. According to the kinetic model of Michaelis-Menten (25), the DM-mediated peptide exchange should obey the equation given in Fig. 3A, which can be described as follows: DR-CLIP complexes serve as the substrate for DM, thereby forming an intermediary DM-DRCLIP complex (step I). The association of DM leads to release of CLIP while DM stays bound to DR (step II). The DM-DR complex is formally equivalent to an enzyme-substrate complex. At this stage, rebinding of CLIP is still possible, accounting for the observation that also the binding of CLIP is accelerated by DM (16). However, in the presence of sufficient amounts of other peptides, loading of the latter will be catalyzed (step III). To fulfill the Michaelis-Menten model (25), the formation of the DR-peptide complex has to proceed unidirectionally, meaning that the final DR-peptide complex should not serve as a substrate for DM-facilitated dissociation. Since the DQw6 peptide loaded onto DR2a revealed to be DM-insensitive (Fig. 2), this peptide is suitable to study Michaelis-Menten kinetics. DM-Mediated Loading Follows Michaelis-Menten Kinetics. In general, the rate of catalysis is given by the linear

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FIG. 2. Influence of DM on the dissociation of DR2a-CLIP(81-105) and DR2a-DQw6(43-58) complexes. Half-time of dissociation (tl/2,off) of DR2a-peptide complexes (100 nM) was determined at pH 5.0 and 37°C in the presence and absence of DM (10 nM) as described (10). Error bars indicate the errors from two or three independent experiments.

increase of product formation during the initial phase of the reaction. Thus, the rate of DM-mediated loading of DQw6 peptide onto DR2a was measured by determining the amount of peptide bound to DR2a during a 3-min incubation period in the presence of DM. During the first 3 min of incubation the increase in peptide loading was found to be linear with time and, most importantly, negligible binding of DQw6 peptide to DR2a was observed in the absence of DM (data not shown). Increasing amounts of the substrate, DR2a-CLIP were utilized and three different concentrations of DM as the catalyst were chosen. The measurements were performed at pH 5.0, the optimal pH for DM-mediated loading (16, 17). Double reciprocal plots of initial velocity of loading versus the amount of substrate according to Lineweaver-Burk (26) resulted in linear regression curves (Fig. 3B) with a common intercept at the x axis from which the Michaelis constant KM

750 nM could be

calculated (Table 1). KM is the substrate concentration at which the reaction velocity is half-maximal. In the present case the KM is low compared with naturally occurring enzyme/ substrate combinations (27). Variations in the amount of DM led to corresponding changes in the maximal velocity, Vma, where

a

turnover number of

n

4.5 ± 1.5 DR2a molecules per

minute per DM molecule could be deduced (Table 1). These data clearly demonstrate that DM acts as a catalyst of the peptide loading process attaining turnover numbers that are in the range of low-capacity enzymes such as lysozyme (-30 molecules per min; ref. 27) or protein-disulfide-isomerase (28). According to the reaction scheme of Fig. 3A, one might assume that both DR-CLIP as well as the peptide to be loaded would act as substrates for DM. Bisubstrate models involve binding of both substrates to the enzyme. However, the DQw6 peptide, CLIP, and other peptides did not show binding to purified DM under the assay conditions (data- not shown). Therefore, it is more likely that the peptide participates in the reaction indirectly-namely, by binding to DR. In this case,"the affinity of the peptide-DR interaction, quantitated by the KD value, is an important parameter. In agreement with that view, formation of the DR2a-DQw6 peptide complex, displaying a KD of around 50 nM (data not shown), was catalyzed by DM with the same efficiency, no matter whether 2 ,uM or 20 AM DQw6 peptide were used (Fig. 3B). Thus the DM-mediated catalysis does not appear to follow bisubstrate kinetics. Competitive Inhibition. A characteristic of MichaelisMenten reactions is the finding of competitive inhibition by substrate analogues. Therefore, DR3 isolated from T2 cells, which has been shown to contain 50-70% DR3-CLIP complexes (10, 11), was added to the DR2a/DQw6 peptide binding assay. DR3-CLIP complexes also serve as a substrate for DM as previously shown (19), but loading with peptide does not take place here because the DQw6 peptide does not to bind to DR3 (data not shown). Indeed, addition of DR3-CLIP complexes resulted in a twofold increase of the KM value, whereas

