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Originally published In Press as doi:10.1074/jbc.M703436200 on July 11, 2007

J. Biol. Chem., Vol. 282, Issue 37, 26956-26962, September 14, 2007
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Biological Heterogeneity of the Peptide-binding Motif of the 70-kDa Heat Shock Protein by Surface Plasmon Resonance Analysis*

Hideki Maeda{ddagger}§, Hiroeki Sahara1, Yoko Mori, Toshihiko Torigo§, Kenjiro Kamiguchi§, Yutaka Tamura||, Yasuaki Tamura§, Kouichi Hirata{ddagger}, and Noriyuki Sato§

From the Departments of {ddagger}Surgery and §Pathology and the Marine Biomedical Institute, Sapporo Medical University School of Medicine, South 1 West 17, Chuo-ku, Sapporo 060-8556, Japan and the ||Department of Bioinformatics Graduate School of Medicine, Chiba University, 1-8-1, Inohana, Chiba 260-8670, Japan

Received for publication, April 25, 2007 , and in revised form, June 29, 2007.


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
 REFERENCES
 
70-kDa heat shock protein family is a molecular chaperone that binds to a variety of client proteins and peptides in the cytoplasm. Several studies have revealed binding motifs between 70-kDa heat shock protein family and cytoplasmic proteins by conventional techniques such as phage display library screening. However, little is known about the binding motif based on kinetic parameters determined by surface plasmon resonance analysis. We investigated the major inducible cytosolic 70-kDa heat shock protein (Hsp70)-binding motif with the human leukocyte antigen B*2702-derived peptide Bw4 (RENLRIALRY) by using a Biacore system based on surface plasmon resonance analysis. The KD value of Hsp70-Bw4 interaction was 1.8 x 10-6 M. Analyses with truncated Bw4 variant peptides showed the binding motif of Hsp70 to be seven residues, LRIALRY. To further study the characteristics of this motif, 126 peptides derived from Bw4, each with single amino acid substitution, were synthesized and analyzed for Hsp70 binding affinity. Interestingly, the Hsp70 binding affinity was abrogated when the residues were substituted for by acidic (Asp and Glu) ones at any position. In contrast, if the substitute residue was aromatic (Trp, Tyr, and Phe) or an Arg residue at any position, Hsp70 binding affinity was maintained. Thus, this study presents a new binding motif between Hsp70 and peptides derived from the natural protein human leukocyte antigen B*2702 and may also elucidate some characteristics of the Hsp70 binding characteristic, enhancing our understanding of Hsp70-binding determinants that may influence diverse cellular and physiological processes.


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
 REFERENCES
 
Molecular chaperones of the 70-kDa heat shock protein family perform numerous functions in the quality control of cells that mediate protein folding, translocation, assembly/disassembly, and repair of unfolded proteins damaged by environmental stress (1). Chaperone activity of this protein family is necessary to recognize binding sites of a target native protein, referred to as the binding motif. It has been reported that the endoplasmic reticulum 70-kDa heat shock protein Bip (Grp78) binds to a peptide containing at least seven residues with maximal affinity in the presence of ATPase activity (2, 3). It was also suggested that peptide-binding sites of Bip show a preference for sequences rich in aliphatic residues. Subsequently, from a study employing a phage display library, the binding motif for Bip was revealed to be a heptapeptide with high contents of aromatic and hydrophobic residues (4).

On the other hand, the bacterial cytoplasmic 70-kDa heat shock protein homolog DnaK has a substrate-binding region in the C terminus, and repeated binding/release to substrates is dependent on the ATPase cycle at an ATPase domain of the N terminus (5, 6). Zhu et al. (7) reported a crystal structure of the C-terminal substrate-binding domain of DnaK, which was located in a hydrophobic substrate-binding cleft with a central pocket placed between a beta sheet and two {alpha} helices. The consensus motif recognized by DnaK consisted of peptides comprising a hydrophobic core of 4–5 residues flanked by basic residues (8). Moreover, in virus-infected cells, it is well known that a 60-kDa heat shock protein (Hsp60)2 and a 90-kDa heat shock protein (Hsp90) bind to hepatitis B virus-derived proteins such as polymerase and transcriptase (9, 10). Recently, it was reported that the host major inducible cytosolic 70-kDa heat shock protein (Hsp70)-binding motif interacted with a heptapeptide from a nucleoprotein of measles virus (11) and stimulated transcriptional activities of this virus (12, 13). Thus, the 70-kDa heat shock protein family is capable of recognizing and binding not only to the structural region of a protein but also to a specific localized peptide motif.

