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Domain Structures and Immunogenic Regions of the 90-kDa Heat-shock Protein (HSP90)

1997; Elsevier BV; Volume: 272; Issue: 42 Linguagem: Inglês

10.1074/jbc.272.42.26179

1083-351X

Takayuki Nemoto, Nobuko Sato, Hiroko Iwanari, Hisahiko Yamashita, Takashi Takagi,

Exercise and Physiological Responses

Domain structures of the 90-kDa heat-shock protein (HSP90) have been investigated with a library of anti-HSP90 monoclonal antibodies (mAbs) and by limited proteolysis with trypsin and chymotrypsin. Thirty-three mAbs were obtained by immunization with bacterially expressed human HSP90α and HSP90β isoforms. Among them, ten and three mAbs reacted specifically with HSP90α and HSP90β, respectively. Immunoblotting and enzyme-linked immunosorbent analyses revealed that major immunogenic domains were located at two restricted regions of HSP90α, i.e. amino acids 227–310 (designated Region I) and 702–716 (Region II), corresponding to a highly charged region and a region near the C terminus, respectively. Taken together with the characteristics of the amino acid sequences, these two immunogenic regions appeared to be exposed at the outer surface of HSP90. We further investigated the domain structures of HSP90 by limited proteolysis in combination with N-terminal sequencing and immunoblotting analyses. Tryptic cleavages of HSP90α at low concentrations revealed the existence of major susceptible sites at Arg400-Glu401, Lys615-Ala616, and Arg620-Asp621. Proteolysis at higher trypsin concentrations caused successive cleavages only toward the N-terminal direction from these sites, and Region I was included in the region selectively deleted during this process, thereby further suggesting its surface location. From these results, we propose three domain structures of HSP90 consisting of amino acids 1–400, 401–615, and 621–732. Differences in the protease sensitivity and immunogenicity further suggest that every domain is composed of two subdomains. This is the first study describing the domain structures and the immunogenic regions of HSP90. Domain structures of the 90-kDa heat-shock protein (HSP90) have been investigated with a library of anti-HSP90 monoclonal antibodies (mAbs) and by limited proteolysis with trypsin and chymotrypsin. Thirty-three mAbs were obtained by immunization with bacterially expressed human HSP90α and HSP90β isoforms. Among them, ten and three mAbs reacted specifically with HSP90α and HSP90β, respectively. Immunoblotting and enzyme-linked immunosorbent analyses revealed that major immunogenic domains were located at two restricted regions of HSP90α, i.e. amino acids 227–310 (designated Region I) and 702–716 (Region II), corresponding to a highly charged region and a region near the C terminus, respectively. Taken together with the characteristics of the amino acid sequences, these two immunogenic regions appeared to be exposed at the outer surface of HSP90. We further investigated the domain structures of HSP90 by limited proteolysis in combination with N-terminal sequencing and immunoblotting analyses. Tryptic cleavages of HSP90α at low concentrations revealed the existence of major susceptible sites at Arg400-Glu401, Lys615-Ala616, and Arg620-Asp621. Proteolysis at higher trypsin concentrations caused successive cleavages only toward the N-terminal direction from these sites, and Region I was included in the region selectively deleted during this process, thereby further suggesting its surface location. From these results, we propose three domain structures of HSP90 consisting of amino acids 1–400, 401–615, and 621–732. Differences in the protease sensitivity and immunogenicity further suggest that every domain is composed of two subdomains. This is the