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

Srivastava OP, Srivastava K, Chaves JM - Mol. Vis. (2008)

Bottom Line: 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.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.

View Article: PubMed Central - PubMed

Affiliation: Department of Vision Sciences, University of Alabama at Birmingham, Birmingham, AL 35294, USA. srivasta@uab.edu

ABSTRACT

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.

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Time-dependent proteolysis of bovine recombinant γB-, γC-, and γD-crystallins by the human lens membrane proteinase. The incubation periods are shown at the bottom of the gel, and the reaction constituents are shown at the top of the gel. The proteolyzed fragments are identified with numbers, and only the fragments from γD-crystallin were used for partial NH2-terminal sequence analysis.
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f10: Time-dependent proteolysis of bovine recombinant γB-, γC-, and γD-crystallins by the human lens membrane proteinase. The incubation periods are shown at the bottom of the gel, and the reaction constituents are shown at the top of the gel. The proteolyzed fragments are identified with numbers, and only the fragments from γD-crystallin were used for partial NH2-terminal sequence analysis.

Mentions: Proteolysis of the bovine recombinant γB-, γC-, and γD-crystallins was examined by the proteinase (isolated from the lens membrane fraction). As stated above in Methods, individual crystallins were incubated at the ratio of 1:40 (proteinase:crystallin) for up to 48 h at 37 °C. After withdrawing the aliquots at 0 h, 24 h, and 48 h during proteolysis and analysis by SDS–PAGE, the γB-crystallin exhibited resistance to proteolysis by the enzyme, but both γC- and γD-crystallins were proteolyzed producing three and six major fragments, respectively (Figure 10, lanes 7 and 10).The partial NH2-terminal sequence analysis of γD-crystallin fragments showed that the following peptide bonds were cleaved: M1-G2 in fragment #1 (~19-kDa), Q54-Y55 in fragment #2 (~14-kDa), M70-G71 in fragment #3 (~12-kDa), and Q103-M104 in fragment #4 (~10 kDa). The cleavage sites in fragments #5 and #6 could not be determined because of the presence of multiple polypeptides. In summary, the major cleavage sites in γD-crystallin by the proteinase were at M1-G2, Q54-Y55, M70-G71, and Q103-M104.


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

Srivastava OP, Srivastava K, Chaves JM - Mol. Vis. (2008)

Time-dependent proteolysis of bovine recombinant γB-, γC-, and γD-crystallins by the human lens membrane proteinase. The incubation periods are shown at the bottom of the gel, and the reaction constituents are shown at the top of the gel. The proteolyzed fragments are identified with numbers, and only the fragments from γD-crystallin were used for partial NH2-terminal sequence analysis.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC2571948&req=5

f10: Time-dependent proteolysis of bovine recombinant γB-, γC-, and γD-crystallins by the human lens membrane proteinase. The incubation periods are shown at the bottom of the gel, and the reaction constituents are shown at the top of the gel. The proteolyzed fragments are identified with numbers, and only the fragments from γD-crystallin were used for partial NH2-terminal sequence analysis.
Mentions: Proteolysis of the bovine recombinant γB-, γC-, and γD-crystallins was examined by the proteinase (isolated from the lens membrane fraction). As stated above in Methods, individual crystallins were incubated at the ratio of 1:40 (proteinase:crystallin) for up to 48 h at 37 °C. After withdrawing the aliquots at 0 h, 24 h, and 48 h during proteolysis and analysis by SDS–PAGE, the γB-crystallin exhibited resistance to proteolysis by the enzyme, but both γC- and γD-crystallins were proteolyzed producing three and six major fragments, respectively (Figure 10, lanes 7 and 10).The partial NH2-terminal sequence analysis of γD-crystallin fragments showed that the following peptide bonds were cleaved: M1-G2 in fragment #1 (~19-kDa), Q54-Y55 in fragment #2 (~14-kDa), M70-G71 in fragment #3 (~12-kDa), and Q103-M104 in fragment #4 (~10 kDa). The cleavage sites in fragments #5 and #6 could not be determined because of the presence of multiple polypeptides. In summary, the major cleavage sites in γD-crystallin by the proteinase were at M1-G2, Q54-Y55, M70-G71, and Q103-M104.

Bottom Line: 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.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.

View Article: PubMed Central - PubMed

Affiliation: Department of Vision Sciences, University of Alabama at Birmingham, Birmingham, AL 35294, USA. srivasta@uab.edu

ABSTRACT

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.

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