Although 70-kDa heat shock protein family-binding motifs derived from observations of interactions with its many substrate proteins have been proposed, little is known about the binding motif based on kinetic parameters determined by surface plasmon resonance (SPR) analysis. Because target molecules basically bind to 70-kDa heat shock protein in low affinity interactions, it might be difficult to measure binding interaction by conventional methods such as competitive immunoprecipitation.

The use of SPR technology, on which bimolecular interaction analysis (Biacore) is based, provides us with the most expedient approach for measurement of low affinity protein interactions in real time. With the Biacore system, one interaction partner is conjugated on the surface of a sensor chip (ligand), and other binding partners flow over the surface (analyte), facilitating analysis of binding differences between multiple analytes and a single ligand. In this study, we investigated the interaction of Hsp70 with the HLA-B*2702-derived peptide Bw4 (RENLRIALRY, HLA-B*2702 amino acids 75–84) and its various truncated and substituted peptides using a Biacore system. Nössner et al. (14) reported that Bw4 can bind to Hsp70 and 70-kDa heat shock cognate protein (Hsc70) and elicit a suppressive effect on T cell function. Because the Hsp70-binding motif basically is a heptapeptide, the aim of this study was to find a new Hsp70-binding motif and its biologic characteristics, if present, using the Biacore system.

Consequently, we demonstrated that Hsp70 bound to Bw4 with KD = 1.8 x 10-6 M. Using truncated and Ala-substituted variants of Bw4, the Hsp70-binding motif of Bw4 was newly found to be heptapeptide LRIALRY. Moreover, we found that if residues in this motif were substituted for by an aromatic (Trp, Tyr, and Phe) or Arg residue at any position, Hsp70 binding affinity was maintained, whereas with acidic residue substitutions, binding affinity to Hsp70 was markedly decreased. The present study suggests a new binding motif between human Hsp70 and peptides derived from natural protein HLA-B*2702 and also discloses characteristics of the Hsp70-binding motif structure. Our findings may contribute to enhanced understanding of 70-kDa heat shock protein family binding determinants that may influence diverse cellular and physiological processes.


   EXPERIMENTAL PROCEDURES
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
RESULTS
 DISCUSSION
 REFERENCES
 
70-kDa Heat Shock Proteins and Synthetic Peptides—Recombinant human Hsp70 protein (NSP-555), the endoplasmic reticulum 70-kDa heat shock protein Bip (Grp78) (SPP-765), and Escherichia coli DnaK protein (SPP-630) were purchased from StressGen (Victoria, Canada). All of the peptides were synthesized by Sigma-Aldrich. The peptides were dissolved with 5% Me2SO (Merck) in phosphate-buffered saline at the indicated concentrations in each experiment.

Surface Plasmon Resonance Analysis using the Biacore System—SPR experiments were performed with a Biacore 3000 system (Biacore, Inc., Uppsala Sweden). With this system, molecules of interest (ligands) are immobilized on a sensor surface, and binding partners (analytes) can then be passed over it in a mobile aqueous phase. Their interaction on the sensor surface can subsequently be monitored in real time without the use of labels. To investigate Hsp70-, Bip-, and DnaK-peptide interactions, we employed Hsp70, Bip, and DnaK proteins as ligands that were immobilized on the sensor surface, and Bw4 (RENLRIALRY, HLA-B*2702 amino acids 75–84) and Bw6 (RESLRNLRGY, HLA-B*0701 amino acids 75–84) peptides as the analytes. It is known that Bw4, but not Bw6, can bind to Hsp70/Hsc70 and elicit a suppressive effect on T cells (14). In addition, truncated and amino acid-substituted peptides of Bw4 also were used as analytes.