first study describing the domain structures and the immunogenic regions of HSP90. The 90-kDa heat-shock protein (HSP90) 1The abbreviations used are: HSP90, 90-kDa heat-shock protein; GST, glutathione S-transferase; HSP90α and β, the α and β isoforms of HSP90, respectively; H6HSP90, HSP90 tagged with a dodecapeptide (MRGSH6GS); HtpG, an E. coli homolog of mammalian HSP90; ELISA, enzyme-linked immunosorbent assay; mAbs, monoclonal antibodies; PAGE, polyacrylamide gel electrophoresis; BCIP, 5-bromo-4-chloro-3-indolyl phosphate; NBT, nitro blue tetrazolium. is one of the major stress proteins in eukaryotic cells. There are at least two HSP90 genes, and two HSP90 isoform proteins, α and β, are expressed in the cytosolic compartment (1Hickey E. Brandon S.E. Smale G. Lloyd D. Weber L.A. Mol. Cell. Biol. 1989; 9: 2615-2626Crossref PubMed Scopus (243) Google Scholar). The amino acid sequence of HSP90α is 85 (human; see Refs. 1Hickey E. Brandon S.E. Smale G. Lloyd D. Weber L.A. Mol. Cell. Biol. 1989; 9: 2615-2626Crossref PubMed Scopus (243) Google Scholar and 2Rebbe N.F. Ware J. Bertina R.M. Modrich P. Stafford D.W. Gene (Amst .). 1987; 53: 235-245Crossref PubMed Scopus (153) Google Scholar) to 90% (yeast; see Ref. 3Borkovich K.A. Farrelly F.A. Finkelstein D.B. Taulien J. Lindquist S. Mol. Cell. Biol. 1989; 9: 3919-3930Crossref PubMed Scopus (569) Google Scholar) homologous to HSP90β. Either one of the isoforms is indispensable for the growth of yeast cells at higher temperatures (3Borkovich K.A. Farrelly F.A. Finkelstein D.B. Taulien J. Lindquist S. Mol. Cell. Biol. 1989; 9: 3919-3930Crossref PubMed Scopus (569) Google Scholar). Biochemical characterization of purified HSP90 indicates that HSP90α predominantly exists as a homodimer and HSP90β exists mainly as a monomer (4Minami Y. Kawasaki H. Miyata Y. Suzuki K. Yahara I. J. Biol. Chem. 1991; 266: 10099-10103Abstract Full Text PDF PubMed Google Scholar). Dimer formation is mediated by the interaction at the C-terminal 191 amino acids in which the C-terminal region (Met628-Asp732) of one subunit associates with the adjacent region (Val542-Tyr627) of the other subunit (5Nemoto T. Ohara-Nemoto Y. Ota M. Takagi T. Yokayama K. Eur. J. Biochem. 1995; 233: 1-8Crossref PubMed Scopus (163) Google Scholar). The amino acid substitutions at 561–685 between α and β isoforms are responsible for the impeded dimerization of HSP90β (5Nemoto T. Ohara-Nemoto Y. Ota M. Takagi T. Yokayama K. Eur. J. Biochem. 1995; 233: 1-8Crossref PubMed Scopus (163) Google Scholar). HSP90 is believed to have a chaperone-like activity for particular molecules that are involved in signal transduction, such as steroid receptors (6Joab I. Radanyi C. Renoir M. Buchou T. Catelli M.-G. Binart N. Mester J. Baulieu E.-E. Nature. 1984; 308: 850-853Crossref PubMed Scopus (339) Google Scholar), casein kinase II (7Doughtery J.J. Rabideau D.A. Iannotti A.M. Sullivan W.P. Toft D.O. Biochim. Biophys. Acta. 1987; 927: 74-80Crossref Scopus (70) Google Scholar), pp60 v-src (8Brugge J.S. Erikson E. Erikson R.L. Cell. 1981; 25: 363-372Abstract Full Text PDF PubMed Scopus (220) Google Scholar), elF2α kinase (9Rose D.W. Wettenhall R.E.H. Kudlicki W. Kramer G. Hardesty B. Biochemistry. 1987; 26: 6583-6587Crossref PubMed Scopus (97) Google Scholar), and aryl hydrocarbon receptor (dioxin receptor) (10Perdew G.H. J. Biol. Chem. 1988; 263: 13802-13805Abstract Full Text PDF PubMed Google Scholar). HSP90 specifically binds to these proteins; and, in most cases, this interaction is essential for the function of the proteins (9Rose D.W. Wettenhall R.E.H. Kudlicki W. Kramer G. Hardesty B. Biochemistry. 1987; 26: 6583-6587Crossref PubMed Scopus (97) Google Scholar, 11Nemoto T. Ohara-Nemoto Y. Denis M. Gustafsson J.-Å. Biochemistry. 