April 2010
Volume 51, Issue 13
ARVO Annual Meeting Abstract  |   April 2010
Auto-Degradation of Human βA3-Crystallin is Due to Its Serine-Type Protease Activity
Author Affiliations & Notes
  • O. P. Srivastava
    Vision Sciences, University of Alabama at Birmingham, Birmingham, Alabama
  • R. Gupta
    Vision Sciences, University of Alabama at Birmingham, Birmingham, Alabama
  • Footnotes
    Commercial Relationships  O.P. Srivastava, None; R. Gupta, None.
  • Footnotes
    Support  NIH EY-06400 and EyeSight Foundation of Alabama
Investigative Ophthalmology & Visual Science April 2010, Vol.51, 5806. doi:
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      O. P. Srivastava, R. Gupta; Auto-Degradation of Human βA3-Crystallin is Due to Its Serine-Type Protease Activity. Invest. Ophthalmol. Vis. Sci. 2010;51(13):5806.

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      © ARVO (1962-2015); The Authors (2016-present)

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Purpose: : To determine whether the auto-degradation of human βA3-crystallin is due to its proteinase activity.

Methods: : Human βA3-crystallin was cloned, expressed and purified as previously described (Gupta, Srivastava and Srivastava, Biochemistry 2006, 45, 9964-9978). The purified βA3-crystallin preparation was incubated under sterile conditions with and without detergents in 0.05 M Tris-buffer, pH 7.9 at 37°C to determine its detergent-induced auto-degradation. Additionally, the crystallin was incubated under identical conditions with protease inhibitors to examine their effects on its auto-degradation. Partial N-terminal sequencing of three major auto-degraded species with Mr of 25, 27 and 29 kDa was performed to determine specific bonds cleaved in the crystallin during their production.

Results: : On incubation of βA3-crystallin with CHAPS {3-[(3-Cholamidopropyl) dimethylammonio]-1-propanesulfonate}, the crystallin showed three major auto-degradation products with Mr of 25, 27 and 29 kDa. The auto-degradation in the crystallin occurred at much slower rate on incubation alone or with Triton X-100, or with CHAPS plus serine protease inhibitors (phenylmethylsulfonyl fluoride, aprotinin and chymostatin and L-1-tosylamido-2-phenylethyl chloromethyl ketone [TPCK]). However, the degradation occurred in the presence of metallo-protease inhibitors (EDTA and EGTA) and cystein protease inhibitors (E-64, methylmaleimide and iodoacetamide). The results suggested that a serine-type protease activity was responsible for auto-degradation. The specific bond cleaved to produce 25, 27 and 29 kDa species wereLys17-Met18, Gln4-Ala5 and Thr-Gly (in His-tag region), respectively.

Conclusions: : Human βA3-crystallin exhibited auto-degradation on incubation with detergents such as CHAPS, which is due to its serine-type protease activity.

Keywords: crystallins • protein structure/function • proteolysis 

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