s2fieldsofstudy: Biology Chemistry

text:
A novel factor required for the assembly of the DnaK and DnaJ chaperones of Thermus thermophilus.

We previously reported the isolation of T.DnaK·DnaJ chaperone complex from Thermus thermophilus. Here, we show that a novel factor is necessary for the assembly of T.DnaK and T.DnaJ into the complex. A dnaK gene cluster of T. thermophilus contained five genes, dnaK-grpE-dnaJ-orf4-clpB. Interestingly, T.DnaJ lacks the whole “cysteine-rich region” that has been postulated to be necessary to bind unfolded proteins. The orf4 gene encodes a novel 78-amino acid protein. Curiously, T.DnaK and T.DnaJ expressed in Escherichia coli did not form the complex. Careful reexamination of the T.DnaK·DnaJ complex revealed the presence of a small protein in the complex, which turned out to be a product of orf4. As expected, expression of three genes, dnaK-dnaJ-orf4, resulted in production of a T.DnaK·DnaJ complex in E. coli that was indistinguishable from the authentic complex in its ability to interact with nucleotide and denatured protein. The product of orf4 was also required for in vitro reconstitution of the complex and named T.DafA (T.DnaK·DnaJ assembly factor A). The complex comprises three copies each of T.DnaK, T.DnaJ, and T.DafA. Even though a definite homolog of T.DafA has not been found in the data base, this finding raises a possibility that interaction between DnaK and DnaJ chaperones in other organisms is also mediated by a small protein yet unnoticed.

We previously reported the isolation of T.DnaK⅐DnaJ chaperone complex from Thermus thermophilus. Here, we show that a novel factor is necessary for the assembly of T.DnaK and T.DnaJ into the complex. A dnaK gene cluster of T. thermophilus contained five genes, dnaK-grpE-dnaJ-orf4-clpB. Interestingly, T.DnaJ lacks the whole "cysteine-rich region" that has been postulated to be necessary to bind unfolded proteins. The orf4 gene encodes a novel 78-amino acid protein. Curiously, T.DnaK and T.DnaJ expressed in Escherichia coli did not form the complex. Careful reexamination of the T.DnaK⅐DnaJ complex revealed the presence of a small protein in the complex, which turned out to be a product of orf4. As expected, expression of three genes, dnaK-dnaJ-orf4, resulted in production of a T.DnaK⅐DnaJ complex in E. coli that was indistinguishable from the authentic complex in its ability to interact with nucleotide and denatured protein. The product of orf4 was also required for in vitro reconstitution of the complex and named T.DafA (T.DnaK⅐DnaJ assembly factor A). The complex comprises three copies each of T.DnaK, T.DnaJ, and T.DafA. Even though a definite homolog of T.DafA has not been found in the data base, this finding raises a possibility that interaction between DnaK and DnaJ chaperones in other organisms is also mediated by a small protein yet unnoticed.
The ubiquitous Hsp70 stress proteins are major members of the chaperone system, which assists protein folding in the cell (1)(2)(3)(4). The Escherichia coli Hsp70 homolog DnaK participates in widely different processes of protein biogenesis such as reactivation of heat-inactivated proteins (5)(6)(7), refolding of denatured proteins (8,9), and degradation of aberrant proteins (10 -12). A general feature of this chaperone activity is the ability of DnaK to recognize nonnative conformations of other proteins and to release them in an ATP-regulated manner. The ATPase activity of DnaK is under tight control of cofactors allowing coordination of ATP binding/hydrolysis and substrate protein interaction (8,13). The weak ATPase of DnaK is stimulated by DnaJ (41 kDa), which accelerates the rate of ATP hydrolysis, and by GrpE (22 kDa), which promotes nucleotides exchange of ATP and ADP (14). DnaJ also possesses a molec-ular chaperone activity of its own, as revealed by its capacity for binding to denatured proteins to prevent aggregation (9,15). DnaJ and GrpE are essential for chaperone activity of DnaK in vivo and in vitro. For these cofactors to assist DnaK function, they should physically interact each other and, in fact, GrpE can associate with DnaK to form a relatively stable complex in an ATP-dependent manner (16 -18). Interaction between DnaK and DnaJ has been also suggested from experiments of glutaraldehyde cross-linking (19) and immunological detection (20); the formation of a high molecular weight ternary complex of DnaK, DnaJ, and a folding intermediate has been reported (8,15). Nevertheless, attempt to isolate a stable complex of DnaK and DnaJ has been unsuccessful, and physical and chemical aspects of the interaction between DnaK and DnaJ have remained unclear.
DnaK is purified from E. coli cells as monomer with a small amount of dimer or higher oligomer (18,21,22). Both DnaJ and GrpE are purified separately as dimer (18,23). In contrast to the E. coli counterparts, T.DnaK 1 from a thermophilic bacterium, Thermus thermophilus, is isolated as a trigonal ring particle with an approximate molecular size of 300 kDa in which three copies of T.DnaK and T.DnaJ are contained (24). This T.DnaK⅐DnaJ complex is stable during incubation with MgAT(D)P and can bind reduced carboxymethylated ␣-lactalbumin, a model protein of denatured state. Here, we report that the T.DnaK⅐DnaJ complex contains additional three copies of a novel small protein whose presence was not noticed previously. This protein is required for the assembly of T.DnaK and T.D-naJ into a complex and is a product of an adjacent gene of dnaJ.

EXPERIMENTAL PROCEDURES
Bacterial Strains, Plasmids, Media, and Growth Conditions-T. thermophilus strain HB8 (ATCC 27634) was used as a source of genomic DNA. E. coli strains used here were JM109 for preparation of plasmids (25), CJ236 for generating uracil-containing single-stranded DNA for site-directed mutagenesis (26) and BL21 (DE3) pLysS for gene expression of plasmids carrying T7 promoter (27). Plasmid pBlue-scriptII KS(Ϫ) (Stratagene) tailed with dideoxythymidine triphosphate using terminal transferase (28) was used for subcloning of polymerase chain reaction (PCR) 2 products. Plasmid pUC18 (25) was served as a vector for the construction of genomic DNA library and the DNA sequence of T. thermophilus gene. Plasmid pUC118 and helper phage M13KO7 (29) were used for generating uracil-containing singlestranded DNA. Plasmids pET3a (Novagen) were used for construction of expression system. T. thermophilus was grown aerobically at 75°C in a medium containing 10 g of yeast extract, 10 g of polypeptone, and 2 g of NaCl/liter (30). E. coli was grown aerobically at 37°C in 2 ϫ YT * 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.
The nucleotide sequence(s) reported in this paper has been submitted to the GenBank TM /EBI Data Bank with accession number(s) D84222.
‡ Recipient of a research fellowship of the Japanese Society for the Promotion of Science for Young Scientists.
Polymerase Chain Reaction-Mixed oligonucleotide primers used for PCR amplification were 5Ј-GCCAAGGCCGT(C/G)GG(C/G)AT(C/ T)GA-3Ј and 5Ј-ATGGCGTAGTAGTCCTTCTT-3Ј. PCR were performed using either 2 g of chromosomal DNA as a template in a 100 l of PCR mixture (10 mM Tris-HCl, pH 8.3, 50 mM KCl, 1.5 mM MgCl 2 , 0.001% (w/v) gelatin, 200 M each of dNTP) containing 1.0 M of each primer and 2.5 units of recombinant Taq DNA polymerase (Takara). Samples were subjected to 30 cycles of 1 min of denaturation at 98°C, 0.5 min of annealing at 55°C, and 2 min of elongation at 72°C.
DNA Isolation and Cloning and Sequencing of T.dnaK Region-DNA was manipulated by standard methods (31) and chromosomal DNA from T. thermophilus was isolated as described previously (32). Southern blot hybridization (33) to total chromosomal DNA from T. thermophilus was performed using the PCR products as probes, and a 5.5-kbp BamHI fragment of the chromosomal DNA was detected. T. thermophilus chromosomal DNA was digested with BamHI, DNA fragments with about 5.5 kbp were cut out, and clones containing dnaK gene were selected by colony hybridization using the probe. The probe was labeled and detected by ECL™ random prime labeling and detection system (Amersham Corp.). The total DNA sequences of both strands were determined by the dideoxy chain termination method (34) using a fluorescent DNA sequencer (ALF™ DNA sequencer II, Pharmacia Biotech Inc.).

Construction of Expression System for T.DnaK⅐DnaJ Complex-
The construction for the expression plasmid of the T.DnaK⅐DnaJ complex was performed as follows. The pMKJ1 containing a NspI fragment of the dnaK-grpE-dnaJ gene, inserted into SphI site of the pUC18, was digested with a restriction enzyme KpnI, and a 2.3-kbp KpnI fragment containing The dnaK gene was subcloned into KpnI site of the pUC118 (pMDK1-1). In the case of the dnaJ gene, pMKJ1 was digested with KpnI-HindIII and a 2.2-kbp KpnI-HindIII fragment containing dnaJ gene was subcloned into KpnI-HindIII sites of the pUC118 (pMDJ1-1). In order to construct the expression system, a NdeI site was introduced to the translational initiation region of dnaK and dnaJ genes in pMDK1-1 and pMDJ1-1 by the method of Kunkel et al. (26) using synthetic oligonucleotides 5Ј-CTTGGCCATATGAATTCACCTCGC-3Ј (dnaK) and 5Ј-GGCCGCCATATGGAATTCCAGGTCCG-3Ј (dnaJ). Changed bases are underlined. The dnaK gene carrying a NdeI site in the translational initiation region was digested with NdeI-HindIII and was inframed with the corresponding NdeI-HindIII sites of the pET3a, and it was named the pMDK6 (dnaK). The dnaJ gene carrying a NdeI site in the translational initiation region were digested with NdeI-HindIII or NdeI-MscI, and each of the fragments was inframed with the corresponding NdeI-HindIII or NdeI-BamHI (blunted end) sites of the pET3a. They were named as pMDJ8 (dnaJ and dafA) and pMDJ10 (dnaJ), respectively. The dnaK-dnaJ expression system was constructed from pMDK6, pMDJ8, and pMDJ10. pMKJ8 was constructed from pMDK6 digested with BamHI-HindIII and pMDJ8 with BglII-HindIII. The pMDJ8 was digested with BglII-HindIII and a 2.3-kbp BglII-HindIII fragment containing the dnaJ-dafA gene was inframed with the corresponding BamHI-HindIII sites of the pMDK6 (pMKJ8). The pMKJ7 was constructed from pMDK6 digested with BamHI-Hin-dIII and pMDJ10 with BglII-HindIII. The pMDJ10 was digested with BglII-HindIII and a 1.3-kbp BglII-HindIII fragment containing the dnaJ gene was inframed with the corresponding BamHI-HindIII sites of the pMDK6 (pMKJ7).
Purification and Characterization of the T.DnaK⅐DnaJ Complex-The T.DnaK⅐DnaJ complex expressed in E. coli was purified as described previously (24) with the following modifications. A hydroxyapatite column was omitted, a gel filtration column Sepharose CL-6B (Pharmacia) equilibrated with buffer A (25 mM Tris-HCl, pH 7.5, 3 mM MgCl 2 , 100 mM Na 2 SO 4 ) was used instead of TSK G3000SWXL (Tosoh). ATPase activities were assayed at 80°C in a 100-l reaction mixture containing 25 mM Tris-HCl, pH 7.5, 5 mM MgCl 2 , 50 mM KCl, 0.5 mM ATP, and 0.7 M T.DnaK⅐DnaJ complex (as a 300-kDa complex). The reaction was initiated by addition of ATP, and terminated after a 20-min incubation by addition of 15 l of 20% perchloric acid. As a control experiment (0% ATPase activity), T.DnaK⅐DnaJ complex was injected into the reaction mixture containing perchloric acid. Mixtures were centrifuged at 16,000 rpm for 3 min at 4°C, and released P i in the supernatant was measured by malachite green assay (35,36). Interaction of the T.DnaK⅐DnaJ complex with reduced carboxymethylated ␣-lactalbumin was examined by gel filtration HPLC, and nucleotides bound to the T.DnaK⅐DnaJ complex were analyzed by reverse phase HPLC as described previously (24). in vitro reconstitution of the T.DnaK⅐DnaJ complex was performed as follows. The recombinant N-terminal Amino Acid Sequencing-The T.DnaK⅐DnaJ complex purified from T. thermophilus was loaded on Tricine-SDS-PAGE (16.5%), and protein bands were blotted to a polyvinylidene difluoride membrane (Bio-Rad). The blotted membrane was stained with Coomassie Brilliant Blue R-250, and a band of the 8-kDa protein was cut out. The cut piece was treated with cyanogen bromide (37) and analyzed with a gas phase peptide sequencer (PI-2020, Tosoh). To estimate the relative molar stoichiometry of T.DnaK, T.DnaJ, and a novel factor (T.DafA) in the T.DnaK⅐DnaJ complex, the complex was subjected to Edman degradation and PTH-amino acids derived from N termini of each component of the complex were analyzed. The T.DnaK⅐DnaJ complex (9 g) in 4 M guanidine hydrochloride was blotted directly onto a polyvinylidene difluoride membrane. Blotted membrane was dried and stained with Coomassie Brilliant Blue R-250. A protein spot was cut out and analyzed with a gas phase peptide sequencer.
Other Methods-Polyacrylamide gel electrophoresis (PAGE) was carried out on 15% polyacrylamide gel in the presence of 0.1% SDS (SDS-PAGE) (38) or on 7.5% polyacrylamide gel in the absence of SDS (native-PAGE). Tricine-SDS-PAGE (16.5%) was carried out for analysis of low molecular size peptides (39). Gels were stained with Coomassie Brilliant Blue R-250. Protein concentrations were assayed by the method of Bradford with bovine serum albumin as a standard (40).

