A serine-type protease activity of human lens βA3-crystallin is responsible for its autodegradation

PURPOSE: The purpose of the study was to determine whether the autodegradation of human βA3-crystallin is due to its intrinsic protease activity. METHODS: Recombinant His-tagged human βA3-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 °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 βA3-crystallin and its autodegradation products to FFCK [5-carboxyfluorescenyl-1-phenylalaninyl-chloromethyl 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: βA3-crystallin protease activity showed activation in the presence of CHAPS but not in presence of Triton X-100. Upon incubation of βA3-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 NH(2)-terminal sequencing showed cleavage of Lys(17)-Met(18), Gln(4)-Ala(5) and Thr-Gly (in the NH(2)-terminal His-tag region) bonds, respectively. Almost no autodegradation of the βA3-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 metallo-protease inhibitors (EDTA and EGTA) and cysteine protease inhibitors (E-64, N-methylmaleimide and iodoacetamide). The βA3-crystallin also exhibited binding to FFCK, suggesting existence of a chymotrypsin-type active site in the βA3-crystallin protease. CONCLUSIONS: The results suggested that a serine-type protease activity of βA3-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 NH(2)-terminal arm of βA3-crystallin.