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Proteomics analysis of amyloid corneal aggregates from lattice corneal dystrophy patients

ABSTRACT: TGFBI associated Corneal Dystrophies (CD) are a group of inherited protein folding disorders linked to the mutation in the TGFBI gene. The resultant mutant protein (TGFBIp) is deposited as insoluble protein aggregates in various layers of the cornea leading to corneal opacity and poor vision. Depending on the type of mutation the deposits may be classified as amyloid fibrillar type, amorphous globular aggregates or a mixed form of both fibrils and amorphous aggregates. However, the molecular mechanism of the mutant-induced amyloidosis is not fully understood. This study aimsto characterize truncated peptides enriched in the amyloid aggregates and to identify the protein composition of the corneal aggregates derived from dystrophic patients using LC-MS/MS and compare the data with normal control cornea. We have identified several amyloid associated proteins, non-fibrillar amyloid associated proteins and TGFBIp as the major component of the corneal deposits. The results suggest that Apolipoprotein A-IV, Apolipoprotein E and Serine protease HtrA1 to be significantly enriched in the corneal deposits compared to the normal cornea. Comparative analysis of peptides from corneal deposits of patient and control identified several peptides of TGFBIp which are enriched in patient tissue and may form the core of corneal amyloids. Most of the peptides represent the 4th FAS-1 domain of the protein. Biophysical studies of two such peptides (G515DNRFSMLVAAIQSAGLTETLNR533 and Y571HIGDEILVSGGIGALVR588) demonstrate that they readily form amyloid fibrils under physiological conditions, confirming their intrinsic propensity to form amyloid fibrils. The identification of proteins which are involved in other protein misfolding disorders as well as identification of peptides from TGFBIp which form -amyloid core highlight that the mechanism of amyloid formation may share common molecular pathways.

INSTRUMENT(S): Q Exactive

ORGANISM(S): Homo Sapiens (human)

TISSUE(S): Eye

DISEASE(S): Lattice Corneal Dystrophy

SUBMITTER: Bamaprasad Dutta

LAB HEAD: Siu Kwan SZE

PROVIDER: PXD006640 | Pride | 2022-02-28

REPOSITORIES: Pride

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			Action	DRS
	01_150122_Patient_Cornea_Minus01_A.msf	Msf
	01_150122_Patient_Cornea_Minus01_A.raw	Raw
	02_150122_Patient_Cornea_Minus01_B.msf	Msf
	02_150122_Patient_Cornea_Minus01_B.raw	Raw
	03_150122_Patient_Cornea_Minus01_C.msf	Msf

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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.

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Proteomics analysis of amyloid corneal aggregates from lattice corneal dystrophy patients

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