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Nadia Sukusu Nielsen, Ebbe Toftgaard Poulsen, Marie Vestergaard Lukassen, Ida B. Thøgersen, Michael W. Risør, Carsten Scavenius, Kasper Runager, Jan J Enghild; Processing of TGFBIp by HtrA1 in relation to corneal dystrophies. Invest. Ophthalmol. Vis. Sci. 2016;57(12):4912.
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© ARVO (1962-2015); The Authors (2016-present)
Transforming growth factor beta-induced protein (TGFBIp) is one of the most abundant proteins within the human cornea where its physiological role still remains elusive. However, TGFBIp is well known for being implicated in corneal dystrophies and so far more than 60 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. These mutations are primarily located within the first or fourth fasciclin 1 domain (FAS1-1 and FAS1-4) of TGFBIp. Protein profiles of the TGFBIp related deposits in corneal dystrophies have revealed that the High-temperature requirement A serine peptidase 1 (HtrA1) accumulates in amyloid deposits. Here we investigate the role of HtrA1 in the in vitro processing of different TGFBIp genotypes representing both amyloid and non-amyloid phenotypes and HtrA1’s potential role in the disease mechanism.
Different genotypes of recombinant TGFBIp were subjected to proteolysis by the serine protease HtrA1 and analyzed with SDS-PAGE followed by immunoblotting against TGFBIp, and by MS. In addition to full length TGFBIp, different genotypes of recombinant FAS1-4 were likewise processed with HtrA1 and analyzed with SDS-PAGE and by MS. Lastly, the fibrillation propensity of the HtrA1-generated FAS1-4 peptides was investigated by ThT assay, SDS-PAGE and by MS.
In vitro, HtrA1 proteolysis of different TGFBIp and FAS1-4 genotypes showed preference for amyloid forming genotypes. In addition, MS analysis revealed that a fraction of the HtrA1-generated FAS1-4 peptides, containing the previously identified core sequence of FAS1-4 amyloid fibrils, were able to form fibrils of amyloid nature as judge by the ThT assay. No degradation was observed for the non-amyloid genotypes correlating well with previous results showing the association of HtrA1 to only the amyloid phenotype.
We have shown that amyloid forming genotypes of TGFBIp and FAS1-4 can be processed by HtrA1 in vitro. Moreover, the HtrA1-generated FAS1-4 peptides could form amyloid fibrils. Elucidation of the mechanism of TGFBIp processing in vitro could lead to a better understanding of the disease mechanism.
This is an abstract that was submitted for the 2016 ARVO Annual Meeting, held in Seattle, Wash., May 1-5, 2016.
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