Hsp70, Bip, and DnaK were immobilized by amine coupling methods, according to the instruction manual for the Biacore 3000, which utilizes a primary amino group of proteins for covalent attachment to the matrix. Briefly, a dextran layer on a sensor chip that is covalently attached to a carboxymethylated dextran gold surface (CM5 sensor chip; Biacore, Inc.) was activated by injection of a mixture of N-hydroxysuccinmide and carbodiimide, creating a reactive ester on the surface for 10 min. Subsequently, 100 µl of 20 µg/ml ligand in 10 mM sodium acetate (pH 5.5) was injected for 10 min and loaded with 1 M ethanolamine for 10 min to block binding to remaining nonreacted ester groups. Finally, the chip surface was washed with running buffer (5% Me2SO, 2.5 mM magnesium acetate, 2.5 mM ATP (disodium salt; Sigma) in phosphate-buffered saline) to remove any loosely bound ligands. The amounts of immobilized Hsp70, Bip, and DnaK as ligands were 19,000, 14,347, and 7,843 resonance units (RU), respectively, corresponding to sensor surface areas of ~19, 14.3, and 7.8 ng/mm2, respectively.

To estimate these ligand-peptide interactions, the affinity of the interaction, i.e. the equilibrium dissociation constant (KD), was determined from the level of binding at equilibrium as a function of the sample concentrations by BIAevalution version 4.1 software (Biacore, Inc.). All of the peptides were dissolved with running buffer including 5% Me2SO, and binding experiments were performed at 25 °C in running buffer with a flow rate of 20 µl/min. To reduce errors in reference subtraction, solvent effects were compensated for by a Me2SO calibration procedure according to the BIAcore manufacturer's instructions. All of the peptide responses were corrected for Me2SO bulk differences via the Me2SO calibration curves that were obtained by the Me2SO calibration procedures performed at the beginning and end of each experiment.


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
DISCUSSION
 REFERENCES
 
The Biacore assay based on SPR was used to monitor the Hsp70-, Bip-, and DnaK-peptide interactions. SPR detects molecular interactions because there is a corresponding change in the refractive index if binding occurs as a peptide passes over a prepared sensor surface. The responses on the sensorgram are designated RU. We initially attempted to estimate the binding affinity between Hsp70 and the Bw4 or Bw6 peptide. Each peptide was injected at a concentration of 50 µM for 2 min at a flow rate of 20 µl/min to detect the association phase and loaded with running buffer alone on the sensor chip for 2 min at a flow rate of 20 µl/min to detect the dissociation phase. As shown in Fig. 1A, the immobilized Hsp70 on the sensor chip bound to Bw4 peptide, whereas Bw6 exhibited low binding compared with the Hsp70-Bw4 interaction. Bw4 binding to Hsp70 tended to be extremely fast, reaching equilibrium immediately after Bw4 peptide injection, and the complex dissociated just as quickly when the flow was switched to a running buffer, with RU returning to the basal level. Therefore, it was difficult to calculate the kinetic parameters (Ka and Kd) directly. The affinity of interaction was determined from the level of binding at equilibrium as a function of the sample concentration, which is referred to as the equilibrium dissociation constant (KD).

We attempted to estimate the binding affinity in the Hsp70-Bw4 interaction. Bw4 peptide in concentrations ranging from 0.1 to 12.5 µM was injected over an Hsp70 sensor chip for 2 min at a flow rate of 20 µl/min and loaded with running buffer alone for 2 min at the same flow rate. Association and dissociation phases of the Hsp70-Bw4 interaction at each concentration were shown to occur in a dose-dependent manner (Fig. 1B). Subsequently, kinetic analysis based on these data were performed, and the equilibrium dissociation constant (KD) was determined using BIAevalution (version 4.1) software. As shown in Fig. 1C, a specific binding response between Hsp70 and Bw4 was observed, and the estimated KD for binding was 1.8 x 10-6 M. On the other hand, as shown in Fig. 1D, the estimated KD for binding between Hsp70 and Bw6 in the same manner was found to be 4.12 x 10-3 M, indicating that this binding affinity was ~440-fold lower than the Hsp70-Bw4 interaction.