1990; 29: 1880-1886Crossref PubMed Scopus (88) Google Scholar, 12Miyata Y. Yahara I. J. Biol. Chem. 1992; 267: 7042-7047Abstract Full Text PDF PubMed Google Scholar). However, a variety of evidence, i.e.the abundance of HSP90 in cells even under nonstressed conditions, the conserved amino acid sequences from prokaryotic to eukaryotic cells (13Bardwell J.C.A. Craig E.A. Proc. Natl. Acad. Sci. U. S. A. 1987; 84: 5177-5181Crossref PubMed Scopus (151) Google Scholar), and the indispensability in yeast (3Borkovich K.A. Farrelly F.A. Finkelstein D.B. Taulien J. Lindquist S. Mol. Cell. Biol. 1989; 9: 3919-3930Crossref PubMed Scopus (569) Google Scholar), strongly suggests that HSP90 is involved in more fundamental functions of cells. In fact, several studies have recently shown that HSP90 functions as a general chaperone. That is, it interacts with various proteins less specifically and modulates their conformation. For instance, the refolding of citrate synthase is significantly enhanced by the co-presence of HSP90 (14Wiech H. Buchner J. Zimmermann R. Jakob U. Nature. 1992; 358: 169-170Crossref PubMed Scopus (445) Google Scholar). The spontaneous refolding of denatured dihydrofolate reductase and irreversible denaturation of firefly luciferase are prevented by association with HSP90 (15Yonehara M. Minami Y. Kawata Y. Nagai J. Yahara I. J. Biol. Chem. 1996; 271: 2641-2645Abstract Full Text Full Text PDF PubMed Scopus (150) Google Scholar). The latter study further demonstrated that the chaperone-like activity of HSP90 is closely related to the oligomerization of HSP90 at temperatures higher than 46 °C. Although HSP90 possesses an ATPase activity (16Nadeau K. Das A. Walsh C.T. J. Biol. Chem. 1993; 268: 1479-1487Abstract Full Text PDF PubMed Google Scholar), this activity does not appear to be needed for the oligomerization of HSP90. Epitope mapping is a useful approach for investigating the structures of proteins of interest. Several anti-HSP90 monoclonal antibodies (mAbs) have been produced to date (6Joab I. Radanyi C. Renoir M. Buchou T. Catelli M.-G. Binart N. Mester J. Baulieu E.-E. Nature. 1984; 308: 850-853Crossref PubMed Scopus (339) Google Scholar, 10Perdew G.H. J. Biol. Chem. 1988; 263: 13802-13805Abstract Full Text PDF PubMed Google Scholar, 17Riehl R.M. Sullivan W.P. Vroman B.J. Bauer V.J. Pearson G.R. Toft D.O. Biochemistry. 1985; 24: 6586-6591Crossref PubMed Scopus (133) Google Scholar). Although the epitopes of several anti-HSP90 mAbs have been described (18Cadepond F. Jibard N. Binart N. Schweizer-Groyer G. Segard-Maurel I. Baulieu E.-E. J. Steroid Biochem. Mol. Biol. 1994; 48: 361-367Crossref PubMed Scopus (19) Google Scholar), systematic assignment of the immunogenicity of HSP90 has not been conducted. In this study, we developed a library of the mAbs that specifically recognize human HSP90 and with them demonstrated that the major immunogenic domains are located in two restricted regions. The domain structures of HSP90 were further analyzed by limited proteolysis. We finally proposed three domain structures of HSP90. In addition, this is the first report of developing isoform-specific mAbs against HSP90. The expression vectors (pGEX-2T, pGEX-3X, and pGEX-4T-1), glutathione-Sepharose 4B, and low molecular weight markers were purchased from Pharmacia Biotech Inc.(Uppsala, Sweden). The plasmids, pQE-9 and pREP4, were obtained from Qiagen Inc. (Chatsworth, CA). TalonTM metal affinity resin was purchased fromCLONTECH. Phage display system was purchased from New England Biolabs Inc. (Beverly, MA). Alkaline phosphatase-conjugated goat anti-mouse IgG was from Bio-Rad. Alkaline phosphatase-conjugated goat anti-mouse IgM and peroxidase-conjugated goat anti-mouse (IgG + IgM) were from EY Laboratories Inc. (San Mateo, CA). AC88, a mAb against HSP90 (17Riehl R.M. Sullivan W.P. Vroman B.J. Bauer V.J. Pearson G.R. Toft D.O. Biochemistry. 