Advisory Committee Chair
Om P Srivastava
Advisory Committee Members
Roderick J Fullard
Kent T Keyser
Dennis J Pillion
David R Whikehart
Document Type
Dissertation
Date of Award
2008
Degree Name by School
Doctor of Philosophy (PhD) School of Optometry
Abstract
The three specific aims of the project were: (1) compare effects of deamidation alone (i.e., αA-N101D, αA-N123D, αA-N101/123D), truncation alone (αA-NT mutant [deletion of residue no. 1-63], or αA-CT mutant [deletion of residue no. 140-173]), or both truncation plus deamidation on structural and functional properties of human lens αA-crystallin; (2) identify components of complexes in the water soluble-high molecular weight (WS-HMW) proteins of normal aging and cataractous human lenses; and (3) determine interactions among WT-αA, αA-NT and αA-CT crystallin species with filensin and phakinin (beaded filament proteins) in vitro. The comparative structural and functional differences between αA-deamidated and/or truncated mutants and WT-αA crystallin were determined. The components of complexes in the WS-HMW proteins were identified by electrospray tandem mass spectrometry (ES-MS/MS). The interactions among phakinin and filensin with WT-αA, αA-NT, or αA-CT were determined using sedimentation and electron microscopic techniques. Results of the first specific aim showed that the αA-CT mutant became water insoluble with increased oligomerization. The αA-N123D mutant showed a decrease in chaperone activity, possibly due to relatively reduced β-sheet content and increased compact tertiary structure compared to WT-αA crystallin. The heteromers of WT-αB plus αA-CT, αA-NT, αA-N101D-NT, αA-N123D-NT, or αA-N101/123D-NT mutants exhibited increased chaperone activity compared to heteromers of WT-αA and WT-αB iii crystallins, which was due to a general increase in both β-sheet content and subunit exchange rates. In the second specific aim, HMW protein complexes contained species with molecular weights between 10 and 90 kDa in normal and cataractous lenses. The complexes in 20-year-old normal lenses contained α-, β-, and γ-crystallin, filensin and phakinin, and the 60 to 70-year-old normal lenses contained filensin, aldehyde dehydrogenase, and the above crystallins. The age-matched cataractous lenses contained the above crystallins and aldehyde dehydrogenase but lacked filensin and phakinin. The ES-MS/MS data also showed that the phakinin and/or filensin fragments were present with αA-crystallin that contained 1 to 65 N-terminal residues. The results of the third specific aim confirmed the importance of the αA Nterminal domain because, in contrast to WT-αA and the αA-CT mutant, the αA-NT mutant showed reduced or no interaction with phakinin and filensin.
Recommended Citation
Chaves, Jose Mauro, "Structural And Functional Properties Of Human αA-Crystallin" (2008). All ETDs from UAB. 3680.
https://digitalcommons.library.uab.edu/etd-collection/3680
