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 Glu 251 -Ser-X-Asp 254 constituted site Ia and Pro 295 -Ile-Trp-Thr-Arg 299 , site Ic. Site II (Asp 701 -Pro 717 ) 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 Glu 222 -Ala 231 of hHsp90␤ could bind to human 94-kDa glucose-regulated protein (Grp94), an endoplasmic reticulum paralog of Hsp90. Among the 5 amino acids constituting site Ic, Trp 297 and Pro 295 were essential for recognition by all anti-site-Ic mAbs, and Arg 299 was important for most of them. The necessity of Ile 296 , Thr 298 , and Arg 299 , 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 Pro 295 -Ile-Trp-Thr-Arg 299 of hHsp90 functions as an immunodominant epitope common to all eukaryotic Hsp90.

HSC90 is required for nascent hepatitis C virus core protein stability in yeast cells

“…Mutant yeast cells with a disrupted HSC82 or HSP82 gene harboring pRS425‐GAL1‐core were cultured with (+) or without (−) 3% galactose for 72 h (A) or 2 h (C–E) at 30 °C. We used a monoclonal antibody that reacts with the Ic epitope common to Hsc82 and Hsp82 [14].…”

“…We examined the Core by western blotting as described previously [12] and in the Supplemental Information. We used primary antibodies specific for actin (sc‐1616; Santa Cruz Biotechnology, Santa Cruz, CA, USA), Hsp90 (K41110, from Dr. Nemoto) [13,14], and Core (515S) [15].…”

Heat Shock Proteins 90 kDa: Immunomodulators and Adjuvants in Vaccine Design Against Infectious Diseases

“…Hsp90 protein contains three well-defined domains: N-, middle, and C-terminal (Buchner, 1999 ; Krishna and Gloor, 2001 ; Kishimoto et al, 2005 ; Kumar et al, 2007 ; Kadota and Shirasu, 2012 ). The N-terminal domain of Hsp90 is the most conserved among the studied species (Johnson, 2012 ), and additional studies using the N-terminal domain of eukaryotic gp96 demonstrated that this domain has immunomodulatory activity and that residues 34–222 are sufficient to retain the bound peptide (Gidalevitz et al, 2004 ).…”

Discovery of Targeted Material Binding Microorganisms Using a Centrifugal Microfluidic Platform

“…Several variations of this approach exist, utilizing a variety of display libraries, such as phage display, [ 7–9 ] yeast display, [ 10,11 ] and bacterial display. [ 12–16 ] Bacterial display systems, typically created in Escherichia coli (E. coli) , have been extensively utilized due to the relative ease of manipulation, rapid growth rate, and direct cell surface peptide expression characteristics. In contrast to phage display, the genetic material is passed on directly to the next sorting round during cell regrowth, rather than requiring reinfection of bacterial cells.…”