Figure 1
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  		FIGURE 1.
The binding of human Hsp70 to peptides Bw4 and Bw6. A, Biacore 3000 sensorgrams illustrating binding of synthetic peptides Bw4 and Bw6 to immobilized Hsp70. Each 50 µM peptide solution was passed over sensor chips on which 19,000 RU of Hsp70 was immobilized. Retention of a peptide on a sensor chip was indicated by a change in RU over the course of the 200-s injection interval. B, overlay plot of sensorgrams of interaction of Bw4 peptide with Hsp70. Bw4 peptides in concentrations (Conc) of 0.1, 0.2, 0.39, 0.78, 1.56, 3.13, 6.25, and 12.5 µM were injected over the Hsp70 sensor chip, and the magnitude of Bw4 binding to immobilized Hsp70 was analyzed. C and D, a kinetic plot and binding isotherm for binding of Bw4 or Bw6 to the Hsp70 sensor chip in peptide concentrations ranging from 0.1 to 12.5 µM. Binding affinity was determined from the level of binding at equilibrium as a function of the sample concentration using BIAevaluation version 4.1 software and referred to as the equilibrium dissociation constant (KD).

 
We also tried to determine whether peptide binding affinity to isotypes of the 70-kDa heat shock protein family such as mammalian endoplasmic reticulum-resident Bip and prokaryotic DnaK molecules was different from Hsp70-Bw4 or -Bw6 interactions. As shown in Fig. 2 (A and B), it was somewhat surprising that the estimated KD values for binding of Bw4 to Bip and DnaK were 3.72 x 10-3 M and 1.85 x 10-4 M, respectively, indicating that Bip and DnaK had low binding affinity as compared with Hsp70. In terms of interactions with Bw6, the estimated KD values for binding to Bip and DnaK were 9.0 x 10-4 M and 4.83 x 10-3 M, respectively (Fig. 2, C and D).

To investigate characteristics of the binding motif of Hsp70-Bw4 interaction, truncated Bw4 variant peptides were employed. The truncated Bw4 peptides presented in Table 1 were injected over an Hsp70 sensor chip in a similar manner. Truncated 9-, 8-, and 7-mer Bw4 peptides from which N-terminal Arg, Arg-Glu, and Arg-Glu-Asn residues were deleted, respectively, interacted with Hsp70 with high affinity (KD < 5 x 10-6 M), as did the wild type, but 6-mer (or fewer) peptides could not bind (KD > 1 x 10-3 M) to Hsp70 (Table 1). In contrast, truncated Bw4 peptides with deletion of only a C-terminal Tyr did not show interaction with Hsp70 (KD > 1 x 10-3 M), indicating that a C-terminal Tyr residue was critical for binding to Hsp70 and that the end of the binding motif of the C terminus seemed to be Tyr-84 of HLA-B*2702. Therefore, the Hsp70-binding motif interacting with Bw4 was 7-mer amino acid residues, LRIALRY, designated N3-Bw4. We also determined the KD value of this peptide. As shown in Fig. 3, the KD for binding between Hsp70 and N3-Bw4 peptide was estimated to be 2.6 x 10-6 M.


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  		TABLE 1
Binding assay of truncated Bw4 peptides

 
Next, key amino acid residues in the motif of Bw4 binding to Hsp70 were studied using synthetic alanine-substituted peptides. Nine of these peptides were loaded over an Hsp70 sensor chip as above. We found that R1A, E2A, and N3A peptides that substituted for alanine at Arg-1, Glu-2, and Asn-3, respectively, maintained high affinities (KD < 5 x 10-6 M), like the wild type (Table 2), indicating that three N-terminal residues without binding motifs could not influence Hsp70 binding affinity. However, other alanine substitutions at L4A, R5A, I6A, L8A, R9A, and Y10A resulted in loss of binding (KD > 1 x 10-3 M) to Hsp70 (Table 2). Thus, it was demonstrated that each amino acid residue in the binding motif LRIALRY was critical for binding of Hsp70-Bw4 peptides.


