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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 five proteinase preparations purified from five separate α-crystallin fractions. The purification was performed by the three-step procedure described in Methods. Lanes 1-5 show five different purified proteinase preparations from five separate α-crystallin fractions. Note that all five preparations reproducibly showed three bands that are identified as 1, 2, and 3.
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f4: SDS–PAGE analysis of five proteinase preparations purified from five separate α-crystallin fractions. The purification was performed by the three-step procedure described in Methods. Lanes 1-5 show five different purified proteinase preparations from five separate α-crystallin fractions. Note that all five preparations reproducibly showed three bands that are identified as 1, 2, and 3.

Mentions: SDS–PAGE analysis of the purified proteinase preparation showed three protein bands of 22-25 kDa (Figure 4). The five such purified preparations showed identical protein bands. The ES-MS/MS analysis identified band 1 as βA3-crystallin (contained residue #33–45 [ITIYDQENFQGKR], #35–45 [IYDQENFQGKR], #36–45 [YDQENFQGKR], #126–137 [MTIFEKENFIGR], #128–137 [IFEKENFIGR], and #197–211 [EWGSHAQTSQIQSIR]). Band 2 was identified as a mixture of βA3-, βB1-, and βB2-crystallins as it showed peptide sequences of βA3 (residue # 33–44 [ITIYDQENFQGK] and #96-109 [WDAWSGSNAYHIER]), βB1 (residue #60–71 [LVVFELENFQGR], residue #123–131 [WNTWSSSYR], #150–159 [ISLFEGANFK], #187–201 [VSSGTWVGYQYPGYR], and #202–214 [GYQYLLEPGDFR]), and βB2 (residue #110–120 [LYENPNFTGKK], #129–139 [PSFHAHGYQEK], and #145–159 [VQSGTWVGYQYPGYR]). Band 3 was identified as βB1-crystallin (contained residue #60–71 [LVVFELENFQGR], #150–159 [ISLFEGANFK], #187–201 [VSSGTWVGYQYPGYR], #202–214 [GYQYLLEPGDFR], and #233–251 [DKQWHLEGSFPVLATEPPK]).


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 five proteinase preparations purified from five separate α-crystallin fractions. The purification was performed by the three-step procedure described in Methods. Lanes 1-5 show five different purified proteinase preparations from five separate α-crystallin fractions. Note that all five preparations reproducibly showed three bands that are identified as 1, 2, and 3.
© Copyright Policy - open-access
Related In: Results  -  Collection

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

f4: SDS–PAGE analysis of five proteinase preparations purified from five separate α-crystallin fractions. The purification was performed by the three-step procedure described in Methods. Lanes 1-5 show five different purified proteinase preparations from five separate α-crystallin fractions. Note that all five preparations reproducibly showed three bands that are identified as 1, 2, and 3.
Mentions: SDS–PAGE analysis of the purified proteinase preparation showed three protein bands of 22-25 kDa (Figure 4). The five such purified preparations showed identical protein bands. The ES-MS/MS analysis identified band 1 as βA3-crystallin (contained residue #33–45 [ITIYDQENFQGKR], #35–45 [IYDQENFQGKR], #36–45 [YDQENFQGKR], #126–137 [MTIFEKENFIGR], #128–137 [IFEKENFIGR], and #197–211 [EWGSHAQTSQIQSIR]). Band 2 was identified as a mixture of βA3-, βB1-, and βB2-crystallins as it showed peptide sequences of βA3 (residue # 33–44 [ITIYDQENFQGK] and #96-109 [WDAWSGSNAYHIER]), βB1 (residue #60–71 [LVVFELENFQGR], residue #123–131 [WNTWSSSYR], #150–159 [ISLFEGANFK], #187–201 [VSSGTWVGYQYPGYR], and #202–214 [GYQYLLEPGDFR]), and βB2 (residue #110–120 [LYENPNFTGKK], #129–139 [PSFHAHGYQEK], and #145–159 [VQSGTWVGYQYPGYR]). Band 3 was identified as βB1-crystallin (contained residue #60–71 [LVVFELENFQGR], #150–159 [ISLFEGANFK], #187–201 [VSSGTWVGYQYPGYR], #202–214 [GYQYLLEPGDFR], and #233–251 [DKQWHLEGSFPVLATEPPK]).

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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