1985; 24: 6586-6591Crossref PubMed Scopus (133) Google Scholar), was purchased from Stressgen Biotech Corp. (Victoria, B.C., Canada). Trypsin (5,200 USP units/mg protein), α-chymotrypsin (11,000 N-acetyl-l-tyrosine etyl ester-hydrolyzing units/mg protein),N-tosyl-l-phenylalanine chloromethyl ketone, andN-α-tosyl-l-lysine chloromethyl ketone were purchased from Sigma. Rainbow markers were from Amersham Corp. 5-Bromo-4-chloro-3-indolyl phosphate (BCIP) and nitro blue tetrazolium (NBT) were purchased from both Promega (Madison, WI) and Kirkegaard & Perry Lab. (Gaithersburg, MD). All other reagents were of analytical grade. Amino acid numbers are presented as those of human HSP90α (732 amino acids) throughout this study, in which the amino acid sequence of HSP90β is aligned with that of HSP90α in consideration of the eight amino acid deletions corresponding to amino acids 1–5 and 238–240 of HSP90α. Full-length forms of human HSP90α and HSP90β were expressed in Escherichia coliY1090 as glutathione S-transferase (GST)-fusion proteins, and the recombinant proteins were purified as described (5Nemoto T. Ohara-Nemoto Y. Ota M. Takagi T. Yokayama K. Eur. J. Biochem. 1995; 233: 1-8Crossref PubMed Scopus (163) Google Scholar). GST-HSP90α or β encoding amino acids 535–732 and their chimeric proteins, GST-HSP90α458–732 and GST-HSP90α1–43/604–732, were expressed as described previously (5Nemoto T. Ohara-Nemoto Y. Ota M. Takagi T. Yokayama K. Eur. J. Biochem. 1995; 233: 1-8Crossref PubMed Scopus (163) Google Scholar). Other expression vectors with deleted forms of HSP90 isoforms were constructed by the cleavage of pGST-HSP90α or β with appropriate restriction enzymes. H6HSP90α, a recombinant HSP90α tagged with a dodecapeptide (MRGSH6GS), was expressed as described (19Nemoto T. Matsusaka T. Ota M. Takagi T. Collinge D.B. Walter-Larsen H. J. Biochem. 1996; 120: 249-256Crossref PubMed Scopus (31) Google Scholar). pQE-9 series of expression plasmids, i.e.pH6HSP90α542–732, α542–728, α542–720, and α542–697, were prepared by the polymerase chain reaction technique with appropriate primers as described previously (5Nemoto T. Ohara-Nemoto Y. Ota M. Takagi T. Yokayama K. Eur. J. Biochem. 1995; 233: 1-8Crossref PubMed Scopus (163) Google Scholar). The details on the constructions are available on request. H6HSP90αs were purified with TalonTM metal affinity resin according to the manufacturer protocol. Following thrombin cleavage of full-length forms of GST-HSP90α and β, the HSP90 moieties were purified by DEAE ion-exchange HPLC as described (20Nemoto T. Ohara-Nemoto Y. Ota M. J. Biochem. 1987; 102: 513-523Crossref PubMed Scopus (26) Google Scholar). The hybridoma cells producing anti-HSP90 mAbs were prepared according to Oli and Herzenberg (21Oli V.T. Herzenberg L.A. Mishell B.B. Shiigi S.M. Selected Methods in Cellular Immunology. W. H. Freeman Co., San Francisco1984: 351-572Google Scholar). The fusions were performed separately with the mice injected with HSP90α and HSP90β. Anti-HSP90 mAb-producing hybridomas were selected by use of an enzyme-linked immunosorbent assay (ELISA) with purified HSP90α or β (0.5 μg) coated on each well of a 96-well titer plate (Falcon). Peroxidase-conjugated anti-mouse (IgG + IgM) antibodies were used as second antibodies. Absorbance at 492 nm was measured following incubation with O-phenylenediamine for 1 h at 30 °C. Positive hybridomas were cloned by limiting dilution. Large scale production of mAbs was carried out by growing the hybridoma cells in ascites of mice. The immunoglobulin fraction was precipitated with 50% (w/v) ammonium sulfate, and the precipitate was dialyzed against saline