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  		TABLE 2
Binding assay of Ala-substituted peptides

 
Subsequently, characteristics of the Hsp70-peptide-binding motif were investigated using a peptide array. An array of single amino acid-substituted peptides, which consisted of 126 peptides, of Bw4 wild peptide (10-mer peptide), was synthesized. For example, in the case of substituted peptides at position 8 of the N terminus, a Leu residue was substituted for by 18 amino acid residues: L8W, -Y, -F, -R, -K, -H, -M, -I, -V, -T, -A, -Q, -G, -P, -N, -S, -D, and -E. Cys substitutions were not performed in this study to avoid multimerization among substituted peptides by S-S binding. All of the peptides were loaded over an Hsp70 sensor chip in a similar manner. As shown in Fig. 4, single amino acid-substituted peptides in white squares represent the high affinity with Hsp70 (KD < 5 x 10-6 M), whereas peptides in blue squares represent decreased binding affinity (KD > 1 x 10-3 M). Peptides, except for R5Y, with aromatic residues (Trp, Tyr, and Phe) and Arg substitution showed high affinity with Hsp70, like the wild type, at any position, whereas acidic residue (Asp and Glu)-substituted peptides lost their binding affinity (KD > 1 x 10-3 M).


Figure 2
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  		FIGURE 2.
A kinetic plot and binding isotherm for binding of Bw4 to Bip (A) and DnaK (B) sensor chips and of Bw6 to Bip (C) and DnaK (D) sensor chips in concentrations ranging from 0.1 to 12.5 µM. The KD value was determined using BIAevaluation version 4.1. Conc, concentration.

 


Figure 3
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  		FIGURE 3.
A kinetic plot and binding isotherm for binding of the 7-mer peptide variant derived from Bw4 to the Hsp70 sensor chip in concentrations ranging from 0.1 to 12.5 µM. The KD value was determined using BIAevaluation version 4.1. Conc, concentration.

 
The estimated KD values for binding between the Hsp70 and five peptides with aromatic Ile or Arg substituted at position 8 are shown in Fig. 5. These peptides in concentrations ranging from 0.1 to 12.5 µM, were injected over an Hsp70 sensor chip for 2 min at a flow rate of 20 µl/min and loaded with running buffer alone for 2 min at the same flow rate. Specific binding affinity interaction between Hsp70 and these peptides was observed, and the estimated KD values for binding of L8Y, -W, -F, -R, and -I were 6.7, 1.1, 1.4, 1.4, and 1.8 x 10-6 M, respectively, indicating binding affinity mostly similar to the wild type. Thus, as shown in Fig. 6, it appears that aromatic and Arg residues are favored residues for interaction with Hsp70, whereas acidic residues are not favored.


   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
REFERENCES
 
The present study demonstrated, by SPR analysis, a new HLA-derived heptapeptide-binding motif, LRIALRY, interacting with human Hsp70. Nössner et al. (14) reported that the Bw4 peptide bound to Hsp70/Hsc70 and elicited a suppressive effect on T cell function. Bw4, consisting of a 10-mer peptide, is derived from the most polymorphic region in the {alpha}1 domain of HLA-B*2702, where it is important for self and nonself recognition by T cell receptors. We focused on the interactions of human Hsp70 with native Bw4 and its truncated and amino acid-substituted variants, because peptides bound to Hsp70 are postulated to have immunomodulatory effects such as T cell tolerance or activation in transplantation and tumor immunity. For example, it was reported that the 70-kDa heat shock proteins, Hsp70/Hsc70 bound to HLA-DRB1-derived peptide sequences comprising the shared epitope, such as HLA-DRB1*040165–77(KDLLEQKRAAVDT) and -DRB1*010165–77(KDLLEQRRAAVDT), which are considered to be associated with an increased risk for rheumatoid arthritis. This might be attributed to the enhanced activation of CD4+ T cells against Hsp70/Hsc70-chaperoned antigen peptides via autophagy (15–17). Thus, the interaction between 70-kDa heat shock protein family and HLA molecules seems to provide some biological insights (18–21).

