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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.

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Related in: MedlinePlus

Comparison of the amount of recombinant TGFBI secreted from HCE and HeLa cells. HCE and HeLa cells (lane 1–lane 4: HCE, lane 5–lane 8: HeLa cells) were transfected with wild-type (WT) and the two mutant TGFBI plasmids. Control represents HCE or HeLa cells without transfection with plasmids. Media from overexpressed cells were collected and analyzed with an anti-myc antibody. The expression level of the R555W mutant protein was much higher than that of the T538P mutant protein. Moreover, the HCE cells expressed more full-length mutant protein than the HeLa cells. This result indicates the cell type and mutation affect the TGFBIp expression level.
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f5: Comparison of the amount of recombinant TGFBI secreted from HCE and HeLa cells. HCE and HeLa cells (lane 1–lane 4: HCE, lane 5–lane 8: HeLa cells) were transfected with wild-type (WT) and the two mutant TGFBI plasmids. Control represents HCE or HeLa cells without transfection with plasmids. Media from overexpressed cells were collected and analyzed with an anti-myc antibody. The expression level of the R555W mutant protein was much higher than that of the T538P mutant protein. Moreover, the HCE cells expressed more full-length mutant protein than the HeLa cells. This result indicates the cell type and mutation affect the TGFBIp expression level.

Mentions: To compare the expression level of TGFBIp-myc in HCE and HeLa cells, an equal amount of protein (70 μg) was collected from the media of these cells after transfection and subjected to electrophoresis. The blot showed that there was more full-length TGFBI-T538P/R555W-myc protein in HCE cells compared with HeLa cells (Figure 5). In addition, the endogenous levels of the TGFBI gene expressed in both cell lines were checked with quantitative real-time RT–PCR. The results showed the expression levels of the endogenous TGFBI gene of the HCE cells were higher than those of the HeLa cells (data not shown). Moreover, the expression levels of the TGFBI-R555W-myc protein were higher than those of the TGFBI-T538P-myc protein, indicating that the mutation also had an effect on protein expression.


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)

Comparison of the amount of recombinant TGFBI secreted from HCE and HeLa cells. HCE and HeLa cells (lane 1–lane 4: HCE, lane 5–lane 8: HeLa cells) were transfected with wild-type (WT) and the two mutant TGFBI plasmids. Control represents HCE or HeLa cells without transfection with plasmids. Media from overexpressed cells were collected and analyzed with an anti-myc antibody. The expression level of the R555W mutant protein was much higher than that of the T538P mutant protein. Moreover, the HCE cells expressed more full-length mutant protein than the HeLa cells. This result indicates the cell type and mutation affect the TGFBIp expression level.
© Copyright Policy - open-access
Related In: Results  -  Collection

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

f5: Comparison of the amount of recombinant TGFBI secreted from HCE and HeLa cells. HCE and HeLa cells (lane 1–lane 4: HCE, lane 5–lane 8: HeLa cells) were transfected with wild-type (WT) and the two mutant TGFBI plasmids. Control represents HCE or HeLa cells without transfection with plasmids. Media from overexpressed cells were collected and analyzed with an anti-myc antibody. The expression level of the R555W mutant protein was much higher than that of the T538P mutant protein. Moreover, the HCE cells expressed more full-length mutant protein than the HeLa cells. This result indicates the cell type and mutation affect the TGFBIp expression level.
Mentions: To compare the expression level of TGFBIp-myc in HCE and HeLa cells, an equal amount of protein (70 μg) was collected from the media of these cells after transfection and subjected to electrophoresis. The blot showed that there was more full-length TGFBI-T538P/R555W-myc protein in HCE cells compared with HeLa cells (Figure 5). In addition, the endogenous levels of the TGFBI gene expressed in both cell lines were checked with quantitative real-time RT–PCR. The results showed the expression levels of the endogenous TGFBI gene of the HCE cells were higher than those of the HeLa cells (data not shown). Moreover, the expression levels of the TGFBI-R555W-myc protein were higher than those of the TGFBI-T538P-myc protein, indicating that the mutation also had an effect on protein expression.

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