Isolation and characterization of betaA3-crystallin associated proteinase from alpha-crystallin fraction of human lenses

Mol Vis. 2008;14:1872-85. Epub 2008 Oct 20.


Purpose: The purpose was to characterize the properties of a proteinase activity associated with betaA3-crystallin, which was isolated from the alpha-crystallin fraction of human lenses.

Methods: An inactive, Arg-bond hydrolyzing proteinase in the alpha-crystallin fraction, which was isolated from the water soluble (WS) protein fraction of 60- to 70-year-old human lenses, was activated by sodium deoxycholate treatment. The activated enzyme was purified by a three-step procedure that included a size-exclusion Agarose A1.5 m chromatography, non-denaturing preparative gel-electrophoresis, and size-exclusion HPLC. The purified proteinase was characterized for the proteinase type, proteolysis of bovine recombinant gammaB-, gammaC-, and gammaD-crystallins, and its presence in three different protein fractions of human lenses (i.e., alpha-crystallin, beta(H)-crystallin, and membrane fractions).

Results: An inactive, Arg-bond hydrolyzing proteinase present in the alpha-crystallin fraction showed activity on treatment with detergents such as sodium deoxycholate, Triton X-100, octyl beta-D-glucopyranoside, and CHAPS (3-[(3-cholamido propyl) dimethylammonio]-1-propanesulfonate). The sodium deoxycholate-activated enzyme was released from the alpha-crystallin fraction since it eluted at a lower molecular weight species than alpha-crystallin during size-exclusion Agarose A1.5 m chromatography. Following a three-step purification procedure, the enzyme showed three species between 22 kDa and 25 kDa during sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) analysis. The three protein bands were identified as betaA3-, betaB1-, and betaB2-crystallin by the matrix-assisted laser desorption/ionization-time of flight (MALDI-TOF) and tandem mass spectrometric (ES-MS/MS) methods. Inhibitor studies revealed that the enzyme was a serine-type proteinase. Among the recombinant betaA3-, betaB1-, or betaB2-crystallins, only the betaA3-crystallin exhibited the proteinase activity following detergent treatment and size-exclusion chromatography. The proteinase also exhibited proteolysis of gammaC- and gammaD- crystallins, and the cleavage of gammaD-crystallin at M(1)-G(2), Q(54)-Y(55), M(70)-G(71), and Q(103)-M(104) bonds. Further, the enzyme was also present in three fractions of human lenses (alpha-crystallin, beta(H)-crystallin, and membrane fractions).

Conclusions: A serine-type betaA3-crystallin proteinase existed in an inactive state in the alpha-crystallin fraction and was activated by detergents. The enzyme proteolyzed alphaA-, alphaB-, gammaC-, and gammaD-crystallins and was present in three fractions (alpha-crystallin, beta(H)-crystallin, and membrane-fractions) of 60 to 70-year-old human lenses.

Publication types

  • Research Support, N.I.H., Extramural
  • Research Support, Non-U.S. Gov't

MeSH terms

  • Aged
  • Animals
  • Arginine / metabolism
  • Cattle
  • Chromatography, High Pressure Liquid
  • Deoxycholic Acid / pharmacology
  • Electrophoresis, Polyacrylamide Gel
  • Endopeptidases / isolation & purification*
  • Endopeptidases / metabolism
  • Enzyme Activation / drug effects
  • Humans
  • Hydrolysis / drug effects
  • Lens, Crystalline / drug effects
  • Lens, Crystalline / enzymology*
  • Middle Aged
  • Protease Inhibitors / pharmacology
  • Protein Processing, Post-Translational / drug effects
  • Recombinant Proteins / analysis
  • Subcellular Fractions / drug effects
  • Subcellular Fractions / enzymology
  • Time Factors
  • alpha-Crystallins / metabolism*
  • beta-Crystallin A Chain / analysis
  • beta-Crystallin A Chain / chemistry
  • beta-Crystallin A Chain / metabolism


  • Protease Inhibitors
  • Recombinant Proteins
  • alpha-Crystallins
  • beta-Crystallin A Chain
  • Deoxycholic Acid
  • Arginine
  • Endopeptidases