In searching for the peptide-binding motifs for the 70-kDa heat shock protein family, it was first reported that an endoplasmic reticulum protein, Bip, could bind to peptides containing at least seven residues with maximal affinity in the presence of ATPase activity (2, 3). It was also indicated that the peptide-binding site of Bip showed a preference for sequences rich in aliphatic residues. Subsequently, from a study employing phage display library screening, the binding motif for Bip was revealed to be a heptapeptide with high contents of aromatic and hydrophobic residues (4). In the heptapeptide sequence, it was suggested that aromatic and hydrophobic residues occupied alternating positions. On the other hand, bacterial cytoplasmic 70-kDa heat shock protein homolog DnaK has a substrate-binding region in the C terminus, and repeated binding/release to substrates is dependent on the ATPase cycle at the ATPase domain of the N terminus (5, 6). Zhu et al. (7) reported the crystal structure of the C-terminal substrate-binding domain of DnaK, which was located in a hydrophobic substrate-binding cleft with a central pocket placed between a beta sheet and two {alpha} helices. Basically, this domain is considered to bind to substrates by making hydrogen bonds (22). In addition, it was reported that a consensus motif recognized by DnaK consisted of peptides comprising a hydrophobic core of 4–5 residues flanked by basic residues (8). In the current study, the binding motif of the HLA-derived protein LRIALRY consisted of a hydrophobic core of 3 residues flanked by basic Arg residues.


Figure 4
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  		FIGURE 4.
Illustration of the Hsp70-peptide-binding motif. An array of 126 single amino acid-substituted peptides was synthesized based on the Bw4 sequence (10-mer peptide). For example, in the case of substituted peptides at position 8 of the N terminus, Leu was substituted for by 18 different amino acid residues. These are represented as L8W, -Y, -F, -R, -K, -H, -M, -I, -V, -T, -A, -G, -P, -N, -S, -Q, -D, and -E in this figure. White blocks represent amino acids with preserved binding affinity for Hsp70 interaction (KD < 5 x 10-6 M), whereas blue blocks indicate loss of binding affinity (KD > 1 x 10-3 M). The blocks with asterisks indicate sequential peptides the same as the wild type.

 


Figure 5
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  		FIGURE 5.
A kinetic plot and binding isotherm for binding of five substituted peptides: L8Y (A), L8W (B), L8F (C), L8R (D), and L8I (E) to on Hsp70 sensor chip in concentrations ranging from 0.1 to 12.5 µM. The KD value was determined using BIAevaluation version 4.1. Conc, concentration.

 
Although such characteristics appeared to be similar to the consensus motif of DnaK, it was interesting that DnaK-Bw4 interaction was ~500-fold lower than Hsp70-Bw4 interaction. The substrate-binding domain of Hsp70 shares ~50% homology in its amino acid sequence with DnaK (18, 23). These structural differences might result in differences of binding affinity. For example, Fourie et al. (24) investigated the binding of 36 peptides to Hsc70, Bip, and DnaK by competition assay, demonstrating that there was little common peptide bound to these three proteins with similar binding affinity. It was also reported that mutant p53 protein-derived V10 peptide (substitution of Cys-126 by Tyr) was bound to Bip and DnaK but that V10-Hsc70 interaction was weaker than Bip and DnaK interaction (24). In our Biacore study, this appeared to be true, because the KD values for binding of Bip and DnaK to V10 peptide were estimated to be 4.28 x 10-5 M and 2.26 x 10-5 M, respectively. In contrast, that of Hsp70-V10 peptide was markedly lower (KD > 1 x 10-3 M) than the Bip-and DnaK-V10 interaction (data not shown), which was consistent with their data (24). It is known that Hsp70/Hsc70 have very similar biochemical functions and are highly homologous proteins in their primary amino acid sequence except for a short heterologous C-terminal portion with an unknown biochemical function (18, 23, 25–27). Therefore, although we did not confirm the interaction between Hsc70 and V10 peptide, it is highly likely that this interaction could occur similarly to that of Hsp70 and V10 peptide in our current Biacore system.


Figure 6
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  		FIGURE 6.
Schematic illustration of the peptide-binding motif to human Hsp70. Aromatic and Arg residues are substitutable residues that could preserve high affinity to Hsp70, suggesting that they are favored residues. In contrast, substitutions with acidic residues resulted in loss of binding affinity, suggesting that they are disfavored residues.