RESULTS
Cloning of the dnaK Gene Cluster of T. thermophilus-In order to clone the dnaK gene cluster of T. thermophilus using a PCR fragment as a probe, a set of primer oligonucleotides were designed from N-terminal amino acid sequences of T.DnaK and T.DnaJ (24), assuming that, as observed for most bacterial dnaK gene clusters (41), dnaK and dnaJ genes of this bacterium are present in the same gene cluster with dnaK being upstream of dnaJ. A 2.5-kbp DNA fragment was amplified by PCR, sequenced, and confirmed to contain the dnaK gene (data not shown). This DNA fragment was used as a probe to clone a BamHI 5.5-kbp DNA fragment from T. thermophilus that contained dnaK gene, and the DNA sequence was determined for both strands. The region contains at least five genes in the order dnaK-grpE-dnaJ-orf4-clpB from upstream (Fig. 1, first  line). N-terminal amino acid sequences of T.DnaK and T.DnaJ determined from peptide sequencing (24) agreed with the deduced amino acid sequences of the genes. An operon including the dnaK gene appears to start with the dnaK gene since, at upstream of the dnaK gene, there is a promoter-like sequence, and also an open reading frame in the reverse direction whose deduced amino acid sequence is homologous to that of FtsH of E. coli (data not shown). We have not determined the sequence of downstream of clpB gene. Intergenic sequences between dnaK and grpE and between grpE and dnaJ are 55 and 6 bases, respectively. Reading frames of dnaJ and orf4 genes overlap 11 bases. clpB gene starts after a 61-base intergenic sequence. With respect of the arrangement of dnaK, dnaJ, and grpE genes, bacterial dnaK gene clusters are grouped into three types. The order observed for T. thermophilus, dnaK-grpE-dnaJ, is the same to that of Streptomyces coelicolor (Fig. 1, second line) (42,43) but is different from that of Bacillus subtilis (Fig. 1, third line), where the order of the genes is grpE-dnaK-dnaJ (44). grpE gene of E. coli is not included in the dnaK operon (41) (Fig. 1, fourth line). The presence of clpB gene is a unique feature of the dnaK gene cluster of T. thermophilus. orf4 encodes a novel 78-amino acid protein ( Fig. 2A). No definite homolog of this protein has been found in the GenBank data base.
T.DnaJ Lacks Whole Cysteine-rich Region-Deduced amino acid sequences of T.DnaK, T.GrpE, and T.DnaJ are 55%, 27%, and 34% identical with those of E. coli, respectively (45)(46)(47)(48). Their molecular sizes are in general smaller than E. coli homologs. T.DnaK, T.GrpE, and T.DnaJ consist of 615, 177, and 280 amino acid residues, respectively, while those of E. coli homologs are 638, 197, and 376 amino acid residues, respectively. Among them, it should be noted that T.DnaJ is 96 amino acid residues shorter than the E. coli homolog. Alignment of T.DnaJ and E. coli DnaJ sequences has revealed that, in addition to the shorter C terminus (24 residues), whole "cysteinerich region" (69 residues) that contains four repeats of the sequence CXXCXGXG (49, 50) is lacking in T.DnaJ (Fig. 2B). This is a surprise since the cysteine-rich region has been found in all known bacterial DnaJ molecules functioning in the DnaK chaperone system and most eukaryotic DnaJ homologs. Another feature of T.DnaJ is an unusually high content of proline residues. There are 28 proline residues in a molecule that occupies 10% of total amino acid residues. Particularly, there is a consecutive six-proline sequence from position 75 to 80. E. coli DnaJ contains 15 proline that is 4% of total amino acid residues.
Recombinant T.DnaK and T.DnaJ Cannot Assemble into the Complex by Themselves-We constructed the pMKJ7 plasmid to express recombinant T.DnaK and T.DnaJ in E. coli under the control by the T7 promoter of pET system. Both T.DnaK and T.DnaJ were successfully expressed in E. coli (BL21(DE3) pLysS) and recovered in a soluble fraction from cell lysate (Fig.  3A, lanes 1 and 2). To remove heat-labile proteins derived from host cells, a soluble fraction of the lysate was treated at 65°C for 10 min. Since the authentic T.DnaK⅐DnaJ complex isolated from T. thermophilus is stable under the above heat treatment, we expected that expressed T.DnaK and T.DnaJ, if they formed a complex, would have remained soluble after heat treatment. However, most T.DnaJ was precipitated while T.DnaK remained in the soluble fraction after heat treatment (Fig. 3A,  lanes 3 and 4). This indicated that the recombinant T.DnaK and T.DnaJ by themselves were unable to assemble into the complex and free T.DnaJ, somehow heat-labile unless it is incorporated into the complex, precipitated during heat treatment. Indeed, the T.DnaK⅐DnaJ complex band was not seen when soluble fraction after heat treatment was examined with native-PAGE (Fig. 3B, lane 1).
The Authentic T.DnaK⅐DnaJ Complex Contains a Novel 8-kDa Protein Unnoticed Before-In order to search the reason why the recombinant T.DnaK and T.DnaJ could not form the complex, we carefully repeated analysis of the authentic T.DnaK⅐DnaJ complex purified from T. thermophilus. Although only DnaK and DnaJ appeared to be components of the T.DnaK⅐DnaJ complex from SDS-PAGE under the conditions described in the previous paper (24), a small peptide band with molecular size about 8 kDa was clearly seen when the same specimen was analyzed on Tricine-SDS-PAGE (16.5%), which gives better resolution than the usual SDS-PAGE for the analysis of low molecular size peptides (Fig. 4A, lane 1). When the purified T.DnaK⅐DnaJ complex was subjected to two-dimen-  3. Expression of dnaK, dnaJ, and orf4 (dafA)  sional PAGE, with native-PAGE (7.5%) as the first dimension and Tricine-SDS-PAGE (16.5%) as the second dimension, three protein bands, T.DnaK, T.DnaJ, and a 8-kDa protein, were developed in the second dimension from a single band of the T.DnaK⅐DnaJ complex in the first dimension (data not shown). Based on these results, we concluded that the 8-kDa protein is the component of the T.DnaK⅐DnaJ complex. Edman degradation of this protein did not produce PTH-amino acid, probably because of a blocked N terminus. The 8-kDa protein was then extracted from the electrophoresed gel, treated with cyanogen bromide, and subjected to Edman degradation. Two sequences were obtained. The major sequence was AKAERIRXDLGA (where X is undetermined residue), and another sequence with minor amount was LARS. These amino acid sequences were present in orf4 of the dnaK gene cluster ( Fig. 2A, underlined); therefore, the 8-kDa protein was identified as the product of orf4.
Assembly of the T.DnaK⅐DnaJ Complex Requires the orf4 Product-We constructed an expression plasmid (pMKJ8) containing dnaK, dnaJ, and orf4 genes to examine contribution of the 8-kDa protein to the assembly of the T.DnaK⅐DnaJ complex. T.DnaK, T.DnaJ, and the 8-kDa protein (a product of orf4) expressed in E. coli cells harbored by the pMKJ8 were recovered in a soluble fraction of cell lysate (Fig. 3A, lanes 5 and 6). In contrast with the pMKJ7 plasmid, which contained dnaK and dnaJ genes but not orf4, all of T.DnaK and a significant amount of T.DnaJ expressed in E. coli cells harbored by the pMKJ8 remained in the soluble fraction after heat treatment (Fig. 3A, lanes 7 and 8), and native-PAGE of this fraction showed a clear band corresponding to the T.DnaK⅐DnaJ complex (Fig. 3B, lane 2). The T.DnaK⅐DnaJ complex was purified to homogeneity from E. coli cells harbored by pMKJ8. The presence of the 8-kDa protein in the complex was clearly demonstrated when thus purified recombinant T.DnaK⅐DnaJ complex was analyzed with Tricine-SDS-PAGE (Fig. 4A, lane 2). The N terminus of the 8-kDa protein expressed in E. coli was not blocked, and the sequence MLARSGWLSLEALSE was obtained by Edman degradation. This sequence agreed with the predicted N-terminal amino acid sequence of the orf4 gene ( Fig.  2A).
The characteristics of the recombinant complex purified from E. coli were compared with those of the authentic T.DnaK⅐DnaJ complex purified from T. thermophilus. Tricine-SDS-PAGE analysis gave an indistinguishable electrophoretic pattern for both complexes (Fig. 4A). The recombinant complex has a weak ATPase activity (2 ϫ 10 Ϫ2 mol/min/mg at 80°C), which was exactly the same as measured for the authentic complex. In addition, the ability of the recombinant complex to bind the reduced carboxymethylated ␣-lactalbumin, which is a model protein of denatured state, was demonstrated with a gel filtration HPLC assay (24). When the mixture of reduced carboxymethylated ␣-lactalbumin and the complex was applied to a G3000SWXL gel filtration column, reduced carboxymethylated ␣-lactalbumin was co-eluted with the complex (data not shown). Nucleotide content in the complex differed apparently between the authentic and recombinant complex. The authentic complex has no endogenously bound nucleotide, while the recombinant one has 0.6 -1.7 (dependent on preparations) mol of ADP/mol of complex. We have employed hydroxyapatite chromatography as a step of purification of the authentic complex and the proteins were exposed to 10 -200 mM sodium phosphate buffer (pH 7.5) during this procedure. Dialysis of the recombinant complex against 10 mM sodium phosphate buffer resulted in loss of all bound ADP from the complex. Therefore, the difference of nucleotide content between authentic and recombinant complexes is attributable to the different purification procedures of the two complexes. When the authentic complex was incubated with MgATP and subjected to a gel filtration column, about 3 mol of ADP/mol of complex remained bound (24). Similarly, when the recombinant complex, previously dialyzed to the phosphate buffer, was incubated with MgATP and gel-filtrated, about 3 mol of ADP/mole of complex remained bound. Altogether, aside from the unblocked N terminus of the 8-kDa protein, the recombinant T.DnaK⅐DnaJ complex is not distinguishable from the authentic T.DnaK⅐DnaJ complex purified from T. thermophilus.
The 8-kDa Protein Is Also Required for in Vitro Reconstitution of the Complex-When the complex, either authentic or recombinant, was denatured in 8 M urea and then urea was removed by dialysis, the complex was reconstituted at almost perfect recovery (data not shown). Reconstitution was achieved even when recombinant T.DnaK⅐DnaJ complex, which contained the 8-kDa protein as a component, was denatured in 8 M urea and proteins were separated from urea by direct loading of the 8 M urea solution on native-PAGE (Fig. 4B, lane 2). However, when a 8 M urea solution containing T.DnaK and T.DnaJ (but not T.DafA) at the same concentrations as the above experiment was applied to native-PAGE, no complex was formed (Fig. 4B, lane 1). Slow removal of urea by dialysis did not change the result (data not shown). Thus, the 8-kDa protein is required for in vitro reconstitution of the complex.
Three Copies of the 8-kDa Protein Are Contained in the Complex-To estimate molar stoichiometry of the 8-kDa protein to T.DnaK and T.DnaJ in the complex, the purified recombinant T.DnaK⅐DnaJ complex was subjected to Edman degradation and the yield of PTH-amino acids derived from each component of the complex was compared. The amounts of PTH-amino acids averaged for the first four cycles were 34.3 Ϯ 5.1 pmol for T.DnaK (the sequence is AKAV), 33.9 Ϯ 5.8 pmol for T.DnaJ (AAKK), 32.0 Ϯ 6.9 pmol for the 8-kDa protein (MLAR). 3 Even though the yield of Edman degradation may not always be quantitative, the result allowed us to argue that an equal number of copies of T.DnaK, T.DnaJ and the 8-kDa protein is present in the T.DnaK⅐DnaJ complex. Since the molecular size of the T.DnaK⅐DnaJ complex has been estimated to be 300 kDa from gel filtration and Ferguson plots and trigonal ring structures were observed with electron microscopy (24), it was concluded that the complex comprises nine polypeptides: three copies of each of the components. DISCUSSION It is evident from this study that a novel protein factor is necessary for the T.DnaK and T.DnaJ of T. thermophilus to assemble into the complex. This protein, now named here as T.DafA (T.DnaK⅐DnaJ assembly factor A), is encoded by a gene that is located at immediate downstream of the dnaJ gene in the dnaK gene cluster. T.DnaJ molecule alone is heat-labile, but it becomes heat-stable when assembled into the complex, suggesting the indispensable role of T.DafA for the growth of this bacterium at optimum temperature (75°C). In addition to T.DnaK, T.DnaJ and T.GrpE, T.DafA is a fourth member of the DnaK chaperone system of T. thermophilus.
The DnaK chaperone system of T. thermophilus has a distinct feature from other organisms; T.DnaK and T.DnaJ assemble each other with aid of T.DafA to form a stable T.DnaK⅐DnaJ complex. Such a stable complex of DnaK and DnaJ has not been reported for DnaKs from other organisms. However, there are some indications that DnaK (Hsp70) chaperone system functions as a large oligomeric complex. Rhodanese or luciferase in nonnative structure forms a large ternary complex of 230 -250 kDa with DnaK and DnaJ (8,15). In reticulocyte lysate from which TRiC (a eukaryotic cytosolic chaperonin) was depleted, nascently synthesized luciferase is bound to Hsp70 and Hsp40 (DnaJ homolog) to form a 230-kDa complex (51). In spite of these works, only little is known about physical and chemical aspects of the interaction of DnaK and DnaJ in chaperone activity. Direct interaction of E. coli DnaK and DnaJ was suggested only from glutaraldehyde cross-linking (19) or immunological detection (20). Interaction of mammalian Hsp70 with Hsp40 has been shown from immunoprecipitation (52). The finding of T.DafA raises a possibility that an unnoticed factor, possibly a small peptide, is generally required for the stable assembly of DnaK and DnaJ. Since we cannot find a definite homolog of T.DafA in GenBank, the functional counterpart of T.DafA in other organisms, if any, might have amino acid sequence with only limited similarity.
As mentioned, organizations of genes in the dnaK gene cluster of T. thermophilus and S. coelicolor are similar to each other (Fig. 1). Corresponding location of the dafA gene in the gene cluster of T. thermophilus, immediate downstream of dnaJ, is occupied by orfX in S. coelicolor, which encodes a 151-amino acid protein (43). There is a weak amino acid sequence similarity between T.DafA and the N-terminal half of the S. coelicolor OrfX (21% of amino acid residues in T.DafA sequence are identical to S. coelicolor OrfX). However, since OrfX of S. coelicolor has been assigned to be a transcriptional regulator of the dnaK gene cluster (43), it is still undetermined whether S. coelicolor OrfX is really related to T.DafA.
Another finding worth mentioning in this report is that DnaJ of T. thermophilus lacks whole cysteine-rich region. All other bacterial DnaJ molecules identified as a member of the DnaK chaperone system so far reported have cysteine-rich region. The T.DafA does not show amino acid sequence similarity to the cysteine-rich region and functional substitution of the missing cysteine-rich region of T.DnaJ by T.DafA is impossible. The various DnaJ homologs contain one or more conserved sequences that may correspond to individual functional domains (49,50). The N-terminal 70 amino acids or "J-region," conserved among all DnaJ family proteins, is responsible for the interaction with DnaK (Hsp70). The 35-amino acid glycine/ phenylalanine-rich region next to the J-region has been proposed to be involved in interaction with DnaK and modulating substrate binding of DnaK (9,53). The 70-amino acid cysteinerich region contains four CXXCXGXG sequences, which take two zinc-finger-like structures where two zinc atoms are really bound (9). It has been demonstrated from binding experiments and cross-linking studies using various DnaJ fragments that the cysteine-rich region is required for the DnaJ to specifically recognize and bind to proteins in denatured state (9). The finding of T.DnaJ without a cysteine-rich region is in apparent contradiction to the above postulation. There are two possible explanations for the contradiction. We do not know yet whether T.DnaJ alone can bind denatured protein.
If not under any conditions, it can be due to the lack of the cysteine-rich region. Another explanation assumes that the polypeptide binding domain of DnaK is sufficient for usual chaperone activity and the role of cysteine-rich region of DnaJ to bind denatured protein is only optional. Some of eukaryotic DnaJ homologs such as Saccharomyces cerevisiae Sec63 lack the cysteine-rich region, but they still function as molecular chaperones cooperating with their Hsp70 partners (54,55). The fact that the DnaJ fragment, containing J-region and glycine/phenylalanine-rich region but not cysteine-rich region, can substitute for intact DnaJ in an assay measuring phage replication (56) also favors the latter explanation. Further analysis of the function of the T.DnaK⅐DnaJ complex may clarify the above argument.


