April 2010
Volume 51, Issue 13
ARVO Annual Meeting Abstract  |   April 2010
Methylglyoxal Increases the Solubility and Decreases the Aggregation Propensity of a Cataract Causing A-Crystallin (G98R) Mutant Protein
Author Affiliations & Notes
  • P. Santhoshkumar
    Ophthalmology, University of Missouri-Columbia, Columbia, Missouri
  • M. Raju
    Ophthalmology, Univ of Missouri Mason Eye Institute, Columbia, Missouri
  • K. Sharma
    Ophthalmology and Biochemistry, University of Missouri, Columbia, Missouri
  • Footnotes
    Commercial Relationships  P. Santhoshkumar, None; M. Raju, None; K. Sharma, None.
  • Footnotes
    Support  NIH Grant EY11981 and Research to Prevent Blindness
Investigative Ophthalmology & Visual Science April 2010, Vol.51, 5800. doi:
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      P. Santhoshkumar, M. Raju, K. Sharma; Methylglyoxal Increases the Solubility and Decreases the Aggregation Propensity of a Cataract Causing A-Crystallin (G98R) Mutant Protein. Invest. Ophthalmol. Vis. Sci. 2010;51(13):5800.

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

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Purpose: : The G98R mutation in human αA-crystallin (αAG98R ) causes cataract. The mutant protein exhibits increased oligomeric mass, decreased stability and altered chaperone function. The objective of this study was to determine effects of methylglyoxal (MGO), a metabolic dicarbonyl compound on the solubility and aggregation properties of αAG98R protein.

Methods: : Wild-type αA-crystallin and G98R mutant proteins were expressed in E. Coli BL21 DE3 cells and purified by anion exchange chromatography. The purified proteins (0.2 mg) were incubated with (50 µM) or without MGO in 0.2 ml of 0.05 M sodium phosphate buffer (pH 7.2) at 37oC for one week. After incubation, the samples were filtered through 0.2 µm filter and 25 µl of the filtrate was injected to HPLC fitted with TSK5000PWXL column and a refractive index detector coupled to multi-angle and dynamic light scattering instruments. The molar mass (Mw), hydrodynamic radius (Rh) and protein concentrations of the samples were determined using ASTRA software developed by Wyatt technologies.

Results: : In the absence of MGO, 18% of αAG98R and 76% of wt-αA were present in soluble form. However, upon reaction with MGO, nearly 80% of αAG98R and wt-αA were present in soluble form suggesting that the reaction of MGO increased the solubility of αAG98R. Reaction of MGO reduced the average Mw of soluble wild-type protein oligomers from 5.62 e5 to 4.67e5 g/mol (17% decrease) and that of αAG98R from 9.68 e6 to 3.36 e6 g/mol (65% decrease). The reaction of MGO reduced the Rh value of αAG98R from 21 to 16 nm but had no effect on the Rh value of wtαA.

Conclusions: : Our data suggests that MGO treatment increases the stability and decreases aggregation of αAG98R protein and thus MGO may have therapeutic role in preventing protein aggregation diseases.

Keywords: crystallins • protein structure/function • cataract 

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