Suppression of DTT-induced aggregation of abrin by αA- and αB-crystallins: a model aggregation assay for α-crystallin chaperone activity in vitro11This work was presented at the Annual Meeting of Association for Research in Vision and Ophthalmology held in Florida (USA) during May 5–10, 2002

The eye lens small heat shock proteins (sHSP), αA- and αB-crystallins, have been shown to function like molecular chaperones, both in vitro and in vivo. It is essential to assess the protective effect of αA- and αB-crystallins under native conditions to extrapolate the results to in vivo conditions. Insulin and α-lactalbumin have widely been used to investigate the chaperone mechanism of α-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 α-lactalbumin and binding of α-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 αA- and αB-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 α-crystallin and abrin B-chain. Interestingly, in contrast to insulin, there is no significant difference between αA- and αB-crystallin in suppressing the aggregation of abrin B-chain at two different temperatures (25 and 37°C). HSP26, an another small heat shock/α-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 α-crystallin and as well as other sHSP.