Abstract
Purpose: :
To determine whether the auto-degradation of human βA3-crystallin is due to its proteinase activity.
Methods: :
Human βA3-crystallin was cloned, expressed and purified as previously described (Gupta, Srivastava and Srivastava, Biochemistry 2006, 45, 9964-9978). The purified βA3-crystallin preparation was incubated under sterile conditions with and without detergents in 0.05 M Tris-buffer, pH 7.9 at 37°C to determine its detergent-induced auto-degradation. Additionally, the crystallin was incubated under identical conditions with protease inhibitors to examine their effects on its auto-degradation. Partial N-terminal sequencing of three major auto-degraded species with Mr of 25, 27 and 29 kDa was performed to determine specific bonds cleaved in the crystallin during their production.
Results: :
On incubation of βA3-crystallin with CHAPS {3-[(3-Cholamidopropyl) dimethylammonio]-1-propanesulfonate}, the crystallin showed three major auto-degradation products with Mr of 25, 27 and 29 kDa. The auto-degradation in the crystallin occurred at much slower rate on incubation alone or with Triton X-100, or with CHAPS plus serine protease inhibitors (phenylmethylsulfonyl fluoride, aprotinin and chymostatin and L-1-tosylamido-2-phenylethyl chloromethyl ketone [TPCK]). However, the degradation occurred in the presence of metallo-protease inhibitors (EDTA and EGTA) and cystein protease inhibitors (E-64, methylmaleimide and iodoacetamide). The results suggested that a serine-type protease activity was responsible for auto-degradation. The specific bond cleaved to produce 25, 27 and 29 kDa species wereLys17-Met18, Gln4-Ala5 and Thr-Gly (in His-tag region), respectively.
Conclusions: :
Human βA3-crystallin exhibited auto-degradation on incubation with detergents such as CHAPS, which is due to its serine-type protease activity.
Keywords: crystallins • protein structure/function • proteolysis