Limits...
Investigation of the influence of Arg555Trp and Thr538Pro TGFBI mutations on C-terminal cleavage and cell endoplasmic reticulum stress.

Zhu M, Yu P, Jiang B, Gu Y - Mol. Vis. (2012)

Bottom Line: Arg555Trp and Thr538Pro mutant TGFBIp were detected with the anti-c-myc and anti-TGFBI antibodies, while wild-type TGFBIp was detected only with the anti-TGFBI antibody, indicating that the Arg555Trp and Thr538Pro mutations prevent the C-terminal cleavage of TGFBIp.Moreover, no significant differences were seen in the expression levels of GRP78/BiP between the mutant and wild-type TGFBIp groups, suggesting that mutations in TGFBIp are unlikely to disrupt protein folding or induce cell ER stress.This is the first time that the influence of TGFBI mutants on C-terminal cleavage and cell ER stress has been illustrated.

View Article: PubMed Central - PubMed

Affiliation: Department of Ophthalmology, Ophthalmic Genetic Research Centre, The First Affiliated Hospital, School of Medicine, Zhejiang University, Hangzhou, China.

ABSTRACT

Purpose: To gain insight into the mechanisms underlying the transforming growth factor-beta induced (TGFBI)-related corneal dystrophies and the influence of the Arg555Trp and Thr538Pro, TGFBI mutations on C-terminal cleavage and cell endoplasmic reticulum (ER) stress were investigated.

Methods: The Arg555Trp and Thr538Pro mutations known to be associated with corneal dystrophy granular type I and lattice corneal dystrophy, respectively, were introduced with the two-sequential PCR site-directed mutagenesis technique. Wild-type and mutant TGFBI DNAs were cloned into the pcDNA3.1(-)/myc-his expression vector and overexpressed in HeLa and human corneal epithelial cells (HCE) with transient transfection. Transfection efficiency was measured by the expression of green fluorescent protein. Expression of the fusion proteins was measured with western blot analysis with anti-c-myc-tag and anti-TGFBI antibodies. For cell ER stress studies, the expression levels of GRP78/BiP in HeLa cells were analyzed with western blot analysis using an anti-GRP78 monoclonal antibody at 12, 24, and 48 h after either the wild-type or mutant plasmid was transfected.

Results: Arg555Trp and Thr538Pro mutant TGFBIp were detected with the anti-c-myc and anti-TGFBI antibodies, while wild-type TGFBIp was detected only with the anti-TGFBI antibody, indicating that the Arg555Trp and Thr538Pro mutations prevent the C-terminal cleavage of TGFBIp. Moreover, no significant differences were seen in the expression levels of GRP78/BiP between the mutant and wild-type TGFBIp groups, suggesting that mutations in TGFBIp are unlikely to disrupt protein folding or induce cell ER stress.

Conclusions: This is the first time that the influence of TGFBI mutants on C-terminal cleavage and cell ER stress has been illustrated. Corneal dystrophy-related mutations are more likely to disrupt the interaction of TGFBI with critical binding proteins than affect the whole protein structure.

Show MeSH

Related in: MedlinePlus

Schematic of proposed mechanism of TGFBI-related corneal dystrophies in present study. The hypothesis of pathogenesis of TGFBI-related corneal dystrophies mainly consists of two aspects: altering the binding interactions of TGFBIp with other critical proteins and interfering with the TGFBIp folding. The influence of TGFBI mutations on C-terminal cleavage and cell endoplasmic reticulum stress investigated in the present study was based on the aspects of the hypothesis. The mechanism by which these mutations cause disease remains unknown, as indicated by the dashed arrow.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
getmorefigures.php?uid=PMC3351430&req=5

f1: Schematic of proposed mechanism of TGFBI-related corneal dystrophies in present study. The hypothesis of pathogenesis of TGFBI-related corneal dystrophies mainly consists of two aspects: altering the binding interactions of TGFBIp with other critical proteins and interfering with the TGFBIp folding. The influence of TGFBI mutations on C-terminal cleavage and cell endoplasmic reticulum stress investigated in the present study was based on the aspects of the hypothesis. The mechanism by which these mutations cause disease remains unknown, as indicated by the dashed arrow.

Mentions: The TGFBI protein (TGFBIp, also known as keratoepithelin or βig-H3), which is encoded by the TGFBI gene, is composed of 683 amino acids and has a molecular mass of approximately 68 kDa. The protein is an extracellular matrix (ECM) protein that contains an N-terminal secretory signal peptide (1–23 aa), a cysteine-rich EMI domain (45–99 aa), four repeats of fasciclin-1-like (FAS1) domain (136–634 aa), and a C-terminal arginine-glycine-aspartic acid (RGD) sequence (642–644 aa). Most of the mutations that have been identified are located in the fourth FAS1 domain [3], such as p.Arg555Trp and p.Thr538Pro. Although the mechanism by which these mutations cause disease remains unknown, the hypothesis of pathogenesis mainly consists of two aspects: altering the binding interactions of TGFBIp with other critical proteins and interfering with TGFBIp folding (Figure 1).


