Target molecules of molecular chaperone (HSP70 family) in injured gastric mucosa in vivo.
OBJECTIVE
Several recent studies, including ours, have indicated the importance of heat shock proteins (HSPs) in cytoprotection against cytotoxic agents and environmental stresses mediated by the chaperone function of HSPs (molecular chaperones). However, the target molecule that is recognized by HSPs in damaged cells currently remains unknown. As HSPs rapidly recognize and bind to degenerated protein in cells, target molecules of HSPs might be key molecules for the initiation and pathogenesis of cellular damage. In the present study, gastric mucosal proteins that specifically bind to the HSP70 family (HSC70) were analyzed using HSC70-affinity chromatography.


METHODS
The gastric mucosa was removed from Sprague-Dawley rats after exposure to water immersion-stress for 0, 1, 3 or 5 h. Soluble fractions of each gastric mucosa were applied to the HSC70-affinity column separately. After washing off non-specific binding proteins, specific binding proteins were eluted by ATP-containing buffer. Binding proteins were analyzed by SDS-polyacrylamide gel electrophoresis. In addition, the amino acid sequence of purified proteins was also analyzed.


RESULTS
Specific HSC70-binding proteins with a molecular weight of 200-kDa and 45-kDa were eluted from an affinity column when gastric mucosal homogenate of 1-h stress exposure was applied. The amino acid sequencing showed that these binding proteins were cytoskeletal myosin (heavy chain) and actin, respectively.


CONCLUSIONS
During the pathogenesis of stress-induced gastric mucosal damage, structurally degenerated cytoskeletal myosin (heavy chain) and actin may be key or initiation molecules which structural changes were firstly recognized by molecular chaperone.
Otaka
Michiro
M
Department of Gastroenterology, Juntendo University School of Medicine, 2-1-1 Hongo, Bunkyo-Ku, Tokyo, Japan. mootaka@juntendo.ac.jp
Odashima
Masaru
M
Izumi
Yuko
Y
Nagahara
Akihito
A
Osada
Taro
T
Sakamoto
Naoto
N
Takada
Makiko
M
Takahashi
Taiji
T
Shimada
Yuji
Y
Tamaki
Kumiko
K
Asaoka
Daisuke
D
Itoh
Hideaki
H
Watanabe
Sumio
S
D000595:Amino Acid Sequence; D000818:Animals; D005753:Gastric Mucosa / Q000737:chemistry / Q000293:injuries / Q000378:metabolism; D050883:HSC70 Heat-Shock Proteins / Q000235:genetics / Q000302:isolation & purification* / Q000378:metabolism*; D018840:HSP70 Heat-Shock Proteins / Q000235:genetics / Q000378:metabolism*; D008969:Molecular Sequence Data; D008970:Molecular Weight; D011485:Protein Binding; D051381:Rats; D017207:Rats, Sprague-Dawley; D012149:Restraint, Physical; D013312:Stress, Physiological
D050883:HSC70 Heat-Shock Proteins; D018840:HSP70 Heat-Shock Proteins