extfieldsofstudy: Medicine Biology

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Ras, Rap, and Rac Small GTP-binding Proteins Are Targets for Clostridium sordellii Lethal Toxin Glucosylation*

Lethal toxin (LT) from Clostridium sordellii is one of the high molecular mass clostridial cytotoxins. On cultured cells, it causes a rounding of cell bodies and a disruption of actin stress fibers. We demonstrate that LT is a glucosyltransferase that uses UDP-Glc as a cofactor to covalently modify 21-kDa proteins both in MAP Kinase Activation— Experiments examining the effects of LT on epidermal growth factor (EGF)-stimulated MAP kinase phosphorylation performed as 3T3 cells cultured fetal calf cells they fetal calf activity cytopathogenic Cells were then into polyacryl- gel electrophoresis sample and 30 (cid:109) g of total each experiment, was electrophoresed on a 12.5% SDS-polyacrylamide gel. The gel was blotted onto nitrocellulose and incubated with a monoclonal antibody directed against MAP kinases (anti-ERK1 and anti- ERK2). Immune complexes were detected by horseradish peroxidase-conjugated secondary antibody followed by the ECL kit (Amersham International). Fluorescence Measurements— LT-catalyzed glucosylation of Y64W Ras-GDP was performed in 50 m M triethanolamine HCl buffer (pH 7.5) 140 m M KCl, 1 m M MgCl 2 , and 0.1 (cid:109) M dithiothreitol. Y64W Ras-GDP (50 (cid:109) M was incubated with 100 (cid:109) M UDP-Glc and 2.5 (cid:109) g/ml LT at 37 °C for 2 h. Control experiments were performed in the absence of LT.Guanine nucleotide exchange and GTP hydrolysis of glucosylated versus unmodified Y64W Ras (0.5 (cid:109) were 37 °C in 50 m M Hepes

