Dataset Information

Protein composition of TGFBI-R124C and TGFBI-R555W associated aggregates suggests multiple mechanisms leading to Lattice and Granular corneal dystrophy

ABSTRACT: Transforming Growth Factor Beta-induced (TGFBI)-related dystrophies constitute the most common heritable forms of corneal dystrophy worldwide. However, other than the underlying genotypes of these conditions, a limited knowledge exists of the exact pathomechanisms of these disorders. This study expands on our previous research investigating dystrophic stromal aggregates, with the aim of better elucidating the pathomechanism of 2 conditions arising from the most common TGFBI mutations: granular corneal dystrophy (GCD1; R555W), and lattice corneal dystrophy (LCD1; R124C). GCD1 and LCD1 patient corneas were stained with H&E and Congo red to visualise stromal non-amyloid and amyloid deposits, respectively. Laser capture microdissection was used to isolate aggregates and extracted protein was analyzed by mass spectrometry. Proteins were identified and their approximate abundances were determined. Spectra of TGFBIp peptides were also recorded and quantified. In total, 3 proteins were found within GCD1 aggregates that were absent in the healthy control corneal tissue. In comparison an additional 18 and 24 proteins within stromal LCD1 and Bowmanâ??s LCD1 deposits, respectively, were identified. Variances surrounding the endogenous cleavage sites of TGFBIp were also noted. An increase in the number of residues experiencing cleavage was observed in both GCD1 aggregates and LCD1 deposits. The study reveals previously unknown differences 1 between the protein composition of GCD1 and LCD1 aggregates, and confirms the presence of the HtrA1 protease in LCD1-amyloid aggregates. In addition, we find mutation specific differences in the processingof mutant TGFBIp species, which may contribute to the variable phenotypes noted in TGFBI-related dystrophies.

INSTRUMENT(S): TripleTOF 5600

ORGANISM(S): Homo Sapiens (ncbitaxon:9606)

SUBMITTER: Dr Jan J. Enghild

PROVIDER: MSV000079985 | MassIVE | Fri Jul 22 15:58:00 BST 2016

SECONDARY ACCESSION(S): PXD002236

REPOSITORIES: MassIVE

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Protein Composition of TGFBI-R124C- and TGFBI-R555W-Associated Aggregates Suggests Multiple Mechanisms Leading to Lattice and Granular Corneal Dystrophy.

Courtney David G DG Poulsen Ebbe Toftgaard ET Kennedy Susan S Moore Johnny E JE Atkinson Sarah D SD Maurizi Eleonora E Nesbit M Andrew MA Moore C B Tara CB Enghild Jan J JJ

Investigative ophthalmology & visual science 20150701 8

<h4>Purpose</h4>Transforming growth factor beta-induced (TGFBI)-related dystrophies constitute the most common heritable forms of corneal dystrophy worldwide. However, other than the underlying genotypes of these conditions, a limited knowledge exists of the exact pathomechanisms of these disorders. This study expands on our previous research investigating dystrophic stromal aggregates, with the aim of better elucidating the pathomechanism of two conditions arising from the most common TGFBI mut ...[more]

PMID: 26207300

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Project description:*Abstract:* In this study, we investigated whether the phenotypic difference observed between the two siblings with an atypical variant of lattice corneal dystrophy type 1 (LCD type 1) caused by a single A546D substitution and a A546D/P551Q double substitution in TGFBIp, can be ascribed to (i), a difference in plaque proteomes (ii), altered proteolysis of TGFBIp or (iii), is a consequence of the P551Q amino acid substitution. Amyloid deposits were isolated from two corneal specimens with atypical LCD type 1 due to a A546D/P551Q mutation in TGFBI using laser capture microdissection and a subsequent analysis by liquid chromatography-tandem mass spectrometry (LC-MS/MS). The findings were compared to the previous findings of a A546D LCD type 1 plaque proteome. Proteolytic processing of TGFBIp was addressed by counting peptide spectra obtained during the mass spectrometry (MS) analyses. Lastly, to study the effect of the P551Q substitution, recombinant FAS1-4 variants (wild type, A546D, A546D/P551Q, and P551Q) were expressed, purified and subjected to in vitro stability assays including circular dichroism, transverse urea gradient gel electrophoresis, and limited proteolysis. Plaque proteomes of the two A546D/P551Q LCD type 1 cases were similar to each other as well as to A564D plaque proteome previously reported. Major proteins associated with the deposits were TGFBIp, serum amyloid P, clusterin, HtrA1, Apolipoproteins A-1, A-IV, D, and E. Proteolytic processing of TGFBIp were also similar in all LCD type 1 cases regardless of the mutation indicating that a C-terminal TGFBIp polypeptide had accumulated in the deposits. The stability assays revealed a minor destabilization of FAS1-4 upon the addition of the P551Q mutation, however, enough to result in different accessibility to proteolytic processing by trypsin for the A546D and A546D/P551Q FAS1-4 variants. Difference in A546D and A546D/P551Q LCD type 1 phenotypes is not ascribed to altered plaque formation or altered proteolytic processing of TGFBIp. Instead, a small decrease in thermodynamic stability introduced by the P551Q mutation is believed to affect TGFBIp fibrillation and proteolytic accessibility of TGFBIp, thereby resulting in the different phenotypes observed in vivo. *Data handling:* Raw data were converted to mgf format using Protein Pilot software (AB Sciex) and searched using an in-house Mascot search engine (Matrix Science, London, UK; version: 2.3.02) against the Swiss-Prot database (version: 2012_05). Enzyme was set to semi-trypsin, and missed cleavage was 1. No fixed modifications were added. Instead oxidation of methionine and proline were included as variable modifications. The MS tolerance and MS/MS tolerance were 10 ppm and 0.4 Da, respectively. Duo, triple, and quadric charges of precursor ions were selected and an ESI-QUAD-TOF system was set as the instrument used. All data sets were simultaneously searched against the decoy database to apply a false discovery rate (FDR) between 1.5 to 7% and a p-value < 0.01. Peptides with an ion score lower than 30 were rejected. Peptide spectra for protein hits identified by only one peptide were manually inspected, thereby reducing the FDR value. In these cases, only spectra with at least three consecutive y or b ions with high intensities were considered valid.