title: A serine-type protease activity of human lens βA3-crystallin is responsible for its autodegradation.
abstract: OBJECTIVE
  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 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 Mr of 22, 27 and 30 kDa, which on partial NH2-terminal sequencing showed cleavage of Lys17-Met18, Gln4-Ala5 and Thr-Gly (in the NH2-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 (Gln4-Ala5 and Lys17-Met18), were localized in the NH2-terminal arm of βA3-crystallin.
authors.affiliation: Department of Vision Sciences, University of Alabama at Birmingham, Birmingham, AL 35294, USA.
authors.forename: R
authors.initials: R
authors.lastname: Gupta
authors.forename: J
authors.initials: J
authors.lastname: Chen
authors.forename: O P
authors.initials: OP
authors.lastname: Srivastava
mesh_terms: D002793:Cholic Acids / Q000494:pharmacology; D002846:Chromatography, Affinity; D003840:Deoxycholic Acid / Q000494:pharmacology; D003902:Detergents / Q000494:pharmacology; D004591:Electrophoresis, Polyacrylamide Gel; D004789:Enzyme Activation / Q000187:drug effects; D005453:Fluorescence; D006801:Humans; D007908:Lens, Crystalline / Q000187:drug effects / Q000378:metabolism*; D011480:Protease Inhibitors / Q000494:pharmacology; D011499:Protein Processing, Post-Translational / Q000187:drug effects; D011994:Recombinant Proteins / Q000302:isolation & purification / Q000378:metabolism; D057057:Serine Proteases / Q000378:metabolism*; D038205:beta-Crystallin A Chain / Q000302:isolation & purification / Q000378:metabolism*
chemical_list: C489108:CRYBA1 protein, human; D002793:Cholic Acids; D003902:Detergents; D011480:Protease Inhibitors; D011994:Recombinant Proteins; D038205:beta-Crystallin A Chain; D003840:Deoxycholic Acid; D057057:Serine Proteases; C028213:3-((3-cholamidopropyl)dimethylammonium)-1-propanesulfonate