containing 0.1% sodium azide. K3700 and K41000 number series indicate the mAbs produced from the hybridoma cells of mice immunized with HSP90β and HSP90α, respectively. When more than one hybridoma colony grew up in a culture well, they were separated at the following screening step. Those mAbs were distinguished by addition of alphabets A–D. Their independence was confirmed by characterization of the biochemical properties. Class and subclass of the heavy chain of the mAbs were determined by the micro-Ouchterlony method. Proteins were subjected to SDS-PAGE at a polyacrylamide concentration of 12.5 or 15%. Separated proteins were stained with Coomassie Brilliant Blue or transferred to a polyvinylidene difluoride membrane (Millipore Corp.). The membrane was incubated with anti-HSP90 mAbs (0.1–0.5 μg/ml) at 25 °C for 2 h. Alkaline phosphatase-conjugated goat anti-mouse IgG or IgM was used as the second antibody at a 1:5000 dilution in 50 mm Tris-HCl (pH 7.6), 0.15 m NaCl (TBS) containing 0.05% Tween 20 and 0.25% bovine serum albumin. After having been washed with TBS containing 0.1% Tween 20, blots were visualized by incubation with BCIP and NBT. Low molecular weight markers and rainbow markers were used as standards for Coomassie staining and immunoblotting, respectively. The regions recognized by the mAbs were investigated by ELISA and immunoblotting with various forms of HSP90s fused to GST. The purified protein (0.5 μg) coated on each well of a 96-well titer plate was incubated with 0.3 μg of purified mAbs in phosphate-buffered saline (pH 7.3). Alkaline phosphatase-conjugated goat anti-mouse IgG or anti-mouse IgM was used as the second antibody at a 1:5000 dilution. BCIP and NBT in a soluble buffer system (Kirkegaard & Perry Lab.) were used as the substrates, and absorbance at 600 nm was measured following a 1-h incubation at 25 °C. A noncleavable octapeptide library covering amino acids 212–312 of HSP90α was prepared according to the manufacturer method (Chiron Mimotopes Pty. Ltd. Clayton, Victoria, Australia). Octapeptides with their N terminus shifted sequentially to each amino acid residue from 212 to 305. The reactivity of the mAbs to the peptides was determined by ELISA as described above. Proteins in 50 mm Tris-HCl (pH 8.0) containing 100 mm NaCl and 10% glycerol were incubated at various concentrations of trypsin (N-tosyl-l-phenylalanine chloromethyl ketone treated) or chymotrypsin (N-α-tosyl-l-lysine chloromethyl ketone treated) at 30 °C for 6 h. Proteolytic peptides (13 μg/lane) were denatured, separated by SDS-PAGE, and transferred to a polyvinylidene difluoride membrane (Bio-Rad). The protein bands visualized with Coomassie Brilliant Blue were subjected to N-terminal sequencing with a model 477A protein sequencer (Applied Biosystems) equipped with an on-line model 120A analyzer. Amino acid sequence with an affinity to an anti-HSP90 mAb (K41007) was determined by use of the phage display system with a heptapeptide library according to the manufacturer protocol. At each step of panning, 2 × 1011 phages were incubated with 15 μg of the mAb precoated on a well of a 96-well Maxisorp plate (Nunc). Following four pannings, the phages were amplified and purified, and then the amino acid sequence of the heptapeptide was deduced from the nucleotide sequence. We obtained 33 independent mAbs against human HSP90 isoforms. For determination of the isoform specificity of the mAbs, ELISA was performed with various amounts of HSP90α and HSP90β (0.07–150 ng). According to the results, we tentatively classified the mAbs into the following three classes: mAbs with cross-reactivity to the other isoform of less than 1% were isoform-specific; mAbs with the cross-reactivity to the other isoform from 1 to 66.7% were