Vma. was unaffected (Fig. 3C and Table 1). This is indicative of competitive inhibition. In contrast, DR3-peptide complexes isolated from a DMpositive B-cell line were found to be only weak competitive inhibitors (Fig. 3C and Table 1), although they were added in higher concentrations than DR3-CLIP. It can be assumed that in DM-positive cells the majority of DR molecules are occupied with stably bound peptides that have been selected by DM. Therefore, these data suggest that DM does not efficiently interact with long-lived DR3-peptide complexes. The small inhibitory effect seen in Fig. 3C can be attributed to DR3-CLIP complexes that have been previously shown to be present in this preparation (10), and to other DM-sensitive DR-peptide complexes. Indeed, mass spectrometric analysis of the self-peptide pool eluted from DR3 after preincubation with DM showed that about 10-20% of the self-peptides were released by DM (H.K., unpublished data). Two important conclusions can be drawn from the inhibition experiments: (i) intrinsically long-lived DR-peptide complexes do not inhibit the action of DM, because they are obviously bad substrates for DM; they probably interact less well with DM; and (ii) DM-sensitive complexes like DR3-CLIP serve as competitive inhibitors of DM-mediated peptide exchange. For the in vivo situation our findings imply that the time span it takes to load a certain class II allele with kinetically stable peptides is not only dependent on the DM concentration and the peptide supply, but also on the amount of other class II alleles in the respective compartment where loading takes place. Kinetic Parameters for Loading of Different Peptides onto DR1 or DR2a. To see if the kinetic parameters of DM catalysis were influenced by the respective DR-peptide combination, loading of DR1 molecules isolated from T2 cells was investigated (Fig. 3D). Like DR2a from T2 cells (see Fig. 1 Upper) the self-peptide profile of DR1 molecules from T2 cells consisted also exclusively of CLIP variants (data not shown). In comparison to DR2a, loading of the DQw6 peptide onto DR1 proceeds

with

a

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turnover number

(n

12).

These data

indicate that the kinetic parameters of the DM catalysis may be influenced by the combination of allele and peptide. However, for the loading of the HA(307-319) peptide, only small differences between DR1 and DR2a were found (Fig. 3D and Table 1). Interestingly, when loading of DR1 with MBP(87-99) was studied, no KM and Vma,, values could be delineated from the Lineweaver-Burk plot (Fig. 3D). This was to be expected according to the Michaelis-Menten model, because the DR1-MBP(87-99) complex formed is known to serve as a substrate for DM-mediated disassembly (16). Similar to CLIP, MBP(87-99) revealed to bind to DR2a with low 30 min) in the absence of DM (data not stability (ti/2,off shown). Therefore, MBP(87-99) is obviously not suitable to study the Michaelis-Menten kinetics of loading.

