May 2006
Volume 47, Issue 13
ARVO Annual Meeting Abstract  |   May 2006
Abnormal Aggregations of Mutant BIGH3 Proteins in vitro
Author Affiliations & Notes
  • C. Yuan
    Department of Ophthalmology, University of Minnesota, Minneapolis, MN
  • A.J. W. Huang
    Department of Ophthalmology, University of Minnesota, Minneapolis, MN
  • Footnotes
    Commercial Relationships  C. Yuan, None; A.J.W. Huang, None.
  • Footnotes
    Support  None
Investigative Ophthalmology & Visual Science May 2006, Vol.47, 5552. doi:
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      C. Yuan, A.J. W. Huang; Abnormal Aggregations of Mutant BIGH3 Proteins in vitro . Invest. Ophthalmol. Vis. Sci. 2006;47(13):5552.

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

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Purpose: : Recent advances in molecular genetics have linked the mutations in BIGH3 gene with various types of corneal dystrophy characterized by the abnormal amyloid and/or non–amyloid deposits in corneal stroma. To understand the pathogenesis of BIGH3–related corneal dystrophies, in vitro studies with recombinant BIGH3 mutant proteins were conducted to investigate the molecular properties responsible for amyloid/fibril formation.

Methods: : A serum–free medium system was used for the large–scale expression of recombinant wild–type, R124C and R555W BIGH3 proteins in 293 cells. To facilitate protein purification, a (His)6–tag motif was inserted after the sequence encoding the signal peptide of BIGH3 in expression plasmids and a strep–tag II motif was inserted before the RGD sequence in the C–terminus. Ni+–NTA and strep–tactin chromatographies were used to purify expressed proteins. Limited proteolysis, intrinsic fluorescence and circular dichroism (CD) spectroscopy were used to characterize the molecular properties of purified recombinant proteins. Thioflavin T (Tht) and Congo red were also used to compare the fibril formation among wild–type and mutant proteins in vitro.

Results: : Both recombinant R124C and R555W proteins displayed distinct degradation patterns by SDS–PAGE gels and Western blots when compared with wild–type BIGH3, suggesting that conformational changes in mutants may result in differential protease susceptibility. CD spectroscopy revealed conversions of secondary structures in R124C and R555W proteins. These mutant proteins were also aggregation–prone and readily denatured by changes of pH, temperature and ionic strength in intrinsic fluorescence studies. Compared with wild–type BIGH3, R124C showed enhanced propensity for fibrillar aggregations, while R555W preferentially formed amorphous aggregations when incubated in vitro.

Conclusions: : Our results indicate that distinct molecular properties exist among the wild–type and dystrophy–related mutant BIGH3 proteins. The difference in protease susceptibility, protein stability and aggregation may contribute to the untoward amyloid and/or non–amyloid aggregations in BIGH3–related corneal dystrophies.

Keywords: cornea: basic science • proteins encoded by disease genes • anterior segment 

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