isoform-preferential; and mAbs reacting with the other isoform with more than 66.7% efficiency were equivalently recognizing ones (TableI). Unexpectedly, K3725A, obtained from a mouse immunized with HSP90β, was specific for HSP90α on the basis of ELISA.Table ICharacteristics of the mAbs against human HSP90No.Name1-aAccording to the results, isoform specificities of mAbs except K3725A are represented as follows: bold letters, HSP90α specific; not modified, HSP90α preferential; bold italic, HSP90β specific; italic, HSP90β preferential; underlined, bound to both isoforms equivalently.AntigenELISAImmunoblottingSpecificity1-bJudged by ELISA and immunoblotting.ClassReactivity to HSP90αReactivity to HSP90βReactivity to HSP90αReactivity to HSP90β1K41007HSP90α1000++−αIgG12K41346HSP90α1000.1++−αIgG13K41241HSP90α1000.2++−αIgG14K41233HSP90α1000.3++−αIgG15K41320HSP90α1000.3++−αIgG2a6K41122AHSP90α1000.4++−αIgG2b7K41020HSP90α1000.4++−αIgG18K41116AHSP90α1000.6++−αIgG2b9K41107HSP90α1000.9++−αIgG2b10K41009HSP90α1001++−αIgG2a11K41315HSP90α1001.9++±α > βIgG2b12K41102HSP90α1005.1++±α > βIgG2a13K41028HSP90α1005.5++±α > βIgG114K41016HSP90α10030.3+++α > βIgG2a15K41218HSP90α10039.7++++α > βIgG116K41002HSP90α10090.5++++α = βIgG117K41116CHSP90α10094.9++++α = βIgM18K41220HSP90α10097.1++++α = βIgG2019K41110HSP90α88.8100++++α = βIgM20K41338HSP90α87.2100++++α = βIgG121K41322HSP90α81.3100++++α = βIgG2b22K41331HSP90α75.1100++++α = βIgG2b23K41122BHSP90α62.9100++++α < βIgG124K3725A1-cNot defined because the results of ELISA and immunoblotting were not compatible.HSP90β1000+++ND1-cNot defined because the results of ELISA and immunoblotting were not compatible.IgM25K3720HSP90β10079.2++++α = βIgG126K3725DHSP90β10090++++α = βIgG127K3738HSP90β53.5100++++α < βIgM28K3729HSP90β49.6100++++α < βIgM29K3716HSP90β4.5100±++α < βIgM30K3714HSP90β2.8100±++α < βIgM31K3705HSP90β0.5100−++βIgM32K3701HSP90β0100−++βIgM33K3725BHSP90β0100−++βIgM1-a According to the results, isoform specificities of mAbs except K3725A are represented as follows: bold letters, HSP90α specific; not modified, HSP90α preferential; bold italic, HSP90β specific; italic, HSP90β preferential; underlined, bound to both isoforms equivalently.1-b Judged by ELISA and immunoblotting.1-c Not defined because the results of ELISA and immunoblotting were not compatible. Open table in a new tab The isoform specificity was further investigated by immunoblotting with bacterially expressed HSP90α and HSP90β. The immunoblotting data were in accord with the isoform specificities of the mAbs estimated by ELISA with the exception of K3725A (Table I). K3725A bound to both HSP90α and HSP90β on immunoblotting. As a result, isoform specificities of the mAbs were assigned as shown in Table I. We obtained ten (numbers 1–10) and three (numbers 31–33) mAbs that specifically interacted with HSP90α and β, respectively; five mAbs (numbers 11–15) that preferentially interacted with HSP90α; five mAbs (numbers 23 and 27–30) that preferentially interacted with HSP90β; and nine mAbs (numbers 16–22, 25, and 26) that were equivalently reactive to both isoforms. The isoform specificity of K3725A (No. 24) remained to be established. Immunoglobulin class and IgG subclass of the mAbs were also determined (Table I). Next we determined the regions of HSP90 recognized by the mAbs by using various deletion mutants of HSP90α (Fig. 1 a) and HSP90β. Most forms were purified to homogeneity by affinity chromatography on SDS-PAGE (data not shown). However, several forms revealed additional bands on SDS-PAGE. Even in such cases, the positions of the expressed proteins were defined by comparison with calculated molecular masses. Bands with molecular masses lower than expected seemed to be proteolytic products of the recombinant proteins. The purified