Lethal toxin (LT) from Clostridium sordellii is one of the high molecular mass clostridial cytotoxins. On cultured cells, it causes a rounding of cell bodies and a disruption of actin stress fibers. We demonstrate that LT is a glucosyltransferase that uses UDP-Glc as a cofactor to covalently modify 21-kDa proteins both in vitro and in vivo. LT glucosylates Ras, Rap, and Rac. In Ras, threonine at position 35 was identified as the target amino acid glucosylated by LT. Other related members of the Ras GTPase superfamily, including RhoA, Cdc42, and Rab6, were not modified by LT. Incubation of serumstarved Swiss 3T3 cells with LT prevents the epidermal growth factor-induced phosphorylation of mitogen-activated protein kinases ERK1 and ERK2, indicating that the toxin blocks Ras function in vivo. We also demonstrate that LT acts inside the cell and that the glucosylation reaction is required to observe its dramatic effect on cell morphology. LT is thus a powerful tool to inhibit Ras function in vivo.
Several different species of the genus Clostridium produce large molecular mass (ϳ250 -300 kDa) cytotoxins that cause effects on the actin cytoskeleton, including disruption of actin stress fibers and rounding of cell bodies. This subgroup of clostridial cytotoxins includes toxins A and B from Clostridium difficile, lethal toxin (LT) 1 and hemorrhagic toxin from Clostridium sordellii, and Clostridium novyi ␣-toxin (1). Recently, toxins A and B from C. difficile, the causative agent of antibiotic-associated diarrhea (2), were shown to covalently modify the mammalian protein Rho by UDP-Glc-dependent glucosylation of threonine 37 (3,4). Rho is a small Ras-related GTPbinding protein involved in the control of actin polymerization (5). Glucosylation of threonine 37 of Rho by C. difficile toxin A or B apparently inactivates this protein and results in a loss of actin stress fiber assembly.
C. sordellii produces two toxins, LT and hemorrhagic toxin, two major virulence factors inducing gas gangrene and hemorrhagic diarrhea in humans and animals (6). These C. sordellii toxins have some similarities to toxins A and B from C. difficile in terms of amino acid sequences and immunological epitopes (7). Despite these similarities, it seems that LT and toxins A and B affect different intracellular target proteins. LT causes morphological and cytoskeletal effects different from those elicited by the C. difficile toxins. The effects consist of the rounding of cell bodies with the reorganization of F-actin structures into numerous cell-surface filopodia and a loss of actin stress fibers (8,9). In addition, we have recently shown that overexpression of RhoA, RhoB, or RhoC cDNA in HeLa cells protects these cells from the effects of toxins A and B, but not from those of LT (9). These observations clearly pointed out that Rho small GTPbinding proteins were the main substrate for the C. difficile toxins and that the targets of LT were distinct.
A mutant hamster fibroblast cell line has been described that is resistant to toxins A and B from C. difficile (10). This resistance was attributed to a low intracellular UDP-Glc content, and the fact that this mutant cell line was not intoxicated by LT indicated that LT is a glucosyltransferase (11).
In this paper, we report that LT modifies and inactivates p21 ras by glucosylation of threonine 35. In addition, LT was also found to glucosylate Rap and Rac proteins. No activity was found on other Ras-related proteins including Ral, Rho, Cdc42, Arf1, and Rab.
EXPERIMENTAL PROCEDURES Materials C. sordellii LT was obtained from culture supernatants of the pathogenic C. sordellii IP82 strain and purified to homogeneity as described previously (8). Recombinant Ha-Ras, RhoA, Rac1, Rap2, Ral, Rab6, and Cdc42 proteins were made either in baculovirus (Rac1, Rab6, and Arf1) or in Escherichia coli through either GST fusions (Ha-Ras, Rap1, Ral, RhoA, and Rac1) or a histidine-tagged fusion (His-Cdc42). The Ral-GST fusion could not be processed by thrombin to yield a 21-kDa protein due to the fact that Ral itself was proteolyzed. Ral was therefore tested for LT glucosylation as a 47-kDa fusion protein. The Rho protein, used in this study, could be fully ADP-ribosylated by exoenzyme C3 or glucosylated by C. difficile toxin B. Rac1 and Cdc42, used in this work, could be fully glucosylated by C. difficile toxin B. Rabbit polyclonal antibodies against fimbrin were a gift of Monique Arpin (Unité de Biologie des Membranes, Institut Pasteur, Paris). The monoclonal antibody MK12 (Zymed Laboratories, Inc., S. San Francisco, CA) was used for immunoblotting MAP kinases. The recombinant Y64W Ras mutant used for tryptophan fluorescence experiments was produced in E. coli and purified as described previously (12).

Methods
Glucosylation Reactions-Incorporation of LT-catalyzed [ 14 C]Glc in the GTPases or cell lysates was performed as described by Just et al. (3) in the case of C. difficile toxin B. Briefly, 10 l of UDP-[ 14 C]Glc in ethanol (0.2 Ci, 300 mCi/mmol; DuPont NEN, Les Ulis, France) was dried down under vacuum. Recombinant proteins (2 g) dissolved in 15 l of 50 mM triethanolamine HCl buffer (pH 7.5) containing 2 mM MgCl 2 , 150 mM KCl, 100 M dithiothreitol, and 2 M GDP were added to the dried UDP-[ 14 C]Glc. LT (2 g/ml) was then added to start the reaction, which was carried out for 1 h at 37°C. The reaction was stopped by adding 5 l of 2 ϫ SDS sample buffer, boiled, and electrophoresed on a 15% SDS-polyacrylamide gel. Upon staining with Coomassie Blue followed by destaining, the gel was dried, and radioactivity was recorded and counted using a PhosphorImager system (Molecular Dynamics, Inc., Sunnyvale, CA). Glucosylation of HeLa cell lysates by LT was performed as follows. HeLa cells (5 ϫ 10 6 ) were homogenized by three cycles of freeze-thawing in 200 l of 50 mM triethanolamine HCl buffer (pH 7.5) containing 100 M dithiothreitol, 1 g/ml leupeptin, and 1 g/ml pepstatin (glucosylation buffer). Cell lysates (20 l) were added to 10 l of dried UDP-[ 14 C]Glc, and 2 g/ml LT was added to start the enzymatic reaction. After 1 h at 37°C, further processing and imaging were done as described above.
In Vivo Glucosylation by LT of Small GTP-binding Proteins in Rat Fibroblasts-Rat-1 fibroblasts (Rat-1-EJ-Rap2.31.A8) stably transfected with G12V Ras and Rap2 (13) were grown in 60-mm Petri dishes to a subconfluent density. LT was added to the cells at the indicated concentration in 5 ml of fresh medium containing 10% fetal calf serum. After 2 h, the cells were removed from the dishes with a rubber policeman and washed in 10 ml of PBS followed by centrifugation at low speed. Washing was repeated five times to remove residual LT, and finally, the cell pellets were resuspended in 50 l of glucosylation buffer. Cells were then lysed by four cycles of freeze-thawing. After homogenization, the amount of protein in each cell lysate was estimated. For in vitro glucosylation of small GTP-binding proteins with LT, 40 l of cell lysate was added to 15 l of dried UDP-[ 14 C]Glc with 5 g/ml LT. This mixture was incubated for 1 h at 37°C. Then, 5 l of each reaction was added to 10 l of sample buffer, boiled, and electrophoresed on a 15% SDS-polyacrylamide gel. The gel was stained, destained, dried, and analyzed for radioactivity by autoradiography.
Localization of the Glucosylated Amino Acid in Ha-Ras-This experiment was performed by microsequencing the radioactively labeled protein. The Ha-Ras protein (10 g) was first radioactively glucosylated by LT with 40 l of dried UDP-[ 14 C]Glc (0.8 Ci) for 1 h (under reaction conditions as described above). Then, 10 mM unlabeled UDP-Glc was added; the reaction was further incubated for an additional 1 h at 37°C; and the proteins were separated on a 12.5% SDS-polyacrylamide gel. After migration and staining with Amido Black, the band observed at 21 kDa was cut out from the gel and digested with 1 g of trypsin in 200 l of 100 mM Tris-HCl buffer (pH 8.8) containing 0.01% Tween 20. The reaction was incubated for 18 h at 35°C. The resulting peptides were separated by HPLC using a hydrophobic C 18 column with an acetonitrile/trifluoroacetic gradient. Fractions eluted from the column were each analyzed for radioactivity. Radioactive peptides were repurified by HPLC using the C 18 column with a sodium acetate buffer (pH 6.00) gradient. The eluted peptides were analyzed for radioactivity. The radioactive peaks were sequenced with an Applied Biosystems microsequencer, and the product of each Edman degradation cycle was collected and counted for radioactivity.
Immunofluorescence Experiments-Cells grown on coverslips were treated with LT and then fixed with 3% paraformaldehyde for 20 min. After fixation, monolayers were washed three times with PBS, and free aldehyde groups were quenched by incubation with 50 mM NH 4 Cl in PBS for 10 min. Cells were permeabilized for 5 min at room temperature in PBS containing 0.2% Triton X-100 and then incubated for 30 min at room temperature with the first antibody. Coverslips were then washed extensively with PBS and incubated with the secondary antibodies (Texas Red-conjugated sheep anti-mouse, Amersham International, Buckinghamshire, United Kingdom) together with fluorescein isothiocyanate-labeled phalloidin (Sigma, L'Isle-d'Abeau, France) for 30 min. After three washes in PBS, coverslips were mounted in Moviol (Calbiochem-Novabiochem GmbH, Bad Soden, Germany), and fluorescence was observed with a confocal microscope.
MAP Kinase Activation-Experiments examining the effects of LT on epidermal growth factor (EGF)-stimulated MAP kinase phosphoryla-tion were performed as follows. Swiss 3T3 cells were cultured according to routine procedures in H21 medium supplemented with 10% fetal calf serum. When the cells reached confluency, they were serum-starved overnight in 0.1% fetal calf serum. After 3 h of incubation with LT (1.7 g/ml) in serum-free medium (the activity of LT was monitored by the cytopathogenic effect on cells), EGF was added (or not) at a 50 ng/ml final concentration for 5 min. Cells were then scraped into polyacrylamide gel electrophoresis sample buffer, and 30 g of total protein, for each experiment, was electrophoresed on a 12.5% SDS-polyacrylamide gel. The gel was blotted onto nitrocellulose and incubated with a monoclonal antibody directed against MAP kinases (anti-ERK1 and anti-ERK2). Immune complexes were detected by horseradish peroxidaseconjugated secondary antibody followed by the ECL kit (Amersham International).
Fluorescence Measurements-LT-catalyzed glucosylation of Y64W Ras-GDP was performed in 50 mM triethanolamine HCl buffer (pH 7.5) containing 140 mM KCl, 1 mM MgCl 2 , and 0.1 M dithiothreitol. Y64W Ras-GDP (50 M) was incubated with 100 M UDP-Glc and 2.5 g/ml LT at 37°C for 2 h. Control experiments were performed in the absence of LT.
Guanine nucleotide exchange and GTP hydrolysis of glucosylated versus unmodified Y64W Ras (0.5 M) were measured at 37°C in 50 mM Hepes (pH 7.5), 1 mM MgCl 2 , and 1 mM dithiothreitol by monitoring tryptophan fluorescence at 340 nm upon excitation at 292 or 300 nm (12). When needed, 2 mM EDTA was added to reduce free magnesium to 0.8 M.
Cell Microinjections-Diploid Chinese hamster lung fibroblasts (Don cells; ATCC CCL16, Don-wt (where "wt" indicates wild-type)) and the C. difficile toxin A-and B-resistant mutant of this cell line, Cdt R -Q (10, 11), referred to here as Don-Q (a UDP-Glc-deficient mutant of these cells), were grown on 13-mm slides for 48 h. Semiconfluent wild-type and mutant cells were microinjected (Eppendorf microinjector) with the indicated concentrations of LT, UDP-Glc, or anti-LT antibodies with fluorescein isothiocyanate-labeled dextran (Sigma) in calcium-free PBS. Approximately 100 cells were microinjected in each experiment. The cultures were further incubated for 30 min at 37°C and fixed with 3.7% paraformaldehyde for 10 min. Cells were visualized by phase-contrast and fluorescence microscopy.

