July 2018
Volume 59, Issue 9
Open Access
ARVO Annual Meeting Abstract  |   July 2018
Structural characterization of corneal dystrophy-related TGFBIp mutants
Author Affiliations & Notes
  • Nadia Sukusu Nielsen
    Molecular Biology and Genetics, Aarhus University, Aarhus, Denmark
    The Interdisciplinary Nanoscience Center, Aarhus University, Aarhus University, Denmark
  • Tania A. Ammitzbøll
    Molecular Biology and Genetics, Aarhus University, Aarhus, Denmark
    The Interdisciplinary Nanoscience Center, Aarhus University, Aarhus University, Denmark
  • Ida B. Thøgersen
    Molecular Biology and Genetics, Aarhus University, Aarhus, Denmark
  • Ebbe Toftgaard Poulsen
    Molecular Biology and Genetics, Aarhus University, Aarhus, Denmark
  • Jan J. Enghild
    Molecular Biology and Genetics, Aarhus University, Aarhus, Denmark
    The Interdisciplinary Nanoscience Center, Aarhus University, Aarhus University, Denmark
  • Footnotes
    Commercial Relationships   Nadia Nielsen, None; Tania Ammitzbøll, None; Ida Thøgersen, None; Ebbe Poulsen, None; Jan Enghild, None
  • Footnotes
    Support  The Synoptik Foundation
Investigative Ophthalmology & Visual Science July 2018, Vol.59, 2254. doi:
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      Nadia Sukusu Nielsen, Tania A. Ammitzbøll, Ida B. Thøgersen, Ebbe Toftgaard Poulsen, Jan J. Enghild; Structural characterization of corneal dystrophy-related TGFBIp mutants. Invest. Ophthalmol. Vis. Sci. 2018;59(9):2254.

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

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Abstract

Purpose : Transforming growth factor beta-induced protein (TGFBIp) is one of the most abundant proteins within the human cornea where its physiological role remains elusive. However, TGFBIp is well known for being implicated in corneal dystrophies and so far, almost 70 different mutations in the TGFBI gene, is link to the occurrence of various types of protein aggregates in the cornea including both amyloid deposits and non-amyloid deposits.
Previously it has been shown that mutations in TGFBIp induce thermodynamic stability changes in TGFBIp. These analyses have mainly been performed on the FAS1-4 domain of TGFBIp. Here we investigate whether the proteolytic stability of TGFBIp mutants is affected, and we observe a correlation between reduced thermodynamic stability and susceptibility to proteolysis. Specific sites of differential proteolysis between wild-type and mutants was identified and correlated to the recently determined structure of wild-type TGFBIp.

Methods : Some of the most frequently observed genotypes of TGFBIp were cloned, expressed, and purified. Subsequently, the TGFBIp mutants were subjected to limited proteolysis by trypsin, analyzed by SDS-PAGE, and the degradation products identified by mass spectrometry and N-terminal sequencing.

Results : The differences in the degradation pattern of wild-type and mutant TGFBIp indicated that some of the mutations alter the fold of TGFBIp. Mutations leading to amyloid formation, especially those where an amino acid buried in the structure of TGFBIp was mutated, showed to be more prone to proteolysis. Cleavage sites were identified in the structure of wild-type TGFBIp and structural differences between wild-type TGFBIp and its mutants were discovered.

Conclusions : Comparison of wild-type and mutant TGFBIp by limited proteolysis indicated that there is a difference in the overall proteolytic stability between TGFBIp wild-type and its mutants. This could support the hypothesis of altered processing of TGFBIp mutants to be part of the disease mechanism. Furthermore, identification of cleavage sites revealed specific regions with structure differences between wild-type and mutants of TGFBIp.

This is an abstract that was submitted for the 2018 ARVO Annual Meeting, held in Honolulu, Hawaii, April 29 - May 3, 2018.

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