Identification of the pentapeptide constituting a dominant epitope common to all eukaryotic heat shock protein 90 molecular chaperones

Abstract

We previously reported that, in human heat shock protein (Hsp) 90 (hHsp90), there are 4 highly immunogenic sites, designated sites Ia, Ib, Ic, and II. This study was performed to further characterize their epitopes and to identify the epitope that is potentially common to all members of the Hsp90 family. Panning of a bacterial library carrying randomized dodecapeptides revealed that Glu251-Ser-X-Asp254 constituted site Ia and Pro295-Ile-Trp-Thr-Arg299, site Ic. Site II (Asp701-Pro717) was composed of several epitopes. When 19 anti-hHsp90 monoclonal antibodies (mAbs) were subjected to immunoblotting against recombinant forms of 7 Hsp90-family members, 2 mAbs (K41110 and K41116C) that recognized site Ic bound to yeast Hsp90 with affinity identical to that for hHsp90, and 1 mAb (K3729) that recognized Glu222-Ala23, of hHsp90beta could bind to human 94-kDa glucose-regulated protein (Grp94), an endoplasmic reticulum paralog of Hsp90. Among the 5 amino acids constituting site Ic, Trp297 and Pro295 were essential for recognition by all anti-site-Ic mAbs, and Arg299 was important for most of them. The necessity of Ile296, Thr298, and Arg299, which are replaced by Leu, Met/Leu, and Lys, respectively, in some eukaryotic Hsp90, was dependent on the mAbs, and K41110 and K41116C could react with Hsp90s carrying these substitutions. From these data taken together, we propose that the pentapeptide Pro295-Ile-Trp-Thr-Arg299 of hHsp90 functions as an immunodominant epitope common to all eukaryotic Hsp90.

Fig. 1.

Epitope map of the mAbs developed against hHsp90β - Recognition sites of 33 and anti-hHsp90 mAbs are represented along with the 732 amino acid hHsp90α. N, M, and C represent N-terminal, middle, and C-terminal domains, respectively (Nemoto et al 1997). The hatched box (residues 223–289) represents highly charged region localized in the N-ternimal domain. Isoform specificity of mAbs except for K3725A is indicated by their letters as follows: bold, hHsp90α specific; not modified, hHsp90α preferential; bold italic, hHsp90β specific; italic, hHsp90β preferential; and underlined, bound to both isoforms equivalently. The recognition sites of mAbs were identified as epitopes (indicated by boxes) or regions carrying the epitopes (indicated by bars). Highly immunogenic regions, sites Ia, Ib, Ic and II correspond to residues 247–257, 263–270, 291–304, and 702–716, respectively. Thirty mAbs were tested for panning as described in “MATERIALS AND METHODS”: Amino acids responsible for the recognition were identified for 14 mAbs (indicated by *); Nine mAbs did not yield positive clones on immunoblotting (−); Seven mAbs yielded clones positive on immunoblotting, but the sequences they recognized either resembled each other nor matched the sequence of hHsp90s (±). Two mAbs without marks were not subjected to the panning. ^aprecisely defined by the panning experiment in this study (see Tables 5–8). ^bdetermined by phage display system in a previous study (Nemoto et al 1997 and see Table 4)

Fig. 2.

ELISA of mAbs that could interact with yHsc82 or hGrp94 - (a) Bindings of the 4 mAbs that could blot yHsc82 as intensively-stained bands were quantified by ELISA. (b) The bindings of K3729 was quantified by ELISA. Symbols for the 7 Hsp90-family proteins are the following: hHsp90α (open circle); hHsp90β (closed circle); hGrp94 (open square); hTrap1 (closed square); yHsc82 (open triangle); EcHtpG (closed triangle); and PgHtpG (closed inverted triangle)

Fig. 3.

Effect of mutation at Ile₂₉₆, Trp₂₉₇, Thr₂₉₈, and Arg₂₉₉ on the antibody binding - (a) Aliquots of ourified hHsp90α-N wt (lane 1), Trp 297Gly (lane 2), Thr298Met (lane 3), Ile296LeuThr298Met (lane 4), and Afg299Gly (lane 5) were separated on SDS-PAGE and then stained with Coomassie brilliant blue (CBB) or subjected to the immunoblotting with K41110 or K41116C. Lane M, molecular markers. (b–d) ELISA was performed with hHsp90α-N wt (open circle), Trp297Gly (closed circle), Thr298Met (open square), and Ile296Leu/Thr298Met (open triangle) as antigens. Groups 1–3 were categorized based on the reactivity toward yHsc82 (see “RESULTS”)

Fig. 4.

Effect of mutation at Arg₂₉₉ on the antibody binding - ELSIA was performed with hHsp90α-N wt (open circle) and Arg299Gly (closed circle) as antigens by using 8 anti-site-Ic mAbs. Groups 1– 3 were categorized based on the reactivity toward yHsc82

Fig. 5.

Immunoblotting analysis of the Hsp90-family proteins endogenously expressed in eukaryotes - Tissue or cell pellets of several species were denatured in SDS-sample buffer, and aliquots (5 μl) were loaded onto SDS-PAGE gels. Separated proteins were immunoblotted with K41110, K41116C, K41220 or AC88/SPA-830 (Stressgen). Lane 1, rat liver; lane 2, budding yeast (S. cerevisiae); lane 3, fission yeast (S. pombe); lane 4, rice (O. sativa) and lane 5, P. caudatum. Lane M, kaleidoscope prestained standards