Disruption of Actin Stress Fibers and Formation of Filopodia Induced in HeLa Cells by LT-
The cytopathic effect of C. sordellii LT consists of the rounding of cell bodies and profound alteration of F-actin-containing structures (8,9). After a 3-h incubation with 2 g/ml LT, HeLa cells became round, displayed F-actin structures rearranged into cell-surface filopodia, and exhibited a loss of actin stress fibers (Fig. 1). Using a polyclonal rabbit antibody that reacts against all known isoforms of the actin-bundling protein fimbrin/plastin (14), we observed that fimbrin/plastin was present in LT-induced filopodia (Fig. 1).
LT Catalyzes the UDP-Glc-dependent Glucosylation of 21-23-kDa Proteins in HeLa Cell Lysates-Incubation of HeLa cell lysates with LT in the presence of UDP-[ 14 C]Glc followed by gel electrophoresis of the reaction products showed that the toxin induced labeling of proteins in the range of 21-23 kDa (Fig. 2). This reaction could be displaced by adding an excess of nonradioactive UDP-Glc, but not UDP-glucuronic acid (Fig. 2). No modification of proteins by LT was found with [ 14 C]Glc alone (data not shown).
LT Glucosylates 21-kDa Proteins in Vivo-To demonstrate that small GTP-binding proteins were glucosylated by LT in vivo, Rat-1-EJ-Rap2.31.A8 fibroblasts were incubated with increasing amounts of LT (from 0.005 to 5 g/ml). The highest concentration of toxin caused the characteristic cytopathogenic effect of LT in 100% of the cells within 1 h. All cells were then lysed, and the lysates were glucosylated with LT a second time, now in vitro in the presence of radioactive UDP-Glc. If LT acts from inside the cell, there should be an inverse correlation between the LT dose used for in vivo pretreatment of cells and the amount of [ 14 C]Glc incorporated into small G-proteins in vitro. As shown in Fig. 3, the highest rate of glucosylation by LT of a 23-kDa protein was observed in control cells. Two minor bands of 21 and 25 kDa glucosylated by LT were also noticed in control lysates (Fig. 3). Fig. 3 also demonstrates that a clear decrease to a total absence of labeling of these bands was observed when the cells had been preincubated in vivo with increasing concentrations of LT prior to the in vitro LT glucosylation. Assuming that LT reacts with small G-proteins, in accordance with its homology to C. difficile toxin B (15), this dose-dependent activity of LT suggests that the toxin exerts its action from within the cell.
LT Glucosylates Ras, Rap, and Rac Small GTP-binding Proteins in Vitro-Specificity of LT was studied by incubating UDP-[ 14 C]Glc and LT with different members of the p21 ras superfamily of small GTP-binding proteins. As shown in Fig. 4, Ha-Ras, Rap2, and Rac1 were substrates for LT-catalyzed glucosylation. In contrast, RhoA, Cdc42, and Rab6 were not modified in vitro by LT. Since Ral was only available as a GST fusion protein, we tested a possible influence of the fusion with GST by adding a Rac-GST construct to the series. As evidenced by Fig. 4, Rac1-GST was a substrate for LT glucosylation, whereas Ral-GST was not modified by the toxin. This suggests that Ral is not modified by LT. Finally, no incorporation of glucose catalyzed by LT could be found on Arf1 (data not shown).
LT Glucosylates Threonine 35 of Ha-Ras-To identify the acceptor amino acid glucosylated by LT, Ha-Ras protein was modified by LT in the presence of UDP-[ 14 C]Glc, electrophoresed on SDS-polyacrylamide gel, and digested with trypsin, and the resulting peptides were separated as described under "Experimental Procedures." As shown in Fig. 5A, 47 fractions were obtained. The radioactivity was exclusively associated with fractions 39 and 40 (Fig. 5A). As shown in Fig. 5 (B and C), repurification of fraction 39 or 40 gave rise to a major peptide (peptide D for fraction 39 and peptide E for fraction 40) containing the radioactivity and several other small peptides. Peptides D and E were microsequenced and gave exactly the same amino acid sequence. Each cycle of Edman degradation was collected and counted for radioactivity. We found the following unambiguous sequence for these peptides: SALTIQLIQN-HFVDEYDPTIEDSYR. Cycle 19 corresponding to a threonine gave a very small signal. The small amount of threonine detected in position 19 may be the consequence of the LT-catalyzed glucosylation of most of the Ras molecules present in the reaction. A decrease in or absence of threonine 37 of RhoA in automated amino acid sequencing, after glucosylation by toxin A or B, has been already reported (3,4). The amino acid sequence found for both peptides D and E corresponds exactly to a sequence found in the Ha-Ras protein between amino acids 17 and 41 (16). Radioactivity was associated first with cycle 19 and decreased thereafter (Fig. 5E). The rise in radioactivity at cycle 19 establishes threonine 35 (of the Ha-Ras molecule) as the unique amino acid glucosylated by LT.

Inhibition of EGF-induced Phosphorylation of MAP Kinases in Swiss 3T3 Cells by LT-
In serum-starved Swiss 3T3 cells, the mitogenic signaling pathway involving tyrosine phosphorylation of growth factor receptors such as the EGF receptor and the subsequent Ras-dependent activation of MAP kinase phosphorylation is reduced to a basal level (17). After incubation with EGF, Ras-dependent activation of MAP kinases ERK1 and ERK2 can be followed by a shift in electrophoretic mobility resulting from phosphorylation (18). If the toxin blocks Ras activity, serum-starved Swiss 3T3 cells incubated with LT before the addition of EGF should not activate MAP kinases. As shown in Fig. 6, serum-starved Swiss 3T3 cells incubated with EGF had MAP kinases shifted toward higher molecular mass compared with MAP kinases of cells not incubated with EGF. In contrast, when serum-starved Swiss 3T3 cells were incubated with LT, prior to incubation with EGF, the growth factor was not able to induce a shift in electrophoretic mobility of the MAP kinases (Fig. 6).
LT Acts in the Cytosol by Glucosylation-To further substantiate the notion that LT reaches the cytosol and acts by glucosylation of small GTP-binding proteins, a series of microinjection experiments was performed. Don-wt cells were incubated with LT in medium containing nonimmune rabbit serum. The expected characteristic cytopathogenic effect was observed in the whole cell population (Fig. 7A). When rabbit anti-LT antibodies were added to the medium, the same amount of LT as used in Fig. 7A did not affect the cells (Fig.  7B). Drugs blocking the endocytic pathway acidification (bafilomycin A 1 , chloroquine, or monensin), known to prevent many bacterial toxins from penetration into the cytosol (19), blocked the activity of LT on cells (data not shown). When Don-wt cells in medium containing anti-LT antibodies were microinjected with LT, they rapidly exhibited the cytopathogenic effect characteristic of LT (Fig. 7, C and D). Successful microinjection was monitored by a yellow-green fluorescence of fluorescein-labeled dextran added to the solutions microinjected (see "Experimental Procedures"). This showed that LT can exert its activity from the cytosol.
To demonstrate that the activity of LT is mediated through glucosylation (of G-proteins), we took advantage of a mutant Don cell (Don-Q). This cell has a low content of UDP-Glc, which renders it resistant to the glucosylating toxins A and B from C. difficile and also to LT (11). Don-Q cells were incubated with LT, followed by microinjection of UDP-Glc into some of them (those lighting up under fluorescence microscopy). As shown in Fig. 7 (E and F), only cells that were microinjected with UDP-Glc exhibited the characteristic cytopathogenic effect of the toxin, suggesting that the toxin and the cofactor act at the same side of the cell membrane. The specificity of the effect was confirmed by microinjecting, instead of UDP-Glc, UDP-Gal or UDP-GlcUA (100 mM) into cells similarly treated with LT. Neither of the additionally used activated sugars promoted any cytopathogenic effect. Finally, none of the three UDP-sugars used in this study had any effect if the cells were not pretreated with toxin (data not shown). Knowing that our rabbit anti-LT serum neutralized the toxin, we microinjected Don-wt cells with this serum and then incubated them with LT added to the medium. As shown in Fig. 7 (G and H), microinjection of anti-LT antibodies protected against LT, clearly indicating that the neutralizing antibody and the toxin meet each other in the cytosol. Accordingly, cells not injected exhibited the cytopathogenic effect typical of LT (Fig. 7, G and H), as did cells microinjected with nonimmune rabbit serum (data not shown). The experiments shown in Fig. 7, together with those presented in Fig. 3, strongly suggest that LT acts from the cytosol by glu-cosylating small GTP-binding proteins using UDP-Glc as a cofactor.

LT Glucosylation of Ras Enhances the GTP Dissociation Rate and Reduces GTP Hydrolysis of the GTP-binding Protein-
The effects of LT glucosylation on the intrinsic properties of Ras was studied using the Y64W Ras mutant. This mutant has the same intrinsic biochemical properties as wild-type Ras, but its activation-deactivation cycle can be followed in real time by monitoring changes in the fluorescence of tryptophan 64 (12). In Fig. 8A, Y64W Ras-GDP, glucosylated or not, was first activated by the addition of GTP. After several minutes, the protein was converted again to the GDP-bound form by addition of a large excess of GDP. This experiment was performed at a low magnesium concentration in order to favor the dissociation of the bound nucleotide (the rate-limiting step of nucleotide exchange) and to prevent GTP hydrolysis. Similar fluorescence changes were observed for the nonglucosylated and  15-l aliquot). B and C, purification by HPLC of fractions 39 and 40. Radioactivity associated with each peptide was counted on 50-l aliquots. D, radioactivity associated with each Edman degradation cycle (each Edman cycle of peptides D and E was combined and counted).

