A serine-type protease activity of human lens beta A3-crystallin is responsible for its autodegradation

作者：Gupta R; Chen J; Srivastava O P^*

来源：Molecular Vision, 2010, 16(239-40): 2242-2252.

Purpose: The purpose of the study was to determine whether the autodegradation of human beta A3-crystallin is due to its intrinsic protease activity.
Methods: Recombinant His-tagged human beta A3-crystallin was expressed in E. coli and purified by a Ni+2-affinity column chromatographic method. To determine protease activity, the purified crystallin was incubated for 24 h with either sodium deoxycholate, Triton X-100, or CHAPS {3-[(3-Cholamidopropyl) dimethylammonio]-1-propanesulfonate} and with benzoyl DL-arginine p-nitroanilide (BAPNA), a colorimetric protease substrate. The autodegradation of the crystallin at 0 h and 24 h on incubation at 37 degrees C with and without detergents (CHAPS/Triton X-100) was also determined by sodium dodecylsulfate-PAGE (SDS-PAGE) method. To examine whether the autodegradation of the crystallin was due to its protease activity, the crystallin was incubated with inhibitors of serine-, metallo-and cysteine-proteases. The binding of the intact beta A3-crystallin and its autodegradation products to FFCK [5-carboxyfluorescenyl-1-phenylalaninylchloromethyl ketone], an analog of TPCK [1-Chloro-3-tosylamido-4-phenyl-2-butanone, a chymotrypsin-type serine protease inhibitor] was also determined by their incubation followed by SDS-PAGE and scanning for fluorescence using a Typhoon 9400 (TM) scanner.
Results: beta A3-crystallin protease activity showed activation in the presence of CHAPS but not in presence of Triton X-100. Upon incubation of beta A3-crystallin for 24 h with CHAPS or sodium deoxycholate and BAPNA as a substrate, a time-dependent increase in the Arg-bond hydrolyzing activity was observed. SDS-PAGE analysis exhibited autodegradation products with M-r of 22, 27 and 30 kDa, which on partial NH2-terminal sequencing showed cleavage of Lys(17)-Met(18), Gln(4)-Ala(5) and Thr-Gly (in the NH2-terminal His-tag region) bonds, respectively. Almost no autodegradation of the beta A3-crystallin occurred during its incubation alone or with CHAPS plus serine protease inhibitors (phenylmethylsulfonyl fluoride [PMSF], approtinin, and chymostatin). In contrast, the autodegradation occurred in the presence of metalloprotease inhibitors (EDTA and EGTA) and cysteine protease inhibitors (E-64, N-methylmaleimide and iodoacetamide). The beta A3-crystallin also exhibited binding to FFCK, suggesting existence of a chymotrypsin-type active site in the beta A3-crystallin protease.
Conclusions: The results suggested that a serine-type protease activity of beta A3-crystalllin was responsible for its autodegradation. The specific bonds cleaved during autodegradation (Gln(4)-Ala(5) and Lys(17)-Met(18)), were localized in the NH2-terminal arm of beta A3-crystallin.

• 出版日期2010-11-2