Identification of the pentapeptide constituting a dominant epitope common to all eukaryotic heat shock protein 90 molecular chaperones.
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.
Kishimoto
Jun
J
Division of Oral and Maxillofacial Surgery, Department of Developmental and Reconstructive Medicine, Course of Medical and Dental Sciences, Nagasaki University Graduate School of Biomedical Sciences, Japan.
Fukuma
Yutaka
Y
Mizuno
Akio
A
Nemoto
Takayuki K
TK
D000595:Amino Acid Sequence; D000818:Animals; D000906:Antibodies / Q000378:metabolism; D001426:Bacterial Proteins / Q000235:genetics / Q000276:immunology; D000939:Epitopes; D005656:Fungal Proteins / Q000235:genetics / Q000276:immunology; D018841:HSP90 Heat-Shock Proteins / Q000235:genetics / Q000276:immunology*; D006801:Humans; D008297:Male; D018832:Molecular Chaperones / Q000235:genetics / Q000276:immunology*; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D010455:Peptides / Q000235:genetics / Q000276:immunology*; D010940:Plant Proteins / Q000235:genetics / Q000276:immunology; D051381:Rats; D017208:Rats, Wistar; D011994:Recombinant Proteins / Q000235:genetics / Q000378:metabolism; D016415:Sequence Alignment; D014020:Tissue Extracts
D000906:Antibodies; D001426:Bacterial Proteins; D000939:Epitopes; D005656:Fungal Proteins; D018841:HSP90 Heat-Shock Proteins; D018832:Molecular Chaperones; D010455:Peptides; D010940:Plant Proteins; D011994:Recombinant Proteins; D014020:Tissue Extracts