samples were used for ELISA; whereas, the immunoblotting analysis was done with the bacterial lysates containing the expressed proteins to avoid proteolysis during the purification step. Twelve forms of recombinant HSP90αs were immunoblotted with K41107, a mAb specific for HSP90α. As shown in Fig. 1 b(left), specific bands and their degraded ones were observed in lanes 1, 3, and 6-8. Blots of HSP90α1–285, HSP90α121–312, and HSP90α216–312 (lanes 6–8) indicated that the epitope for K41107 resided within the sequence of amino acids 216–285. This result was further confirmed by ELISA (Fig. 1 b, right, columns 6-8). K41122B, a mAb that bound to the two isoforms equivalently, reacted with HSP90α216–312 and HSP90α1–47/290–312 (Fig. 1 c, lanes 8 and 9), indicating its binding to a short segment of 23 amino acids (290–312). We performed the epitope mappings with the rest of the mAbs by the same procedures. The full-length form and four deleted forms of HSP90β were used for the investigation of the mAbs specifically or preferentially bound to HSP90β. In consequence, their recognition sites were defined as shown in Table II. These observations demonstrated that the epitopes defined by most mAbs were located in particular regions: the epitopes recognized by 25 out of the 33 mAbs (76%) were found within amino acids 185–335, most probably in 216–312. We tentatively designated this region as Region I. The second one, designated Region II, seemed to be found at the C-terminal region at 533–732. As expected, the isoform specificity of mAbs seemed to be dependent on the degree of amino acid substitutions in the recognized region: mAbs recognizing conserved sites, such as amino acids 290–312, had no or little isoform preference; and amino acids 216–285 containing the least conserved region (amino acids 238–273, 47% homology between HSP90α and HSP90β) were recognized by isoform-specific or highly isoform-preferential mAbs (Table II).Table IIRegions of amino acid sequences recognized by anti-HSP90 mAbsHSP90mAbs2-aIsoform specificities of mAbs are presented as described in Table I.Immunogenic region2-bThe regions recognized by multiple mAbs are tentatively designated Regions I and II.48–196K41218185–335K3701K3716K3729K3738|Region I216–285K41020K41107K41116AK41122A|K41233K41241K41320K41346|K41028K41102K41315||290–312K3720K3725DK41002K41110|K41116CK41220K41322K41331|K41338K41122B|313–458K41016533–6032-cDistinct from Region II, the boundary of which (702–716) was defined in Figs. 4 and 5.K3725A2-dThe isoform specificity remains to be determined.535–719K3705K3725BK3714|Region II604–732K41007K1009|The recognition regions described as amino acid numbers of HSP90α were determined by immunoblotting analysis and ELISA using either twelve recombinant HSP90αs or five HSP90βs, according to the specificity of the mAbs.2-a Isoform specificities of mAbs are presented as described in Table I.2-b The regions recognized by multiple mAbs are tentatively designated Regions I and II.2-c Distinct from Region II, the boundary of which (702–716) was defined in Figs. 4 and 5.2-d The isoform specificity remains to be determined. Open table in a new tab The recognition regions described as amino acid numbers of HSP90α were determined by immunoblotting analysis and ELISA using either twelve recombinant HSP90αs or five HSP90βs, according to the specificity of the mAbs. Immunoblotting analysis and ELISA indicated specific localization of the immunogenic region (Region I) most probably within amino acids 216–312. Because the region consisted of ∼100 amino acid residues, we prepared 94 octapeptides of which N termini were shifted in every amino acid from 212 to 305 of HSP90α. ELISA with the octapeptide library was performed with all mAbs recognizing Region I