 
In the peptide array experiments, when a residue in the LRIALRY motif was substituted for by an acidic residue, the binding affinity to Hsp70 was reduced in all cases. In the case of DnaK, acidic residues were excluded from cores and were found to be disfavored in the flanking region when 4360 peptides derived from 37 biologically relevant proteins were screened (8). In addition, Zhang and Oglesbee (13) reported similar results in which substitutions with acidic residues markedly reduced binding affinity to Hsp70. Thus, acidic residues appear to be disfavored in binding to the human 70-kDa heat shock protein family.

Meanwhile, it was reported that Hsp70 and Hsp90 could transfer peptides produced by the ubiquitin-proteasome system from the cytoplasmic milieu to the endoplasmic reticulum via transporters associated with antigen processing but not by natural diffusion (28, 29). These peptides were then bound to an endoplasmic reticulum-resident chaperone, GP96, and loaded onto major histocompatibility complex class I molecules in the endoplasmic reticulum (30–32). It was also reported that peptides bound to Hsp70 in tumor cells were identified as tumor antigens and that the purified tumor-derived Hsp70-peptide complex elicited tumor-specific cytotoxic T cell immunity in vitro and in vivo (33–36). We showed here that the new Hsp70-binding motif was a heptapeptide, LRIALRY. Interestingly, in this motif, aromatic residues or an Arg residue could be substituted at any position. These data might help elucidate the interaction of this motif with tumor antigens and suggest the possibility of construction of immunomodulatory cancer vaccines (35, 36).

Our study may also have several biologic implications. When we searched for homologous proteins with the Swiss-Prot data base, there were many such proteins with the same sequence in their primary amino acids as the LRIALRY motif. The majority of these proteins included HLA class I heavy chain molecules such as A*23, A*24, A*25, A*32, B*38, B*49, B*51, B*52, B*53, B*57, B*58, and B*59. When we extended the search for homologous sequences of proteins with residues favored in the motif as illustrated in Fig. 6, there were many human housekeeping proteins such as Hsp40 homologs, {alpha} adrenergic receptors, adenylate cyclase, protein ariadne-2, collagen {alpha}-3 chain receptor, acetyl-CoA carboxylases, HLA class IIDQ4, DQ-DRW9, tyrosine-protein kinase JAK2, and many other proteins, and the number of such molecules was at least more than 317. In case of Hsp40 homologs among these 317 molecules, recent studies suggested that these protein-derived peptides efficiently activated CD4+CD25+ regulatory T cells (37–39). Therefore, one explanation for these data is that successful induction of immunosuppressive regulatory T cells might be attributable to Hsp40-derived peptides that could bind to Hsp70 with high affinity. Although the biologic significance of the current peptide-binding motif to Hsp70 is not yet clear, one immunological speculation is intriguing. There is a possibility that Hsp70-chaperoned peptides with our current motif may preferentially enter into the endoplasmic reticulum, make complexes with peptide-HLA class I, and subsequently be expressed on the cell surface. If these interactions happen in the thymus, our current observations might be compatible with the efficient induction of the negative selection of T cells, because peptides with this motif can be derived from not only self HLA class I heavy chains themselves but also many sets of self-housekeeping proteins.


   FOOTNOTES
 
* This work was supported by Grants-in-aid for Scientific Research 16209013 and 15659097 from the Ministry of Education, Culture and Science of Japan (to N. S.). 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. Back

1 To whom correspondence should be addressed: South 1 West 17, Chuo-ku, Sapporo 060-8556, Japan. Tel.: 81-11-611-2111; Fax: 81-11-612-5861; E-mail: hsahara{at}sapmed.ac.jp.

2 The abbreviations used are: Hsp, heat shock protein; Hsc, heat shock cognate protein; SPR, surface plasmon resonance; HLA, human leukocyte antigen; RU, resonance unit. Back


   ACKNOWLEDGMENTS
 
We thank Drs. Fumio Sugawara, Satoko Aoki, Mihoko Takami, Takeki Saito, and Yoichi Takakusaki of Science University of Tokyo for helpful assistance in this study.



   REFERENCES
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
 REFERENCES
 

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