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Human phenotypically distinct TGFBI corneal dystrophies are linked to the stability of the fourth FAS1 domain of TGFBIp.

ABSTRACT: Mutations in the human TGFBI gene encoding TGFBIp have been linked to protein deposits in the cornea leading to visual impairment. The protein consists of an N-terminal Cys-rich EMI domain and four consecutive fasciclin 1 (FAS1) domains. We have compared the stabilities of wild-type (WT) human TGFBIp and six mutants known to produce phenotypically distinct deposits in the cornea. Amino acid substitutions in the first FAS1 (FAS1-1) domain (R124H, R124L, and R124C) did not alter the stability. However, substitutions within the fourth FAS1 (FAS1-4) domain (A546T, R555Q, and R555W) affected the overall stability of intact TGFBIp revealing the following stability ranking R555W>WT>R555Q>A546T. Significantly, the stability ranking of the isolated FAS1-4 domains mirrored the behavior of the intact protein. In addition, it was linked to the aggregation propensity as the least stable mutant (A546T) forms amyloid fibrils while the more stable variants generate non-amyloid amorphous deposits in vivo. Significantly, the data suggested that both an increase and a decrease in the stability of FAS1-4 may unleash a disease mechanism. In contrast, amino acid substitutions in FAS1-1 did not affect the stability of the intact TGFBIp suggesting that molecular the mechanism of disease differs depending on the FAS1 domain carrying the mutation.

SUBMITTER: Runager K

PROVIDER: S-EPMC3037607 | biostudies-literature | 2011 Feb

REPOSITORIES: biostudies-literature

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Human phenotypically distinct TGFBI corneal dystrophies are linked to the stability of the fourth FAS1 domain of TGFBIp.

Runager Kasper K Basaiawmoit Rajiv V RV Deva Taru T Andreasen Maria M Valnickova Zuzana Z Sørensen Charlotte S CS Karring Henrik H Thøgersen Ida B IB Christiansen Gunna G Underhaug Jarl J Kristensen Torsten T Nielsen Niels Chr NC Klintworth Gordon K GK Otzen Daniel E DE Enghild Jan J JJ

The Journal of biological chemistry 20101206 7

Mutations in the human TGFBI gene encoding TGFBIp have been linked to protein deposits in the cornea leading to visual impairment. The protein consists of an N-terminal Cys-rich EMI domain and four consecutive fasciclin 1 (FAS1) domains. We have compared the stabilities of wild-type (WT) human TGFBIp and six mutants known to produce phenotypically distinct deposits in the cornea. Amino acid substitutions in the first FAS1 (FAS1-1) domain (R124H, R124L, and R124C) did not alter the stability. How ...[more]

PMID: 21135107

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Project description:PURPOSE: To screen a cohort of corneal dystrophy patients from North India for mutations in the transforming growth factor beta induced (TGFBI) gene, to correlate genotypes to phenotypes, to describe structural implications of various mutations on protein function, and to discuss the implications for diagnosis. METHODS: Eighty affected individuals from 61 unrelated families, who were diagnosed with autosomal dominant granular and/or lattice corneal dystrophy, were recruited for the study. Detailed clinical evaluation was undertaken for these patients to establish their corneal phenotypes. Genomic DNA was isolated from peripheral blood samples and all exons of TGFBI were screened for mutations by polymerase chain reaction (PCR) and direct DNA sequencing. Protein molecular dynamics (MD) simulations were performed for the mutations detected to assess the changes in protein structure. RESULTS: The most common mutations seen were Arg555Trp and Arg124Cys. Two novel mutations, Ser516Arg (c.DNA1548C>G), with a phenotype similar to granular corneal dystrophy I (GCDI), and Leu559Val (c.DNA1675T>G), with an atypical phenotype closely resembling epithelial basement membrane dystrophy/map dot fingerprint dystrophy, were identified. Protein modeling studies involving wild type and mutant protein indicated that the Leu559Val is a destabilizing mutation and that Ser516Arg could adversely affect the specific binding of Fas1 domain 4 with other proteins. In addition, two single-nucleotide polymorphisms, rs4669 and rs11331170, were also identified. Mutations were not identified in 8 affected individuals, 6 of whom were diagnosed with bowman layer dystrophy and 2 with lattice corneal dystrophy. CONCLUSIONS: This is the first comprehensive report of TGFBI mutations covering a large part of North India. Identification of novel mutations, the presence of phenotypic variability, and the genetic heterogeneity seen in our cases stress the need for mandatory screening of TGFBI for precise diagnosis and classification of corneal dystrophies.

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.

Dataset Information

Human phenotypically distinct TGFBI corneal dystrophies are linked to the stability of the fourth FAS1 domain of TGFBIp.

Publications

Human phenotypically distinct TGFBI corneal dystrophies are linked to the stability of the fourth FAS1 domain of TGFBIp.

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