Abstract
Purpose :
To evaluate if minichaperones derived from lens protein, αA-crystallin can suppress the hemolytic activity of mellitin from bee venom.
Methods :
Minichaperones with all L- or D- amino acids and with or without a cell-penetrating sequence were synthesized and tested for their anti-aggregation and anti-hemolytic activities. The chaperone-like activity of the peptides was compared using alcohol dehydrogenase aggregation assay. Mellitin (0 or 25 µM) and minichaperones (0, 25 or 50 µM) were incubated together in PBS at 37o C for 30 min. The anti-hemolytic activity of minichaperones was evaluated by adding 28 µl of the incubation mixture to 1 ml PBS containing 0.25 ml of 2% human RBC at 37oC. Phosphate buffer saline and Triton X-100 (1%) was used as negative and positive control, respectively. After one hr incubation, the tubes were centrifuged at 3000 rpm for 10 min, and the released hemoglobin from RBC was monitored by measuring the absorbance of the supernatant at 540 nm. The relative hemolytic activity of melittin was determined in comparison with the positive control (Triton X-100).
Results :
Minichaperones synthesized with all D-amino acids and having cell-penetrating sequence exhibited 2 – 3 folds higher anti-aggregation activity compared to minichaperones synthesized with all L- amino acids. Under our experimental conditions mellitin showed about 53% hemolysis, and the minichaperones showed a concentration-dependent suppression of hemolytic activity of melittin. The hemolytic activity of mellitin decreased only marginally (13%) in presence all L- minichaperone at 2x molar concentration. Mellitin, however, showed a 43% reduction in hemolytic activity in the presence of all D- minichaperone and 79% suppression in hemolysis in the presence of D- minichaperone having a cell-penetrating sequence.
Conclusions :
Minichaperones synthesized with D-amino acids and having a cell-penetrating sequence has as a therapeutic potential against bee venom.
This abstract was presented at the 2019 ARVO Annual Meeting, held in Vancouver, Canada, April 28 - May 2, 2019.