Isolation and characterization of βA3-crystallin associated proteinase from α-crystallin fraction of human lenses

摘要

Purpose: The purpose was to characterizethe properties of a proteinase activity associated with βA3-crystallin,which was isolated from the α-crystallin fraction of human lenses. Methods: An inactive, Arg-bondhydrolyzing proteinase in the α-crystallin fraction, which was isolatedfrom the water soluble (WS) protein fraction of 60- to 70-year-oldhuman lenses, was activated by sodium deoxycholate treatment. Theactivated enzyme was purified by a three-step procedure that included asize-exclusion Agarose A1.5 m chromatography, non-denaturingpreparative gel-electrophoresis, and size-exclusion HPLC. The purifiedproteinase was characterized for the proteinase type, proteolysis ofbovine recombinant γB-, γC-, and γD-crystallins, and its presence inthree different protein fractions of human lenses (i.e., α-crystallin, βH-crystallin,and membrane fractions). Results: An inactive, Arg-bondhydrolyzing proteinase present in the α-crystallin fraction showedactivity on treatment with detergents such as sodium deoxycholate,Triton X-100, octyl β-D-glucopyranoside, and CHAPS (3-[(3-cholamidopropyl) dimethylammonio]-1-propanesulfonate). The sodiumdeoxycholate-activated enzyme was released from the α-crystallinfraction since it eluted at a lower molecular weight species thanα-crystallin during size-exclusion Agarose A1.5 m chromatography.Following a three-step purification procedure, the enzyme showed threespecies between 22 kDa and 25 kDa during sodium dodecylsulfate-polyacrylamide gel electrophoresis (SDS–PAGE) analysis. Thethree protein bands were identified as βA3-, βB1-, and βB2-crystallinby the matrix-assisted laser desorption/ionization-time of flight(MALDI-TOF) and tandem mass spectrometric (ES-MS/MS) methods. Inhibitorstudies revealed that the enzyme was a serine-type proteinase. Amongthe recombinant βA3-, βB1-, or βB2-crystallins, only the βA3-crystallinexhibited the proteinase activity following detergent treatment andsize-exclusion chromatography. The proteinase also exhibitedproteolysis of γC- and γD- crystallins, and the cleavage ofγD-crystallin at M1-G2, Q54-Y55,M70-G71, and Q103-M104bonds. Further, the enzyme was also present in three fractions of humanlenses (α-crystallin, βH-crystallin, and membranefractions). Conclusions: A serine-typeβA3-crystallin proteinase existed in an inactive state in theα-crystallin fraction and was activated by detergents. The enzymeproteolyzed αA-, αB-, γC-, and γD-crystallins and was present in threefractions (α-crystallin, βH-crystallin, andmembrane-fractions) of 60 to 70-year-old human lenses.