Characterization of a sodium deoxycholate-activatable proteinase activity associated with βA3/A1-crystallin of human lenses

A human lens proteinase was purified by a five-step procedure that included two consecutive size-exclusion agarose A 1.5 m chromatographies, a preparative non-denaturing gel-electrophoretic separation, HPLC on a size-exclusion column (TSK G-3000 PWXL) followed by preparative isoelectric focusing. A 2300-fold purified enzyme showed a major band of 22 kDa during SDS-PAGE, a pH optimum of 7.8, pIbetween 4.5 and 5.0, a loss of activity above 45 degrees C and a serine type nature. The partial N-terminal sequence of the enzyme, i.e. P-M-P-G-S-L-G-P-W, matched with the sequence of human lens beta A3/Al-crystallin starting at residue No. 23. Based on the Western blot results of the enzyme with five different site-specific polyclonal. antibodies raised against beta A3/Al-crystallin, it was concluded that the 22 kDa crystallin enzyme had a cleaved N-terminus but an intact C-terminus. The beta A3/Al-crystallin, isolated from human lenses, also exhibited proteinase activity following detergent activation and size-exclusion chromatography. The mouse recombinant PA3/Al-crystallin proteinase was purified by the above five-step procedure, from a homogenate of Sf-9 cells transfected with baculovirus containing the full length coding sequence of PA3/Al-crystallin. The mouse 22 kDa species also exhibited proteinase activity and immunoreactivity with anti-beta A3/Al-C-terminal antibody. Together, the data suggest that a truncated species of beta A3/Al-crystallin exhibits proteinase activity. (C) 1999 Elsevier Science B.V. All rights reserved.