FIG. 6. EGF-induced mobility shift of MAP kinases in cells pretreated with LT.
Serum-starved Swiss 3T3 cells were treated with EGF and LT as shown. Cells were lysed, and ϳ30 g of total protein/ experiment was electrophoresed, blotted, and stained with the monoclonal antibody MK12 (ERK1, 44 kDa (p44); ERK2, 42 kDa (p42)). glucosylated forms of Ras (Fig. 8A). Indeed, binding of GTP in place of GDP induced a decrease in fluorescence, and conversely, binding of GDP in place of GTP induced an increase in fluorescence. Upon GTP addition, the time course of the fluorescence decrease was similar for the two forms of Ras, indicating that glucosylation did not greatly modify the GDP dissociation rate. In contrast, the increase in fluorescence by GDP addition was four times faster for glucosylated Ras than for unmodified Ras (Fig. 8A). This result demonstrates that glucosylation weakened GTP binding in the nucleotide site of Ras by accelerating its dissociation rate. Similar effects of glucosylation were observed for the dissociation rate of GTP␥S either at low (1 M) or high (1 mM) magnesium concentration (data not shown).
The effect of glucosylation on GTP hydrolysis by Ras is shown in Fig. 8B. Y64W Ras-GDP was incubated with GTP at 1 mM magnesium. Activation was triggered by the addition of 2 mM EDTA, which reduced the free magnesium concentration to Ͻ1 M. The first instantaneous fluorescence decrease reflected the dissociation of magnesium from Y64W Ras-GDP, whereas the slower fluorescence decrease reflected (as in Fig. 8A) the exchange of GTP for GDP. After completion of GDP/GTP exchange, magnesium was added back to the reaction (1 mM free magnesium). Due to the intrinsic GTPase activity of the protein, the fluorescence of the unmodified form of Ras slowly increased toward the level of fluorescence initially observed for Ras-GDP (Fig. 8B). In the case of the glucosylated form of Ras, much slower kinetics of GTPase activity was observed. Indeed, upon glucosylation of threonine 35, Ras had a four times slower intrinsic GTPase activity (Fig. 8B).
Glucosylation of Y64W Ras by LT slightly modified the fluorescence of the protein. As compared with unmodified Y64W Ras, LT-glucosylated Y64W Ras exhibited, on one hand, a larger absolute fluorescence level and, on the other hand, a smaller fluorescence change upon GDP/GTP exchange or GTP hydrolysis (Fig. 8, A and B). Therefore, we looked for a fluorescence signal that could correlate with the glucosylation of the protein. When Y64W Ras-GDP was incubated with LT and isothiocyanate-labeled dextran added to the injected medium. C and D, typical rounding of Don-wt cells microinjected with LT (concentration in the micropipette of 200 g/ml) in medium containing LT antibodies (1:200 dilution) to protect against any LT molecules possibly leaking out from injected cells. E and F, typical rounding of UDP-Glc-deficient Don-Q cells exposed to LT (1.25 g/ml; 3 h) in the medium and then microinjected with UDP-Glc (concentration in the micropipette of 100 mM). G and H, protection from rounding of Don-wt cells microinjected with neutralizing anti-LT antibodies (serum diluted 1:10) and exposed to LT (1.25 g/ml; 3 h) in the medium. The toxin is accessible to neutralizing anti-LT antibodies once it reaches the cytosol. UDP-Glc, fluorescence was enhanced by 2% within 2 h (Fig.  8C). Since this signal required both LT and UDP-Glc, it certainly reflects the time course of UDP-Glc incorporation. DISCUSSION Toxins A and B from C. difficile have been shown to covalently modify and thereby inactivate the small GTP-binding protein Rho, resulting in the disruption of F-actin structures (20,21). In vitro and in vivo evidence indicates that toxins A and B modify RhoA by UDP-Glc-dependent glucosylation of threonine 37 (3,4). In addition to RhoA, toxins A and B from C. difficile also modify in vitro Rac1 and Cdc42 (3,4), two other proteins of the Rho subfamily involved in the control of mem-brane ruffling and filopodia formation, respectively (22)(23)(24). Also, it has recently been reported that the ␣-toxin from C. novyi is a glycosyltransferase that acts on the cytoskeleton through modification of Rho. However, in this case, UDP-Glc was not the cofactor required for modification. 2 We report here that LT, like toxins A and B from C. difficile, is also a glucosyltransferase that uses UDP-Glc to modify small GTP-binding proteins. However, the substrate specificity of LT is different from that of toxins A and B. LT glucosylates Ras, Rap2, and Rac1 in vitro. LT had no effect on Rho or on Cdc42, two of the main substrates for C. difficile toxins A and B.
The effects induced by LT on the HeLa cell actin cytoskeleton are obviously different from those elicited by toxins A and B from C. difficile. The LT effects consist of the disruption of actin stress fibers and the formation of filopodia containing F-actin and fimbrin/plastin (9). Glycosyltransferase activity of both C. difficile and C. novyi toxins is directed toward GTP-binding proteins of the Rho subfamily. With C. sordellii LT, we have the first toxin that mainly acts on the Ras subfamily of GTPases. The specific effect of LT on the HeLa cell actin cytoskeleton is fundamentally different from that observed with toxin A or B from C. difficile (9). Since both toxin B (or A) and LT are able to glucosylate Rac (3,4), the specific activity of LT on the cytoskeleton cannot be attributed to Rac modification alone. Instead, we believe that the combination of the modified GTPases causes LT to induce its cytopathogenic effect. We would like to stress that since the physiological function of Rap is still unknown, Rap modification by LT could be a key event in LTinduced cytoskeletal disruption.
In Swiss 3T3 cells, stimulation of Ras activates membrane ruffling and actin stress fiber organization by Rac-and Rhodependent mechanisms (22). Since LT inactivates both Ras and Rac, this may in turn inhibit Rho, resulting in the collapse of actin stress fibers. It is interesting to note that filopodia induced by LT markedly resemble those generated by microinjection of the activated form of Cdc42 into Swiss 3T3 cells (23,24). It is tempting to speculate that LT is responsible for the formation of filopodia by indirectly activating Cdc42, which in turn is a consequence of a toxin-induced Ras inactivation. An alternative hypothesis to explain the formation of filopodia due to LT could be that Cdc42 is already in an active state, but formation of filopodia is masked by activation of Ras, leading to a dominant phenotype of membrane ruffling and actin stress fibers. Inactivation of Ras and Rac would therefore allow observation of the Cdc42 phenotype.
LT inactivates Ras by glucosylation of threonine 35, which corresponds to threonine 37 of Rho (25), the residue modified by toxins A and B (3,4). In addition, our data strongly suggest that LT acts in the cytosol and glucosylates small 21-kDa molecules in vivo, resulting in the inactivation of Ras, since serum-starved Swiss 3T3 cells intoxicated with LT have no Ras-dependent induced MAP kinase phosphorylation (see Fig. 6).
At the present time, we do not understand the nature of the substrate specificity of LT for Ras, Rap, and Rac. It seems reasonable that amino acid sequences apart from the threonine 35 acceptor site of glucosylation enable LT to specifically recognize the various small G-proteins.
LT glucosylation of Ras at threonine 35 induced a small but significant decrease in the K off of GDP, most likely due to a higher affinity of glucosylated Ras for magnesium. Such a difference in magnesium affinity has not been observed for the T35A mutant of Ras (26). Apart from this small difference, the , by the addition of 10 M GTP (first arrow). Deactivation was achieved by the addition of 500 M GDP while its intrinsic fluorescence at 340 nm was continuously monitored. GDP dissociation rate constants were as follows: glucosylated, 0.0125 s Ϫ1 ; and control, 0.017 s Ϫ1 . GTP dissociation rate constants were as follows: glucosylated, 0.0125 s Ϫ1 ; and control, 0.0033 s Ϫ1 . B, shown is GTP hydrolysis at 1 mM magnesium. Glucosylated or unmodified Y64W Ras-GDP (0.5 M) was incubated with 10 M GTP in the presence of 1 mM magnesium. GDP/GTP exchange was initiated by the addition of 2 mM EDTA. After 6 min, GTP hydrolysis was initiated by the addition of 2 mM MgCl 2 (1 mM free magnesium). Note the change in time scale after magnesium addition. C, glucosylation of Y64W Ras-GDP induces a small increase in the intrinsic fluorescence of the protein. The fluorescence of Y64W Ras-GDP was continuously monitored while 0.8 g/ml LT and 100 M UDP-Glc were sequentially added to the fluorescence cuvette. In A and C, the samples were excited at 300 nm to minimize light absorption due to the large amount of nucleotides used in these experiments. In B, the excitation was set at 292 nm.
Thr-35 glucosylated form of Ras in the GTP-bound form has properties very similar to those of the T35A mutant: a 4-fold increase in the GTP K off and a four to five times slower rate of GTP hydrolysis (26). It is thus extremely likely that the Thr-35 glucosylation of Ras, as the T35A mutant of Ras, has a much decreased affinity for the Raf Ras-binding domain (27). The T35A mutant of Ras has a 200-fold reduced affinity for the Raf Ras-binding domain (27) and represents the mutation that has the most drastic effect on the Ras/Ras-binding domain interaction (27). Threonine 35 contacts both magnesium and ␥-phosphate in the GTP-bound form and a water molecule that also makes a hydrogen bound with aspartic acid 38 in the Rap⅐Raf Ras-binding domain complex (28). Threonine 35 is conserved in all of the small G-proteins and is an essential residue of the switch I region (29). Thus, the modification of threonine 35 either by mutation (T35A) or by glucosylation would result in the inability of Ras to interact with its effector (27). Even the conservative T35S mutation greatly decreases (ϳ20-fold) the transforming potential of an oncogenic Ras, pointing to the importance of this residue in switching to the active conformation and/or interacting with the Raf effector (30).
It is remarkable that four out of five members of the group of these large clostridial cytotoxins (toxins A and B, LT, and ␣-toxin) have glycosyltransferase activities on small GTP-binding proteins. Recently, we have found that C. sordellii hemorrhagic toxin, the fifth member of this large clostridial cytotoxin group, is also a glucosyltransferase. 3 Taking into account that LT is the first toxin that inactivates the Ras small GTP-binding protein, it should soon become a powerful laboratory reagent to explore cellular signaling pathways stimulated by this molecule.


extfieldsofstudy: Biology Medicine

text:
Use of combinatorial peptide libraries to construct functional mimics of tumor epitopes recognized by MHC class I-restricted cytolytic T lymphocytes.

Identification of cytolytic T lymphocyte (CTL) epitopes presented by major histocompatibility complex (MHC) class I molecules on tumor cells is critical for the design of active immunotherapy. We describe the use of combinatorial peptide libraries with defined amino acids in two MHC anchor positions to search for epitopes that are recognized by H-2Db- and Kb-restricted CTL specific for the mouse lymphoma EL4. An iterative strategy was used for screening libraries in which 16 amino acids were divided into 3 groups and 3 subgroups: alpha (AL, VT, FY); beta (GS, P, DE); gamma (KR, H, NQ). The proportions of each group and subgroup at individual peptide positions were changed in the library synthesis, and the effect of these changes on CTL activity was measured in a sensitive RMA-S cell assay. A single H-2Db epitope mimic was deduced from the original library that contained > 2 x 10(8) potential peptides and was at least 9 logs more potent than the original library. Immunization of syngeneic mice with this peptide elicited CTL that lysed EL4 cells as well as RMA-S cells pulsed with peptides isolated from Db molecules of EL4 cells, indicating functional similarity between the mimicking peptide and the naturally processed CTL epitope. Furthermore, adoptive transfer of such a CTL line had a therapeutic effect in mice with EL4 established as an ascites tumor. Two H-2Kb-restricted epitope mimics of the same tumor were also identified. Our method represents a novel approach for the construction of MHC class I-restricted targets that can serve as immunogens for active immunotherapy of cancer.

