Differences in properties between human alphaA- and alphaB-crystallin proteins expressed in Escherichia coli cells in response to cold and extreme pH.
It has been reported that alphaA-crystallin has greater protective effects against apoptosis in lens epithelial cells than alphaB-crystallin [Andley, Song, Wawrousek, Fleming and Bassnett (2000) J. Biol. Chem. 275, 36823-36831]. Because the alphaA-crystallin proteins are specifically expressed in the vertebrate lens, we examine the non-specific properties of both alphaA- and alphaB-crystallins in an Escherichia coli system. E. coli cells were transformed with the inducible protein expression vector pET-11a, harbouring the gene for either human alphaA- or alphaB-crystallin, and two other control plasmids, pET-1la vector alone or pGEX-2T vector encoding GST (glutathione S-transferase). These cells were exposed to various stress conditions, such as cold-shock at 4 degrees C or extremely low or high pH environments (pH 4.7 or pH 8.0) for 6 h, and survival of the host cells and the solubility of the expressed target proteins in the cytosol were examined. Under these stress conditions, the cells expressing alphaB-crystallin protein demonstrated significantly improved survival when compared with the other cells, and the expressed protein in the cytosol was almost soluble, in contrast with the alphaA-crystallin protein. Differences in the amino acid sequence between the proteins in a phenylalanine-rich region next to the N-terminal consensus alpha-crystallin domain was considered to be responsible for chaperone activity and cell survival.
Takeuchi
Satoru
S
Department of Health Promotion and Human Behavior, Kyoto University, Yoshida-konoe, Sakyo-ku, Kyoto 606-8501, Japan.
Mandai
Yumi
Y
Otsu
Akiko
A
Shirakawa
Taro
T
Masuda
Katsuyoshi
K
Chinami
Masanobu
M
D000595:Amino Acid Sequence; D002455:Cell Division / Q000235:genetics; D003080:Cold Temperature; D004591:Electrophoresis, Polyacrylamide Gel; D004926:Escherichia coli / Q000187:drug effects / Q000235:genetics* / Q000254:growth & development; D006358:Hot Temperature; D006801:Humans; D006863:Hydrogen-Ion Concentration; D007544:Isopropyl Thiogalactoside / Q000494:pharmacology; D008969:Molecular Sequence Data; D011994:Recombinant Proteins / Q000187:drug effects / Q000235:genetics / Q000378:metabolism; D017386:Sequence Homology, Amino Acid; D012995:Solubility; D038202:alpha-Crystallin A Chain / Q000235:genetics / Q000378:metabolism*; D038203:alpha-Crystallin B Chain / Q000235:genetics / Q000378:metabolism*
D011994:Recombinant Proteins; D038202:alpha-Crystallin A Chain; D038203:alpha-Crystallin B Chain; D007544:Isopropyl Thiogalactoside