Investigation of the influence of Arg555Trp and Thr538Pro TGFBI mutations on C-terminal cleavage and cell endoplasmic reticulum stress.

Zhu M, Yu P, Jiang B, Gu Y - Mol. Vis. (2012)

Schematic of proposed mechanism of TGFBI-related corneal dystrophies in present study. The hypothesis of pathogenesis of TGFBI-related corneal dystrophies mainly consists of two aspects: altering the binding interactions of TGFBIp with other critical proteins and interfering with the TGFBIp folding. The influence of TGFBI mutations on C-terminal cleavage and cell endoplasmic reticulum stress investigated in the present study was based on the aspects of the hypothesis. The mechanism by which these mutations cause disease remains unknown, as indicated by the dashed arrow.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC3351430&req=5

f1: Schematic of proposed mechanism of TGFBI-related corneal dystrophies in present study. The hypothesis of pathogenesis of TGFBI-related corneal dystrophies mainly consists of two aspects: altering the binding interactions of TGFBIp with other critical proteins and interfering with the TGFBIp folding. The influence of TGFBI mutations on C-terminal cleavage and cell endoplasmic reticulum stress investigated in the present study was based on the aspects of the hypothesis. The mechanism by which these mutations cause disease remains unknown, as indicated by the dashed arrow.
Mentions: The TGFBI protein (TGFBIp, also known as keratoepithelin or βig-H3), which is encoded by the TGFBI gene, is composed of 683 amino acids and has a molecular mass of approximately 68 kDa. The protein is an extracellular matrix (ECM) protein that contains an N-terminal secretory signal peptide (1–23 aa), a cysteine-rich EMI domain (45–99 aa), four repeats of fasciclin-1-like (FAS1) domain (136–634 aa), and a C-terminal arginine-glycine-aspartic acid (RGD) sequence (642–644 aa). Most of the mutations that have been identified are located in the fourth FAS1 domain [3], such as p.Arg555Trp and p.Thr538Pro. Although the mechanism by which these mutations cause disease remains unknown, the hypothesis of pathogenesis mainly consists of two aspects: altering the binding interactions of TGFBIp with other critical proteins and interfering with TGFBIp folding (Figure 1).

Bottom Line: Arg555Trp and Thr538Pro mutant TGFBIp were detected with the anti-c-myc and anti-TGFBI antibodies, while wild-type TGFBIp was detected only with the anti-TGFBI antibody, indicating that the Arg555Trp and Thr538Pro mutations prevent the C-terminal cleavage of TGFBIp.Moreover, no significant differences were seen in the expression levels of GRP78/BiP between the mutant and wild-type TGFBIp groups, suggesting that mutations in TGFBIp are unlikely to disrupt protein folding or induce cell ER stress.This is the first time that the influence of TGFBI mutants on C-terminal cleavage and cell ER stress has been illustrated.

View Article: PubMed Central - PubMed

Affiliation: Department of Ophthalmology, Ophthalmic Genetic Research Centre, The First Affiliated Hospital, School of Medicine, Zhejiang University, Hangzhou, China.

ABSTRACT

Purpose: To gain insight into the mechanisms underlying the transforming growth factor-beta induced (TGFBI)-related corneal dystrophies and the influence of the Arg555Trp and Thr538Pro, TGFBI mutations on C-terminal cleavage and cell endoplasmic reticulum (ER) stress were investigated.

Methods: The Arg555Trp and Thr538Pro mutations known to be associated with corneal dystrophy granular type I and lattice corneal dystrophy, respectively, were introduced with the two-sequential PCR site-directed mutagenesis technique. Wild-type and mutant TGFBI DNAs were cloned into the pcDNA3.1(-)/myc-his expression vector and overexpressed in HeLa and human corneal epithelial cells (HCE) with transient transfection. Transfection efficiency was measured by the expression of green fluorescent protein. Expression of the fusion proteins was measured with western blot analysis with anti-c-myc-tag and anti-TGFBI antibodies. For cell ER stress studies, the expression levels of GRP78/BiP in HeLa cells were analyzed with western blot analysis using an anti-GRP78 monoclonal antibody at 12, 24, and 48 h after either the wild-type or mutant plasmid was transfected.

Results: Arg555Trp and Thr538Pro mutant TGFBIp were detected with the anti-c-myc and anti-TGFBI antibodies, while wild-type TGFBIp was detected only with the anti-TGFBI antibody, indicating that the Arg555Trp and Thr538Pro mutations prevent the C-terminal cleavage of TGFBIp. Moreover, no significant differences were seen in the expression levels of GRP78/BiP between the mutant and wild-type TGFBIp groups, suggesting that mutations in TGFBIp are unlikely to disrupt protein folding or induce cell ER stress.

Conclusions: This is the first time that the influence of TGFBI mutants on C-terminal cleavage and cell ER stress has been illustrated. Corneal dystrophy-related mutations are more likely to disrupt the interaction of TGFBI with critical binding proteins than affect the whole protein structure.

Show MeSH
Related in: MedlinePlus