Proteomic Analysis of Amyloid Corneal Aggregates from TGFBI-H626R Lattice Corneal Dystrophy Patient Implicates Serine-Protease HTRA1 in Mutation-Specific Pathogenesis of TGFBIp

Abstract

TGFBI-associated corneal dystrophies are inherited disorders caused by TGFBI gene variants that promote deposition of mutant protein (TGFBIp) as insoluble aggregates in the cornea. Depending on the type and position of amino acid substitution, the aggregates may be amyloid fibrillar, amorphous globular or both, but the molecular mechanisms that drive these different patterns of aggregation are not fully understood. In the current study, we report the protein composition of amyloid corneal aggregates from lattice corneal dystrophy patients of Asian origin with H626R and R124C mutation and compared it with healthy corneal tissues via LC-MS/MS. We identified several amyloidogenic, nonfibrillar amyloid associated proteins and TGFBIp as the major components of the deposits. Our data indicates that apolipoprotein A-IV, apolipoprotein E, and serine protease HTRA1 were significantly enriched in patient deposits compared to healthy controls. HTRA1 was also found to be 7-fold enriched in the amyloid deposits of patients compared to the controls. Peptides sequences (GDNRFSMLVAAIQSAGLTETLNR and YHIGDEILVSGGIGALVR) derived from the fourth FAS-1 domain of TGFBIp were enriched in the corneal aggregates in a mutation-specific manner. Biophysical studies of these two enriched sequences revealed high propensity to form amyloid fibrils under physiological conditions. Our data suggests a possible proteolytic processing mechanism of mutant TGFBIp by HTRA1 and peptides generated by mutant protein may form the β-amyloid core of corneal aggregates in dystrophic patients.

Biochemical mechanisms of aggregation in TGFBI-linked corneal dystrophies

“…The protein composition of the aggregates was compared with healthy control tissue to identify the accumulating proteins. This approach has been applied in subsequent studies by ourselves and others that determined the protein aggregate compositions related to TGFBI-linked corneal dystrophies (Courtney et al, 2015;Karring et al, 2013;Poulsen et al, 2016;Poulsen et al, 2014b;Venkatraman et al, 2017).…”

“…The protein profiles of amyloid LCD deposits from patients carrying R124C, A546D, A546D/P551Q, V624M, or H626R TGFBIp mutations show the accumulation of other amyloidogenic proteins in addition to TGFBIp, as well as nonfibrillar amyloid-associated proteins. The amyloidogenic proteins include apolipoprotein A-I, apolipoprotein A-IV, lysozyme C, protein S100-A8, protein S100-A9, and lactotransferrin (Courtney et al, 2015;Karring et al, 2013;Karring et al, 2012;Poulsen et al, 2014b;Venkatraman et al, 2017). Whether the amyloidogenic proteins are directly involved in the progression of amyloid deposits or merely innocent coaggregating bystanders is not known.…”

“…Analysis of TGFBIp in amyloid deposits with a mutation in the FAS1-4 domain showed extended accumulation of various ragged isoforms of two polypeptide regions in FAS1-4 (Karring et al, 2013;Karring et al, 2012;Poulsen et al, 2014b;Venkatraman et al, 2017). For simplicity, these two polypeptide regions have been designated as the F515-R533 and Y571-R588 peptides in the literature, referring to the first (e.g., 515) and last (e.g., 533) amino acid number in the protein sequence of TGFBIp.…”

Matrix‐Assisted Laser Desorption Ionization Mass Spectrometry Imaging of Key Proteins in Corneal Samples from Lattice Dystrophy Patients with TGFBI‐H626R and TGFBI‐R124C Mutations

“…There have been several studies that have investigated and reported the protein composition of corneal aggregates from dystrophic patients with different TGFBI mutations . Our group has reported the protein composition of corneal aggregates from lattice corneal dystrophy (LCD) patients with H626R and R124C mutations using liquid chromatography‐mass spectrometry/mass spectrometry (LC‐MS/MS) methods . All studies have identified several amyloidogenic, non‐amyloid fibrillar proteins and TGFBIp to be the major components of the corneal aggregates and suggested the possibility of differences in proteolytic procession of mutant protein compared to wild type (WT) …”

Effect of osmolytes on in-vitro aggregation properties of peptides derived from TGFBIp

“…It has been reported that the peptides derived from the 1 st and 4 th FAS-1 domains of mutant TGFBIp have increased aggregation propensity compared to the WT [21][22][23][24] . We have also previously reported the protein composition of the amyloid fibrils from Lattice Corneal Dystrophy patients (LCD) using mass spectrometric assays 25 . The proteolytic fragments of TGFBIp derived from amyloid fibrils of patients, upon digestion with trypsin showed a higher abundance of peptides from the 4 th FAS-1 domain of TGFBIp compared to healthy controls.…”