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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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Structural changes in sodium deoxycholate-treated, 25 kDa βA3-crystallin species with proteinase activity compared to untreated, 32 kDa WT βA3-crystallin without enzyme activity. These preparations were obtained as described in Figure 6. A: The far UV-CD spectra show that the truncated 25 kDa βA3-species (red line) contains a greater level of alpha helical content than the WT-βA3 (black line). B: The intrinsic Trp fluorescence spectra show a red shift in the truncated 25 kDa βA3-species (red line) as compared to the WT-βA3 (black line). C: The fluorescence spectra after ANS-binding show a blue shift in the truncated 25 kDa βA3-species (red line) as compared to the WT-βA3 (black line).
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f7: Structural changes in sodium deoxycholate-treated, 25 kDa βA3-crystallin species with proteinase activity compared to untreated, 32 kDa WT βA3-crystallin without enzyme activity. These preparations were obtained as described in Figure 6. A: The far UV-CD spectra show that the truncated 25 kDa βA3-species (red line) contains a greater level of alpha helical content than the WT-βA3 (black line). B: The intrinsic Trp fluorescence spectra show a red shift in the truncated 25 kDa βA3-species (red line) as compared to the WT-βA3 (black line). C: The fluorescence spectra after ANS-binding show a blue shift in the truncated 25 kDa βA3-species (red line) as compared to the WT-βA3 (black line).

Mentions: Upon activation of the His-tagged WT βA3-crystallin (Mr ~32 kDa, Figure 6, lane 1, NH2-terminal sequence: MRGSH [the sequence represented His-tag]) with deoxycholate and size-exclusion HPLC, the proteinase activity-containing fraction showed an NH2-terminally cleaved major, 25 kDa βA3 species (Figure 6, lane 2; NH2-terminal sequence: MAQTN) and several aggregated species. The partial NH2-terminal sequencing showed that the 25 kDa species was generated upon cleavage at the K17-M18 bond in the NH2-terminal extension arm of WT βA3-crystallin. This cleavage site was different than the cleavage site (N22-P23) that we have reported earlier [20] during the proteinase activation. This difference could be due to the His-tag in the WT βA3-crystallin. The truncated 25 kDa species predominantly contained an α-helical structure compared to the β-sheet structure in the untruncated 32 kDa WT βA3-crystallin (Figure 7A; WT βA3: β-sheet 51% and α-helix 34%; truncated 25 kDa βA3 with proteinase activity: β-sheet 11% and α-helix 86%). Similarly, compared to the 32 kDa WT βA3-crystallin, the truncated 25 kDa βA3-crystallin species exhibited a red shift in its intrinsic Trp fluorescence spectrum (Figure 7B) and a blue shift during binding to a hydrophobic probe ANS (8-anilino-1 naphthalene sulfate; Figure 7C).


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

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

Structural changes in sodium deoxycholate-treated, 25 kDa βA3-crystallin species with proteinase activity compared to untreated, 32 kDa WT βA3-crystallin without enzyme activity. These preparations were obtained as described in Figure 6. A: The far UV-CD spectra show that the truncated 25 kDa βA3-species (red line) contains a greater level of alpha helical content than the WT-βA3 (black line). B: The intrinsic Trp fluorescence spectra show a red shift in the truncated 25 kDa βA3-species (red line) as compared to the WT-βA3 (black line). C: The fluorescence spectra after ANS-binding show a blue shift in the truncated 25 kDa βA3-species (red line) as compared to the WT-βA3 (black line).
© Copyright Policy - open-access
Related In: Results  -  Collection

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

f7: Structural changes in sodium deoxycholate-treated, 25 kDa βA3-crystallin species with proteinase activity compared to untreated, 32 kDa WT βA3-crystallin without enzyme activity. These preparations were obtained as described in Figure 6. A: The far UV-CD spectra show that the truncated 25 kDa βA3-species (red line) contains a greater level of alpha helical content than the WT-βA3 (black line). B: The intrinsic Trp fluorescence spectra show a red shift in the truncated 25 kDa βA3-species (red line) as compared to the WT-βA3 (black line). C: The fluorescence spectra after ANS-binding show a blue shift in the truncated 25 kDa βA3-species (red line) as compared to the WT-βA3 (black line).
Mentions: Upon activation of the His-tagged WT βA3-crystallin (Mr ~32 kDa, Figure 6, lane 1, NH2-terminal sequence: MRGSH [the sequence represented His-tag]) with deoxycholate and size-exclusion HPLC, the proteinase activity-containing fraction showed an NH2-terminally cleaved major, 25 kDa βA3 species (Figure 6, lane 2; NH2-terminal sequence: MAQTN) and several aggregated species. The partial NH2-terminal sequencing showed that the 25 kDa species was generated upon cleavage at the K17-M18 bond in the NH2-terminal extension arm of WT βA3-crystallin. This cleavage site was different than the cleavage site (N22-P23) that we have reported earlier [20] during the proteinase activation. This difference could be due to the His-tag in the WT βA3-crystallin. The truncated 25 kDa species predominantly contained an α-helical structure compared to the β-sheet structure in the untruncated 32 kDa WT βA3-crystallin (Figure 7A; WT βA3: β-sheet 51% and α-helix 34%; truncated 25 kDa βA3 with proteinase activity: β-sheet 11% and α-helix 86%). Similarly, compared to the 32 kDa WT βA3-crystallin, the truncated 25 kDa βA3-crystallin species exhibited a red shift in its intrinsic Trp fluorescence spectrum (Figure 7B) and a blue shift during binding to a hydrophobic probe ANS (8-anilino-1 naphthalene sulfate; Figure 7C).

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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