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FIG. 3. Michaelis-Menten kinetics of DM-mediated peptide exchange. (A) Equation for the DM-mediated peptide exchange. According to the Michaelis-Menten model, DR-CLIP is the substrate that forms a complex with DM (step I). This step of the reaction achieves equilibrium with the educts and with the DM-DR complex that is formed upon dissociation of CLIP (step II). Step III, where peptide binding is accompanied by release of DM, has to proceed unidirectionally, per definition. (B) Effect of DM on the velocity of DQw6(43-58) peptide loading onto DR2a. Binding of the self-peptide DQw6(43-58) (-, *, and A, 2 jiM; or O, 20 jiM) to DR2a (60-600 nM) from T2 cells in the presence of HLA-DM (- and C, 8 nM; *, 20 nM; *, 60 nM) was measured after 3 min of incubation at pH 5.0 and 37°C by HPSEC. Binding in the absence of DM does not exceed background levels with 2 ,iM peptide; in the case of the 20 jiM peptide, the uncatalyzed binding (attaining up to 25% of catalyzed binding) was subtracted. Initial rates of loading were plotted versus the amount of DR2a in a double reciprocal diagram according to Lineweaver-Burk (26). Catalytic parameters as the Michaelis constant, KM, and the maximal velocity of loading, Vmax, were calculated from the intercept of the linear regression curve y = a + bx with the x axis (= -1/KM) and the x axis (= 1 /Vmax), respectively. Values calculated for KM and Vm are given in Table 1. The correlation coefficient (r2) of the linear regression was r2 = 0.990 (8 nM DM), r2 = 0.988 (20 nM DM), and r2 = 0.996 (60 nM DM). (C) Competitive inhibition of peptide loading of DR2a by DR3-CLIP and DR-peptide complexes. Peptide loading of DQw6(43-58) peptide onto DR2a was measured in presence of 8 nM HLA-DM, as described above, in the absence of inhibitor (-), in the presence of 300 nM DR3 from T2 cells (mainly DR3-CLIP) (0), or in the presence of 600 nM DR3 from DM-positive COX cells (mainly DR3-peptide) (O). Correlation coefficients were r2 = 0.990 (no inhibitor), r2 = 0.999 [300 nM DR3 (T2)], and r2 = 0.987 [600 nM DR3 (COX)]. Values calculated for KM and Vm are given in Table 1. (D) DM-mediated loading of various peptides onto DR1 or DR2a from T2. Loading of DQw6(43-58) onto DR1 (-), HA(307-319) onto DR1 (0), HA(307-319) onto DR2a (0), and MBP(87-99) onto DR1 (A) was determined in presence of 20 nM HLA-DM, as described above.

Real-Time Analysis of the DM-DR Interaction. Surface plasmon resonance is a spectroscopic technique which allows the investigation of protein-protein interactions in real time (29). In particular, this technique is suitable for the kinetic analysis of short-lived or low-affinity complexes. To visualize putative DM-DR complexes, recombinant DM molecules were generated in a baculovirus expression system (16), purified, and covalently immobilized on the chip surface of the biosensor. DR-CLIP complexes were then passed over the immobilized DM molecules at pH 5.1, 25°C. As shown in Fig. 4A and B, DR1-CLIP as well as DR2a-CLIP complexes from T2 cells interacted with DM in a dose-dependent manner.

Since the dissociation of the bound DR molecules slow

(tl/2,off

25

min),

a

was

quite

calculation of the kinetic parameters

for the DM-DR interaction was not possible. Obviously, dissociation of DR molecules from DM is not favored at 25°C and pH 5 in the absence of exogenous peptides. Importantly, DR-peptide complexes isolated from DMpositive B-cell lines WT-100 and LD2B revealed to interact less well with DM compared with DR-CLIP complexes (Fig. 4 C and D). These data reinforce the conclusions drawn from the failure of kinetically stable DR-peptide complexes to act as competitive inhibitors of the Michaelis-Menten kinetics (cf. Fig. 3C) and strongly suggest that DM cannot efficiently

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Table 1. Kinetic parameters of peptide loading mediated by DM c(DM), c(inhibitor) KM,* nf,t Vmaxt DR Peptide nM Inhibitor nM nM nM min-1 minDR2a DQw6(43-58) 8 720 47 5.9 DR2a DQw6(43-58) 20 740 85 4.2 DR2a DQw6(43-58) 60 795 183 3.1 DR2a DQw6(43-58) 8 DR3-CLIP 300 1300 45 5.6 DR2a DQw6(43-58) 8 DR3-peptide 600 820 47 5.9 DR2a HA(307-319) 20 600 57 2.8 DR1 20 DQw6(43-58) 1140 246 12.3 DR1 HA(307-319) 20 510 68 3.4 DR1 MBP(87-99) 20 NA§ NA§ NA§ *KM, Michaelis constant, is calculated from the Lineweaver-Burk plot [Fig. 3 B-D, intercept with the x axis (= - 1/KM)] and gives the concentration of DR molecules at which half-maximal velocity of DM catalysis is reached. tWm , maximal velocity, is calculated from the Lineweaver-Burk plot [Fig. 3 B-D; intercept with the y axis (=1/Vmax)]. Maximal velocity of catalysis is extrapolated for infinite concentrations of substrate. *The turnover number (n) denotes the number of DR molecules that are loaded by one DM molecule per minute and is calculated by Vm. divided by the concentration of DM. §NA, not applicable; no KM, Vmax, or turnover number could be calculated for loading of MBP(87-99) onto DR1 (see text).

