May 2006
Volume 47, Issue 13
ARVO Annual Meeting Abstract  |   May 2006
Oligomeric Structure and Chaperone Function of Human A– Crystallin With Deletions of C–Terminal Residues
Author Affiliations & Notes
  • A. Aziz
    Biochem/Molec, Univ of Arkansas for Medical Sciences, Little Rock, AR
  • E.C. Abraham
    Biochem/Molec, Univ of Arkansas for Medical Sciences, Little Rock, AR
  • Footnotes
    Commercial Relationships  A. Aziz, None; E.C. Abraham, None.
  • Footnotes
    Support  NIH Grant EY11352
Investigative Ophthalmology & Visual Science May 2006, Vol.47, 1998. doi:
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      A. Aziz, E.C. Abraham; Oligomeric Structure and Chaperone Function of Human A– Crystallin With Deletions of C–Terminal Residues . Invest. Ophthalmol. Vis. Sci. 2006;47(13):1998.

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

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Purpose: : Our previous studies have shown enhanced formation of αA–crystallins with C–terminal truncation in diabetic human and rat lenses. The structural and functional properties of the truncated rat αA crystallins were reported earlier. The present study is for determining the oligomeric structure and chaperone function of truncated αA– crystallins which exist in human lenses.

Methods: : Various truncated forms of human αA– crystallins were prepared by Quick Change Site–Directed Mutagenesis Kit (Stratagene). Stop codon was introduced at the appropriate positions. Coding sequences for the αA– wild–type and its C–terminal truncated proteins were confirmed by automated DNA sequencing. The proteins (in pET vector) were expressed in E. coli BL21 (DE3) pLysS cells and were purified by chromatography on Sephacryl S–300 HR columns and identity was confirmed by SDS–PAGE. Molecular masses were determined by molecular sieve HPLC using a 600 mm x 7.8 mm Biosep SEC 4000 column (Phenomenex). The chaperone activity was determined by analyzing the ability of the wild type αA– crystallin and the truncated proteins to prevent EDTA induced aggregation of alcohol dehydrogenase. The secondary and tertiary structural changes were assessed by far– UV and near– UV CD spectra measurements, respectively.

Results: : Molecular masses of truncated human αA1–162, αA1–163 and αA1–172 were 475, 550 and 700 kDa, respectively, as compared to 750 kDa for αA wild–type. The truncated αA1–162 and αA1–163 showed significantly reduced chaperone activity, alterations in secondary and tertiary structures and smaller aggregate sizes. These altered properties were more pronounced in the αA1–162 when compared with the αA1–163. However, αA1–172 was more or less like the wild type αA with no significant change in chaperone activity, oligomeric size and structural parameters. When compared with 75% decrease in oligomeric size and 60% decrease in chaperone activity of rat αA1–162 (Biochemistry, 40:11857–11863, 2003), human αA1–162 showed only 40% decrease in oligomeric size with complete loss of chaperone activity.

Conclusions: : The results suggest that in human lenses, the presence of αA1–172 , which is the major form of the post–translationally modified αA– crystallin, is not expected to affect the overall αA– crystallin chaperone function. However, the deletion of additional C–terminal sequences, as in αA1–162 and αA1–163 , results in loss of chaperone activity and extensive change in structural properties. Structural and functional features of hetero–aggregates of αA and αB mutants are under investigation.

Keywords: crystalline lens • chaperones • protein structure/function 

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