Cloning and characterization of a thermostable catfish alphaB-crystallin with chaperone-like activity at high temperatures.
We have cloned, expressed and characterized catfish alphaB-crystallin (FalphaB). Genomic sequence comparison has revealed conservation of intron splicing sites and coding regions, however, the two intron sequences, 5'- and 3'-untranslated regions of FalphaB gene are shorter than those reported for other vertebrates. In contrast to mammalian homologues with a subunit association ratio (alphaA-crystallin/alphaB-crystallin) of 3:1, alpha-crystallin from catfish lens showed a ratio of 19:1. The biophysical properties and chaperone-like activity of recombinant FalphaB and porcine alphaB-crystallin (PalphaB) were studied and compared by heat denaturation, circular dichroism, intrinsic and dye-binding fluorescence, gel-filtration, and analytical ultracentrifugation. FalphaB shows 50% precipitation occurring at 72 degrees C that is higher than PalphaB at 66 degrees C. Even though FalphaB also possesses more surface hydrophilic regions than PalphaB, FalphaB still possesses higher chaperone activity to prevent aggregation of alcohol dehydrogenase at 60 degrees C. The molecular mass of FalphaB showed a smaller size (450 kDa) than PalphaB (550 kDa), which is also confirmed by analytical ultracentrifugation. In addition, FalphaB possesses better refolding potential after preheating treatment than PalphaB. FalphaB also exhibits higher chaperone-like activity than PalphaB to prevent insulin aggregation induced by dithiothreitol. In contrast to the prevalent notion that fish crystallins generally denature easily, FalphaB with chaperone-like activity appears to be more stable than mammalian homologues towards thermal and chemical denaturation.
Yu
Chung-Ming
CM
Graduate Institute of Life Sciences, National Defense Medical Center, Taipei, Taiwan.
Chang
Gu-Gang
GG
Chang
Hui-Chuan
HC
Chiou
Shyh-Horng
SH
D000595:Amino Acid Sequence; D000818:Animals; D002397:Catfishes / Q000235:genetics*; D003001:Cloning, Molecular; D018076:DNA, Complementary / Q000235:genetics; D016678:Genome; D006358:Hot Temperature; D018832:Molecular Chaperones; D008969:Molecular Sequence Data; D010802:Phylogeny; D017433:Protein Structure, Secondary; D017434:Protein Structure, Tertiary; D016415:Sequence Alignment; D013045:Species Specificity; D013696:Temperature; D038203:alpha-Crystallin B Chain / Q000235:genetics* / Q000502:physiology
D018076:DNA, Complementary; D018832:Molecular Chaperones; D038203:alpha-Crystallin B Chain