interact with DR molecules once they are loaded with stably binding peptides. As discussed above, the binding of DRpeptide complexes to DM seen in Fig. 4 C and D can probably be attributed to the presence of DR-CLIP and other low stability DR-peptide complexes in the respective preparations. These findings appear to be at variance with a recent report, in which DM and DR3 could be coprecipitated by the conformation-dependent mAb 16.23 (15). mAb 16.23 is thought to recognize DR3 molecules loaded with peptides different from CLIP, but as discussed by the authors, the specificity of 16.23 is not absolutely clear. Thus it is possible that mAb 16.23 recognizes DM-bound DR3 immediately after CLIP release. Alternatively, in this experiment, 16.23 bound to a DR3 subpopulation loaded with low-stability peptides different from CLIP. Conclusion. The findings presented here demonstrate that HLA-DM acts as a catalyst for peptide loading onto DR molecules. This process followed the classical Michaelis-

Menten model originally set up for enzymes and showed that one DM molecule was able to convert 3 to 12 DR-CLIP complexes into DR-peptide complexes per minute. In agreement with this model, Michaelis constants and turnover numbers could only be determined if the resulting DR-peptide complex was long-lived and therefore refractory to DMmediated peptide release. This model comprises the existence of a transient enzyme-substrate complex leading to enhanced generation of structurally altered substrate molecules that are no longer substrates for the enzyme. The data presented here support the idea that DM prolongs the half-life of an otherwise short-lived conformer of the class II af3 dimer that is favorable for unloading as well as for loading. Most likely this conformer displays a more "open" state of the peptide binding groove. Consequently, all potentially binding peptides are expected to associate with increased efficiency in the presence of DM. This was indeed found in the present study (cf. Fig. 3) and in previous studies (16, 17).

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FIG. 4. Real-time analysis of DM-DR interaction. BIAcore sensorgrams of DR1-CLIP (A) and DR2a-CLIP (B) complexes binding to immobilized soluble DM. Complexes were applied in various concentrations with a 5-min association phase and a 3-min dissociation phase. The actual concentrations are shown on the sensorgrams. All binding curves are expressed as resonance units (RU) as a function of time. (C and D) Interaction of DR-CLIP complexes from T2 cells (T2.DR1, T2.DR2a) with immobilized DM molecules in comparison with DR-peptide complexes isolated from DM-positive B cells (WT-100, LD2B). One out of two to three representative experiments is shown.

Proc. Natl. Acad. Sci. USA 93 (1996)

Immunology: Vogt et al. However, only high-stability peptides with an optimal distribution of anchor and nonanchor side-chains, such as the DQw6 peptide, will stay firmly bound and lock the groove in a "closed" conformation that does not favor DM binding. On the other hand, suboptimal peptides, such as MBP(87-99) or CLIP, which have an intrinsically fast off-rate, are not able to sufficiently stabilize the groove so that the groove may be kept "open" by DM, and the peptides can easily dissociate (Figs. 2 and 3C). In the plasmon resonance experiments shown in Fig. 4, preferentially low-stability DR-CLIP complexes were able to associate with immobilized DM and to act as a competitor in the Michaelis-Menten studies. However, far fewer DRpeptide complexes isolated from DM-positive cells, which can be assumed to contain a sizable fraction of high-stability complexes, were able to bind to DM and function as competitors. Thus, in acidic endosomal/lysosomal compartments DM favors the generation of long-lived class II-peptide complexes for efficient presentation to T cells at the cell surface. We would like to thank R. Pipkorn for synthesis, labeling, and purification of peptides; S. Schmitt for transfecting T2 cells with DR and for expert technical assistance; and D. M. Zaller for providing us with purified recombinant DM.

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