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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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SDS–PAGE analysis of the column fractions that contained proteinase activity after Agarose A1.5 m chromatography of the sodium deoxycholate-treated α-crystallin fraction. Note that two protein bands (identified as bands 1 and 2) with Mr of 20–25 kDa and the third band (identified as band 3) of 38 kDa were recovered.
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f1: SDS–PAGE analysis of the column fractions that contained proteinase activity after Agarose A1.5 m chromatography of the sodium deoxycholate-treated α-crystallin fraction. Note that two protein bands (identified as bands 1 and 2) with Mr of 20–25 kDa and the third band (identified as band 3) of 38 kDa were recovered.

Mentions: The following three experiments suggested the presence of an Arg-bond hydrolyzing proteinase in the α-crystallin fraction of human lenses. In the first experiment, no proteinase activity was observed in the α-crystallin fraction of human lenses of 60 to 70-year-old donors until the preparation was treated with sodium deoxycholate and fractionated by a size-exclusion Agarose A1.5 m column. Following chromatography, the proteinase eluted later than α-crystallin, which suggests its dissociation and a lower molecular weight than the crystallin. On examination of the fractions that contained proteinase activity by SDS–PAGE, three protein bands, two of about 22–25 kDa and one of about 38 kDa, were observed (Figure 1). Similar results were obtained with the α-crystallin fraction isolated from lenses of 20-year-old donors, suggesting that the association of the enzyme with the crystallin occurred early during aging (results not shown). In the second experiment, the dissociation of the proteinase activity from the α-crystallin fraction following treatment with sodium deoxycholate (anionic, 0.1% final concentration), Triton X-100 (non-ionic, 0.1%), octyl β-D-glucopyranoside (non-ionic, 0.2%), and CHAPS (zwitterionic, 0.1%) was also observed. All other detergents except Triton X-100 were able to dissociate the enzyme at almost the same levels (1.0 enzyme units from 0.8 mg protein per ml from the α-crystallin fraction; Table 1). The third experiment showed that the activation of the enzyme also resulted in the proteolysis of α-crystallin. With the sodium deoxycholate treatment of α-crystallin and incubation under sterile conditions at 37 °C for 10 h, the α-crystallin preparations showed proteolysis, which was absent in the detergent-untreated preparation (Figure 2A,B). Upon MALDI-TOF mass spectrometric analysis, the proteolyzed fragments of both αA- and αB-crystallins were identified (i.e., spot no. 6 [Mr 10 kDa] was of αB-crystallin and spot no. 1-5 and 7-9 [Mr 6–14 kDa] were of αA-crystallin).


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

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

SDS–PAGE analysis of the column fractions that contained proteinase activity after Agarose A1.5 m chromatography of the sodium deoxycholate-treated α-crystallin fraction. Note that two protein bands (identified as bands 1 and 2) with Mr of 20–25 kDa and the third band (identified as band 3) of 38 kDa were recovered.
© Copyright Policy - open-access
Related In: Results  -  Collection

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

f1: SDS–PAGE analysis of the column fractions that contained proteinase activity after Agarose A1.5 m chromatography of the sodium deoxycholate-treated α-crystallin fraction. Note that two protein bands (identified as bands 1 and 2) with Mr of 20–25 kDa and the third band (identified as band 3) of 38 kDa were recovered.
Mentions: The following three experiments suggested the presence of an Arg-bond hydrolyzing proteinase in the α-crystallin fraction of human lenses. In the first experiment, no proteinase activity was observed in the α-crystallin fraction of human lenses of 60 to 70-year-old donors until the preparation was treated with sodium deoxycholate and fractionated by a size-exclusion Agarose A1.5 m column. Following chromatography, the proteinase eluted later than α-crystallin, which suggests its dissociation and a lower molecular weight than the crystallin. On examination of the fractions that contained proteinase activity by SDS–PAGE, three protein bands, two of about 22–25 kDa and one of about 38 kDa, were observed (Figure 1). Similar results were obtained with the α-crystallin fraction isolated from lenses of 20-year-old donors, suggesting that the association of the enzyme with the crystallin occurred early during aging (results not shown). In the second experiment, the dissociation of the proteinase activity from the α-crystallin fraction following treatment with sodium deoxycholate (anionic, 0.1% final concentration), Triton X-100 (non-ionic, 0.1%), octyl β-D-glucopyranoside (non-ionic, 0.2%), and CHAPS (zwitterionic, 0.1%) was also observed. All other detergents except Triton X-100 were able to dissociate the enzyme at almost the same levels (1.0 enzyme units from 0.8 mg protein per ml from the α-crystallin fraction; Table 1). The third experiment showed that the activation of the enzyme also resulted in the proteolysis of α-crystallin. With the sodium deoxycholate treatment of α-crystallin and incubation under sterile conditions at 37 °C for 10 h, the α-crystallin preparations showed proteolysis, which was absent in the detergent-untreated preparation (Figure 2A,B). Upon MALDI-TOF mass spectrometric analysis, the proteolyzed fragments of both αA- and αB-crystallins were identified (i.e., spot no. 6 [Mr 10 kDa] was of αB-crystallin and spot no. 1-5 and 7-9 [Mr 6–14 kDa] were of αA-crystallin).

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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