Identification of cytolytic T lymphocyte (CTL) epitopes presented by major histocompatibility complex (MHC) class I molecules on tumor cells is critical for the design of active immunotherapy. We describe the use of combinatorial peptide hbraries with defined amino acids in two MHC anchor positions to search for epitopes that are recognized by H-2D b-and K brestricted CTL specific for the mouse lymphoma EL4. An iterative strategy was used for screening hbraries in which 16 amino acids were divided into 3 groups and 3 subgroups: ot(AL, VT, FY); [3(GS, P, DE); y(KR, H, NQ). The proportions of each group and subgroup at individual peptide positions were changed in the library synthesis, and the effect of these changes on CTL activity was measured in a sensitive RMA-S cell assay. A single H-2D b epitope mimic was deduced from the original library that contained >2 • 10 s potential peptides and was at least 9 logs more potent than the original hbrary. Immunization of syngeneic mice with this peptide ehcited CTL that lysed EL4 cells as well as RMA-S cells pulsed with peptides isolated from D b molecules of EL4 cells, indicating functional similarity between the mimicking peptide and the naturally processed CTL epitope. Furthermore, adoptive transfer of such a CTL line had a therapeutic effect in mice with EL4 established as an ascites tumor. Two H-2K brestricted epitope mimics of the same tumor were also identified. Our method represents a novel approach for the construction of MHC class I-restricted targets that can serve as immunogens for active immunotherapy of cancer. M ost antigens recognized by CD8 + cytolytic T lymphocytes (CTL) are derived from cytoplasmic proteins whose presentation involves the degradation by a proteasome proteinase complex into small peptides, transportation of peptides to the endoplasmic reticulum by peptide transporter-associated proteins, and binding to MHC class I molecules which then travel through the Golgi complex to reach the cell surface (1)(2)(3). The peptides are usually 8-10 amino acids long, with 2-3 primary anchor residues that interact with the MHC class I molecule (4,5), and 2-3 amino acid residues that bind to the TCR (6). Further analysis of the requirement of MHC binding by a peptide indicates that several positions, besides the primary anchor and TCR binding amino acid residues, can be replaced only by a limited number of amino acids, suggesting a critical role of secondary anchor positions (7,8). Synthetic peptides have been used for the induction of specific CTL by immunization either in vitro or in vivo (9)(10)(11)(12)(13), and vaccination with peptides can induce CTL-mediated immune responses against tumors (14,15) and virus-infected cells (16) in mice. Identification of peptides that can elicit an efficient CTL response is, therefore, of practical importance for the development of immunogens for active immunotherapy of cancer.
Several methods have been used to identify unknown CTL epitopes. If the amino acid sequence of a protein antigen is known, overlapping peptides of 8-10 amino acids in length can be synthesized and screened as CTL targets, an approach that has led to the discovery of a number of CTL epitopes derived from proteins of viral origin (17). CTL epitopes may also be identified subsequent to the search for MHC-binding motis and measurement of the affinity of potential antigenic peptides for MHC molecules (18,19). If the tumor antigen is not known, isolation of tumor-associated peptides from MHC class I molecules followed by purification and Edman sequencing (14,20) or mass spectrometry (21) provides a direct way of identifying CTL epitopes. Genetic approaches, such as the screening of DNA expression libraries, provide another alternative and have led to the discovery of several human and mouse antigens recognized by specific CTL (22,23). In many situations, however, because of the complexity and low abundance of the tumor epitopes presented, the application of these techniques is often time-consuming and sometimes problematic. The epitope recognition by CD8 + and MHC class I-restricted CTL can be degenerate. This opens the possibility of using combinatorial peptide libraries to search for peptides that mimic naturally presented epitopes. Analysis of several well-defined peptides that encode viral CTL epitopes by synthetic peptide libraries indicates that only a certain combination of amino acids is required for the interaction between MHC-peptide and TCR, whereas the amino acids in other positions can be changed without affecting function (8,24). It is unclear from these studies, however, whether the peptide analogues can serve as functional mimics of the native peptide. We report here on combinatorial peptide libraries that were screened based on their ability to render RMA-S cells susceptible to lysis by specific CTL against unknown tumor antigens. The EL4 mouse lymphoma system, a model that we have studied in the past (25,26), was used. Using an iterative strategy, effective peptides within the libraries were selected. Syngeneic mice immunized with these peptides generated K b-and D brestricted CTL recognizing EL4 cells in vitro and in vivo.

Materials and Methods
Mice and Cell Lines. Female C57BL/6 mice, 6-8 wks old, were purchased from Taconic Co. (Germantown, NY). The EL4 lymphoma, which is of C57BL/6 (H-2b) origin, was purchased from American Type Culture Collection (Rockville, MD). An EL4 line (B7 + EL4) expressing the costimulatory molecule B7-1 was previously generated in our laboratory by infection of EL4 cells with pLXSN recombinant retrovirus containing murine B7-1 cDNA (25). RMA-S, a peptide transporter-defective lymphoma line (27), is of C57BL/6 origin. P815.K b and P815.D b are clones from the P815 mastocytoma, which had been transfected to express K b or D b genes, respectively (26). All cell lines were maintained in vitro at 37~ in IMEM containing 10% fetal bovine serum (referred to as "medium").
Peptide Library Synthesis. Peptide libraries were synthesized on either a peptide synthesizer (model 430A; Applied Biosystems, Inc., Foster City, CA) using Boc amino acids or on a multiple peptide synthesizer (model AMS 422; Gilson Electrical Med. Inc., Middleton, WI) using Fmoc amino acids (28). Peptide mixtures corresponding to the D b motif were synthesized only by the Fmoc strategy, whereas peptide mixtures corresponding to the K b motif were synthesized by a mixed Fmoc and Boc strategy. The anchor residue positions (5) for K b peptide libraries were fixed at 5 (F, Y) and 8 (L) or for D b peptide libraries at 5 (N) and 9 (L), while all other positions were variable. 9 The strategy for synthesis was the same as previously described (29). Briefly, the variable X position corresponded to an approximately equimolar incorporation of 16 amino acids by a simultaneous coupling of the appropriate mixture which, when Fmoc amino acids were used, was e~(A/L/V/T/F/Y ratio 1:1. . Peptides were cleaved from the resin and deprotected by a 2-h reaction with TFA/water/thioanisole/ ethanedithiol (100:5:5:2.5). After precipitation from cold ether, the precipitates were dissolved in formic acid and either diluted with water and lyophilized (library mixtures), or they were purified as single peptides by reverse-phase HPLC with a gradient of acetonitrile in 0.1% TFA. A previously described Th epitope, HBVc 128-140/I-A b (TPPAYRPPNAPIL) (30), was synthesized and used for immunization. A Kb-binding peptide, KHYKIRNL, from the murine p56 lck sequence (31) was used as a control. Single peptides were analyzed on a Biolon AB mass spectrometer (Uppsala, Sweden).
Peptide libraries with the K b motif were synthesized by the Boc strategy as described previously (29). Free peptides were obtained from the peptide resin by reaction with 90% hydrogen fluoride/10% anisole for 1 h at 0 ~ After evaporation of hydrogen fluoride, the peptide-resin mixture was washed with ethyl acetate, and the peptides were dissolved in 50% acetic acid and obtained as a dry powder after lyophilization from 5% acetic acid. When the peptide libraries had been reduced to a mixture of 64 peptides (two possibilities at each of six positions; Fig. 1 B), peptide synthesis was done by the Fmoc strategy as described above. All subsequent libraries and individual peptides were synthesized by this method.

Isolation and Purification of MHC Class 1-associated Peptides.
Natural peptide mixtures and purified peptides from EL4 cells were prepared from either H-2K b or D b proteins by a combination of affinity chromatography and reverse-phase HPLC, as described (26).
Assays for CTL Activity. Peptide libraries, individual peptides, and peptide fractions eluted from either K b or D b molecules of EL4 cells (26) were reconstituted in PBS and further diluted in medium. RMA-S cells were incubated at 22~ for 48 h, labeled with SlCr at 22~ for 2 h, and incubated with peptides in 96-well V-shaped plates at 37~ for 1.5 h, after which they were used as targets for lysis by CTL. C2D is a CD8 + CTL line specific for EL4 cells and reacts with a single peptide isolated from D b molecules (26). 4A2 is an EL4-specific CD8 + CTL clone that was generated from a mouse that had been immunized with B7 + EL4 cells (25,26). It recognizes a single peptide isolated from K b molecules (see Fig. 3). The CTL activity was examined in a standard 4-h SlCr release assay with different E/T ratios as indicated.

Generation of CTL by Immunization of Mice with Peptides.
C57BL/6 mice were injected subcutaneously with peptides at 100 Ixg/mouse emulsified in IFA, with or without mixing the peptides with 100 Ixg of the HBVc 128-140/I-Ab Th peptide. 14 d later, spleen cells were prepared and stimulated with B7 + EL4 cells for 5 d as described (25). In some experiments, the bulk-cultured spleen cells were restimulated in vitro every 7-10 d, and their cytolytic activity was examined in a standard 4-h SlCr release assay.
Adoptive Transfer of the Mimicking Peptide-induced CTL for Treatment of Ascitic EL4 Tumor. The method of adoptive transfer of CTL for treatment of established tumors has been described previously (32,33). Briefly, C57BL/6 mice, in groups of five, were injected intraperitoneally with 105 EL4 cells. 3 d later, the mice were injected with 5 • 106 cells of an EL4pepI-induced CTL line, followed on day 4 by an injection of 2,000 U i.p. human rlL-2 (Cetus Corp., Emeryville, CA) every day for 3 d. The control groups were injected with either IL-2 alone or IL-2 with a control CTL line that was specific for EL4peplI-A and lost CTL activity in in vitro cultures. The mice were monitored daily for survival.

Results
Strategy for the Synthesis of Combinatorial Peptide Libraries. We applied an iterative strategy (29) by dividing the 16 amino acids into three groups: ec(AL, VT, FY); [3(GS, P, DE); ~/(KR, H, NQ), and each group was further divided into three subgroups as can be noted by the grouping within the parentheses. Four amino acids, C, M, I, and W, were omitted from the library for reasons previously described (29). In addition, M, I, and W were expected to decrease the solubility of the library, possibly to a concentration too low to enable us to detect biological response. The significance of our three-group strategy is that it allowed a simultaneous decrease (to 0) in the proportion of one group, an increase (two-to threefold, as indicated) in the proportion of a second group, and no change in the proportion of the third group at each of the variable residue positions in the peptide library. The effect of these changes on the biological activity of the peptide library indicated which of the groups, or amino acids within the groups, was contributing the most to the observed biological effect. Thus, a decrease in activity indicated that the first group was most important, an increase in activity indicated that the second group was most important, and an unchanged activity indicated that the third group was the most important. A repeat of the iterative strategy with the corresponding subgroups ultimately allowed the deduction of a single, highly active peptide.

