Suppression of DTT-induced aggregation of abrin by alphaA- and alphaB-crystallins: a model aggregation assay for alpha-crystallin chaperone activity in vitro.
The eye lens small heat shock proteins (sHSP), alphaA- and alphaB-crystallins, have been shown to function like molecular chaperones, both in vitro and in vivo. It is essential to assess the protective effect of alphaA- and alphaB-crystallins under native conditions to extrapolate the results to in vivo conditions. Insulin and alpha-lactalbumin have widely been used to investigate the chaperone mechanism of alpha-crystallin under native conditions. Due to its smaller size, insulin B-chain may not represent the binding of putative physiological substrate proteins. As it stands, the aggregation of alpha-lactalbumin and binding of alpha-crystallin to it varies under different experimental conditions. Abrin, a ribosome inactivating protein isolated from the seeds of Abrus precatorius, consists of a 30 kDa A-chain and a lectin-like B-chain of 33 kDa joined by a single disulfide bond. Reduction of the disulfide link between the two chains of abrin leads to the aggregation of the B-chain. In this study, we demonstrate that dithiothreitol (DTT)-induced aggregation of abrin B-chain could be monitored by light scattering similar to that of insulin. Moreso, this process could be suppressed by recombinant human alphaA- and alphaB-crystallins in a concentration dependent manner, notably by binding to aggregation prone abrin B-chain. SDS-PAGE and HPLC gel filtration analysis indicate that there is a soluble complex formation between alpha-crystallin and abrin B-chain. Interestingly, in contrast to insulin, there is no significant difference between alphaA- and alphaB-crystallin in suppressing the aggregation of abrin B-chain at two different temperatures (25 and 37 degrees C). HSP26, an another small heat shock/alpha-crystallin family protein, was also able to prevent the DTT-induced aggregation of abrin. These results suggest that due to relatively larger size of its B-chain (33 kDa), compared to insulin B-chain (about 3 kDa), abrin may serve as a better model substrate for in vitro chaperone studies of alpha-crystallin and as well as other sHSP.
Reddy
G Bhanuprakash
GB
Biochemistry Division, National Institute of Nutrition, Hyderabad 500 007, India. geereddy@yahoo.com
Narayanan
Sriram
S
Reddy
P Yadagiri
PY
Surolia
Ira
I
D000036:Abrin / Q000302:isolation & purification / Q000378:metabolism*; D002850:Chromatography, Gel; D003459:Crystallins / Q000378:metabolism*; D004220:Disulfides; D004229:Dithiothreitol / Q000494:pharmacology; D006360:Heat-Shock Proteins / Q000378:metabolism*; D006801:Humans; D007328:Insulin / Q000378:metabolism; D008958:Models, Molecular; D018832:Molecular Chaperones / Q000378:metabolism*; D010084:Oxidation-Reduction; D013379:Substrate Specificity; D013696:Temperature
D003459:Crystallins; D004220:Disulfides; D006360:Heat-Shock Proteins; D007328:Insulin; D018832:Molecular Chaperones; D000036:Abrin; D004229:Dithiothreitol