except K3701 and K3716, which did not react with the α isoform (Table I). As shown in Fig.2, two-thirds of the mAbs (15/23) revealed single binding peaks at their respective positions; and accordingly, their epitopes were clearly defined. On the other hand, two binding peaks were observed with K3725D, K3738, and K41331; three peaks with K41110 and K41116C; and four major peaks with K3729 (Fig. 2,c and d). No bindings were observed with K41002 and K41028 (Fig. 2 d). With four mAbs (K3725D, K41110, K41116C, and K41331) among those that revealed multiple peaks, the peaks were close together (Fig. 2, c and d). This suggested that more than eight amino acids were involved in recognition of the mAbs and that several combinations of amino acids less than eight residues were sufficient for the bindings. K3738, an HSP90β-preferential mAb, revealed two peaks relatively distant from each other (Fig. 2 d, open squares). However, the two binding regions (DDEAEEKEDKEEE, 232–244 and EEEKEKEEKESEDK, 242–255) could not be separated when all peptides interacting with the mAb were aligned. That is, the last peptide (amino acids 237–244) forming the first peak overlapped the first peptide (242–249) forming the second peak. In addition, it should be remembered that the hybridoma producing K3738 was derived from a mouse immunized with the β isoform. HSP90β has a three-amino acid deletion corresponding to 238–240 of HSP90α. Moreover, the two binding regions are considerably similar to each other; and thus, we postulate that either the deletion, the similarity between the two regions, or both explain the two binding peaks of K3738. Accordingly, we prudently propose that the epitope of K3738 is localized within amino acids 232–255. Unexpectedly, another HSP90β-preferential mAb, K3729, bound to various peptides at completely distinct positions,i.e. DKEVSDDEAEEK (227–238), IEDVGSDEEEEKK (258–270), EKYIDQEELN (282–291), and PDDITNEEYG (301–310) (Fig. 2 d). Again, there are sequence similarities between amino acids 227–238 and 258–270 and between 282–291 and 301–310. If not all, this may explain the multiple binding peaks of K3729. The results of ELISA with the octapeptide library clearly showed that the boundaries of Region I were amino acids 227 and 310. In addition, three sites, designated Sites Ia, Ib, and Ic, emerged as the most immunogenic regions. Site Ia (247–257) was recognized by K41116A, K41122A, K41320, and K41102; Site Ib (263–270) was recognized by K41020, K41107, K41233, K41241, and K41315; and Site Ic (291–304) was recognized by K3720, K41110, K41116C, K41220, K41322, K41331, K41338, and K41122B. The epitopes of K41346 and K3738 overlapped with Site Ia. K3729 recognized four sites (227–238, 258–270, 282–291, and 301–310) in which overlapping of the second site with Site Ib, the third one with the epitope of K3725D (280–291), and the fourth one with Site Ic occurred. These results indicate that the mAbs recognizing Region I bound to one of Sites Ia–Ic or to the regions that overlapped with them (Fig. 3). We also characterized the recognition sites at the C-terminal region by immunoblotting analyses of both chimeric proteins and deletion mutants. First, because the mAbs recognizing this region were specific or highly preferential to either one HSP90 isoform or the other (Table II), we investigated the recognition sites with chimeric proteins of the two isoforms (Fig.4 a). Although all chimeric proteins migrated at the same position on SDS-PAGE, they were clearly distinguished by the immunoblotting. K41009, an HSP90α-specific mAb, bound to HSP90β535–700/α701–732 but did not do so to the counter chimera (Fig. 4 b, lanes 3 and 4). On the other hand, K3705, an HSP90β-specific mAb, bound to HSP90α535–700/β701–732 (Fig. 4 c, lane 3)