Screening of Peptide Libraries for I~-and I~-restricted Epitopes Recognized by EL4-specific CTL.
We first screened the peptide library by a Db-restricted CTL line, C2D, which lyses EL4 cells (26). The starting library was X-X-X-X-N-X-X-X-L, where X corresponded to any of 16 possible amino acids. This library consisted of a mixture of 167 = 268,435,456 peptides. The mixed peptides from this library were loaded onto RMA-S cells whose sensitivity to lysis by the C2D CTL was examined. No significant lysis was observed ( Fig. 1 A, curve 1). In an attempt to increase the solubility of the library, we examined paired substitutions, DE or KR, at each of the variable X positions. As shown in Fig. 1 A (curve 2), substitution of DE at position 7 gave slightly increased activity. Substitution of DE at the other positions had no effect on activity, as was also observed for the corresponding substitution of KR (data not shown).
The library with the DE substitution at position 7 (33,554,432 peptides), X-X-X-X-N-X-DE-X-L, was subjected to an iterative strategy of variation. Each of the X positions was substituted with c~2. This corresponded to a decrease (to 0) in the 13 group amino acids (G, S, P, D, E), an increase (twofold) in the ~/group amino acids (K, R, H, N, Q), and no change in the o~ group of amino acids (A, L, V, T, F, Y). The results of these substitutions are shown in Table 1. Substitution of ot~/2 at positions 1, 6, and 8 had litde effect, indicating that the ot group was preferred at these positions; substitution of or at position 4 decreased activity, indicating that the 13 group was preferred; substitution of ~x~/2 at positions 2 and 3 increased activity, indicating that the ~/ group was preferred. This suggested that a reduced library of 54,000 peptides corresponding to ot-~/-~/-13-N-ot-DE-ot-L would be more active. When a parallel iterative procedure was conducted by substituting 132~/at each of the X positions, the results were in agreement at positions 1, 4, 6, and 8, but differed for positions 2 and 3, where ot was preferred (data not shown). Two possibilities at each of two positions indicated four combinations that 123 Blake et al. needed to be investigated. Each of these libraries was synthesized and the results (data not shown) showed all the new libraries to be more active than the 33,554,432-peptide library. The library with ~/at positions 2 and 3 was the most active and had an activity several orders of magnitude greater than that of the 33,554,432 library ( Fig. 1 A, curve 3). The 54,000-peptide library (ot-~/-~-13-N-ot-DE-ot-L) was subjected to further modifications (Table 2) by a logical division of each of the et, 13, and ~/ groups into their component subgroups. Substitution of (AL)2VT at positions 1, 6, and 8 increased the activity indicating that AL was preferred at each of these positions. Substitution of GSP3 (threefold increase ofproline) at position 4 also showed increased activity indicating that P was preferred at that posi-  liThe effect of o~',/2 amino acid substitution ofpeptide libraries on CTL activity was evaluated in comparison with the library X-X-X-X-N-X-DE-X-L. A decrease in CTL activity indicated tha the 13 group was most important, an increase in activity indicated that the 3' group was most important, and an unchanged activity indicated that the c~ group was the most important. In this round screening, the deduced library was: oL-~/-y-13-N-o~-DE-o~-L.
tion. The corresponding substitutions at positions 2 and 3 gave equivocal results similar to our observation in the first iteration. As seen in Table 2, it was difficult to distinguish between Kt< and NQ. Other data indicated that histidine was preferred at position 3 (data not shown). Thus, there were 2 • 3 = 6 possible combinations to investigate, and each of the corresponding libraries was synthesized. The results indicated that the 32 (25) peptide library AL-NQ-H-P-N-AL-DE-AL-L was the most active ( Fig. 1 A, curve  4). Finally, single amino acid substitutions (Table 3) indicated that the best single peptide was AQHPNAELL (referred to as EL4pepI; Fig. 1 A, curve 5). A comparison of the ability to sensitize RMA-S for lysis by the C2D CTL line between EL4pepI and the various libraries indicates that the activity of EL4pepI was at least 9 logs higher than that of the starting library ( Fig. 1 A). A similar strategy was used to search for a Kb-restricted CTL epitope. The starting library with the K b motif X-X-X-X-FY-X-X-L contained 33,554,432 (2 • 166) peptides. For screening, we used either the anti-EL4 CTL clone 4A2, which had been obtained by limiting dilution cloning of bulk-cultured spleen cells from C57BL/6 mice immunized with B7 § EL4 cells (26) or bulk-cultured polyclonal T cells (25). Paired substitutions of KR, DE, or NS at position 1 showed that the KR substitution slightly increased the activity of the library. The reduced library KP,-X-X-X-FY-X-X-L was subjected to the iterative process. Substitution of each of the five remaining X positions with [~2Y, fol- lowed by subgroup iterations, ultimately led to a single highly active peptide, KHYLFRNL (referred to as EL4peplI). The results are summarized in Fig. 1 B, and the EL4peplI has a potency "~8 logs higher than that of the starting library.
Induction of CTL Specific for EL4 Cells by Immunization with Epitope Mimics. The two peptides EL4pepI and EL4peplI were used in an attempt to generate CTL against EL4 cells by immunizing C57BL/6 mice. Immunization with EL4pepI alone gave a weak CTL response against EL4 cells. However, injection of a mixture of EL4pepI and the helper peptide HBVc 128-140/I-A b elicited a strong CTL activity against EL4 cells, whereas immunization with HBVc 128-140/I-A b alone did not induce any CTL response. The CTL appeared to be specific for EL4 cells, since the P815 mastocytoma line was not lysed. In contrast, immunization of mice with either EL4peplI alone or a combination of EL4peplI and HBVc 128-140/I-A b did not induce any detectable EL4-specific CTL response ( Table 4).
The failure of EL4peplI to elicit an EL4-reactive CTL suggested that it differed in the structure from the naturally processed peptide. We observed that substitution of position 1 with A, L, G, T, S, or N had no effect on the activity in the tLMA-S assay (data not shown), whereas all of the other positions were sensitive to change. Substitution ofalanine at position 2 reduced activity substantially, substitution ofalanine at position 3-8 reduced it much more, and conservative changes at positions 3, 4, 6, and 7 gave peptides with significantly reduced activity (data not shown). When a mixture of four peptides, EL4peplI-A, EL4peplI-L, EL4peplI-G, and EL4peplI-T (corresponding to EL4pepII with the amino terminal lysine substituted with the respective indicated amino acids), was used to immunize mice, CTL that lysed EL4 cells, but not P815.K b cells, could be generated (Fig. 2 A). Further analysis demonstrated that these CTL recognized EL4peplI-A and EL4peplI-T, but not EL4peplI-G or EL4peplI-L (Fig. 2 B). Immunization with EL4peplI-A or EL4peplI-T individually also induced EL4-specific CTL, whereas immunization with the control mlck peptide, which has a similar amino acid composition to EL4peplI and binds to K b molecules, did not (Fig. 2 C). The potency of the EL4peplI variants was further enhanced by immunization *Spleen cells were prepared from the mice immunized with the peptides and were cocultured with B7 § EL4 cells for two cycles of stimulation (7 d for each cycle). The cells were then tested for their CTL activity on EL4 and P815 cells in a standard 4-h SlCr release assay. *C57BL/6 mice were injected subcutaneously with the indicated peptides at 100 Ixg/mouse with or without 100 Ixg of HBVc 128-140/I-A b Th peptide in IFA. ~Not done.
rally processed peptides on EL4 cells, we purified from EL4 cells both K b-and Db-associated peptides. Lysates were prepared from in vitro-cultured EL4 cells, and the peptide-MHC class I complex was purified by affinity chromatography using mAbs to either K b or D b, after which the eluted peptides were fractionated by reverse-phase HPLC. Individual peptide fractions were loaded onto RMA-S cells to sensitize them for lysis by CTL that had been induced by the mimicking peptides. The EL4pepI-induced CTL lysed RMA-S cells pulsed with fraction 8, 9, and 10 of peptides eluted from D b molecules, while they did not react with the other fractions. This recognition pattern was not identical, but similar to that of the C2D CTL line (Fig. 3 A). The EL4pepII-A-elicited CTL, similarly to the 4A2 CTL clone, specifically recognized fraction 9 of peptides eluted from K t' molecules ( Fig. 3 B). Our results demonstrate that immunization of mice with mimicking peptides can induce CTL that cross-react with natural tumor epitopes presented by EL4 cells. In both cases, a standard 4-h SlCr release assay was used. Mice were also immunized with an individual peptide from the control peptide mlck, EL4peplI-A, or EL4pepI-T at 100 I~g, and spleen cell cultures were prepared as described above. The cytolytic activity of the cultured spleen cells was examined against EL4 cells or P815 cells at an E/T ratio of 2:1 (C).

Treatment of an Established Ascites EL4 Tumor by Adoptive
remaining mice were alive and had no sign of tumor on day 140, when the experiment was terminated (Fig. 4). Similar results were obtained in another experiment with five mice per group.

Discussion
Using combinatorial peptide hbraries, we have identified several functionally active peptides that can mimic the epitopes of tumor-associated antigens recognized by K b-and Db-restricted CTL and are specific for the EL4 mouse lymphoma. The mimicking peptides are recognized by CTL that were raised by immunizing mice with B7 + EL4 cells, and they can induce CTL responses against EL4 cells. In principle, this method can be used to construct functional epitope mimics, provided that the presenting MHC molecules and the peptide-binding motif, are known. It thus represents a novel approach for exploring new targets for T ceUmediated immunotherapy of cancer.
Based on the motif and affinity ofpeptides to MHC class I molecules, a number of them have been successfully predicted from known proteins such as potential CTL epitopes (17)(18)(19). However, additional parameters appear to contribute to what constitutes a CTL epitope. For example, some peptides predicted by their motif are nonimmunogenic (34), or they are immunogenic, but the CTL induced by them do not react with naturally presented peptides (19,35). In addition, not all high affinity peptides are immunogenic (19,36). We showed previously that the C2D CTL fine recognizes a subdominant epitope that can only induce a CTL response in the presence of costimulation by B7-CD28 interaction (26). The apphcation of the iterative strategy using a multiple peptide synthesizer for rapid simultaneous synthesis ofhbraries, and the stable Db-restricted CTL clone C2D for screening, enabled us to find a single active peptide (EL4pepI) in ,'o10 wk. The CTL induced by  EL4pepI are MHC class I restricted, express CD8 (data not shown), and can lyse EL4 cells (Table 4) as well as 1LMA-S cells pulsed with naturally presented peptides eluted from D b molecules of EL4 ceils (Fig. 3 A) Days after Tumor Inoculation Figure 4. Treatment of mice with EL4 ascites tumor by adoptive transfer of EL4pepI-induced CTL. C57BL/6, in groups of five, were injected intraperitoneally with 105 EL4 cells. 3 d later, they were injected intraperitoneally with 5 • 10 6 cells of an EL4pepI-induced CTL line followed the next day by an injection of 2,000 U i.p. of human rlL-2; the IL-2 injection was repeated once a day for three consecutive days. Control groups were injected with either IL-2 or IL-2 combined with a control CTL line. The mice were monitored daily for survival, and the experiment was terminated on day 140 after tumor injection.
However, the use of combinatorial peptide libraries represents a rapid and reliable approach to determine the functional mimicries of unknown CTL epitopes. Although EL4pepI itself did not produce a strong CTL response in syngeneic mice, injection of the peptide together with the Th epitope HBVc 128-140/I-A b (30), however, did induce a potent CTL response against EL4 cells (Table 4). Similarly, a P815 tumor-associated nonapeptide (37) did not induce CTL unless a Th epitope was included in the immunization (38). These results underscore the importance of Th cells in the induction of EL4specific CTL by peptides.
EL4pepII was found to be highly active in sensitizing RMA-S cells for lysis by CTL, indicating that it can trigger the cytolytic machinery through TCP, ligation. However, CTL induced by immunization of mice with this peptide did not lyse EL4 cells (Table 4), although they can lyse EL4peplI-pulsed RMA-S cells (Johnston, J.V., J. Blake, and L. Chen, unpublished data). Furthemaore, at least seven analogues of EL4peplI that differed only at position 1 were capable of sensitizing RMA-S cells, but only two of those (EL4pepII-A and EL4pepII-T) elicited CTL that cross-reacted with EL4 cells (Fig. 2). These results suggest that EL4pepII is immunogenic and argues that the failure of the peptide to induce anti-EL4 CTL was not caused by an inappropriate immunization. In many cases, high affinity ofa peptide to the MHC class I molecule is correlated with its immunogenicity (36). Our preliminary experiments showed that EL4pepII-A binds H-2D t' better than EL4-peplI (Johnston, J.V., J. Blake, and L. Chen, unpublished data), suggesting this possibility.
Adoptive transfer of a CTL line from mice immunized with EL4pepI inhibited the growth of EL4 cells as an ascitic tumor and prolonged the survival of the tumor-bearing mice (Fig. 4), indicating that CTL induced by the mimicking peptide can destroy EL4 cells in vivo. Experiments are underway to investigate the effect of the mimicking peptides in the induction of antitumor immunity in vivo. Preliminary data indicate that a weak protective immunity against challenge of EL4 cells can be achieved by simply immunizing with a mixture of EL4peplI-A with IFA. The efficacy of immunization with mimicking peptides can probably be further increased by presenting them in conjunction with costimulation provided by B7-CD28 interaction (39,40) or by immunizing peptide-pulsed dendritic cells (41).
We thank Ingegerd Hellstr6m for her support, P. H6glund for the RMA-S cell line, Becky Woodworth for peptide synthesis, Alison Malacko for purification of eluted peptides, and Stephanie Ashe Newby for assistance in the animal studies.