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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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Western blot analysis of GRP78/BiP. HeLa cells were harvested at 12 (A), 24 (B), and 48 (C) h after transfection with the pcTGFBI-WT-myc (WT), pcTGFBI-T538P-myc (T538P), and pcTGFBI-R555W-myc (R555W) plasmids. The proteins were separated on 8% SDS-polyacrylamide gels, transferred, and probed with an anti-GRP 78 antibody. The amount of β-actin was analyzed with an anti-β-actin antibody to ensure that equal amounts of protein were loaded in each lane for electrophoresis. The intensity ratio was calculated by the intensity of the GRP78 band divided by the intensity of the β-actin band (D). No significant differences were found between the mutant and wild-type groups at 12 and 48 h (p>0.05). The intensity ratio of the T538P group was slightly lower than the wild-type group at 24 h (p=0.003). This transient fluctuation returned to the same level as the wild-type group after 48 h. The expression of GRP78/BiP at 48 h was significantly higher than that at 12 and 24 h (p<0.05). In the positive control group, GRP78/BiP expression was significantly increased in the tunicamycin-treated cells compared with the untreated cells (E).
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f6: Western blot analysis of GRP78/BiP. HeLa cells were harvested at 12 (A), 24 (B), and 48 (C) h after transfection with the pcTGFBI-WT-myc (WT), pcTGFBI-T538P-myc (T538P), and pcTGFBI-R555W-myc (R555W) plasmids. The proteins were separated on 8% SDS-polyacrylamide gels, transferred, and probed with an anti-GRP 78 antibody. The amount of β-actin was analyzed with an anti-β-actin antibody to ensure that equal amounts of protein were loaded in each lane for electrophoresis. The intensity ratio was calculated by the intensity of the GRP78 band divided by the intensity of the β-actin band (D). No significant differences were found between the mutant and wild-type groups at 12 and 48 h (p>0.05). The intensity ratio of the T538P group was slightly lower than the wild-type group at 24 h (p=0.003). This transient fluctuation returned to the same level as the wild-type group after 48 h. The expression of GRP78/BiP at 48 h was significantly higher than that at 12 and 24 h (p<0.05). In the positive control group, GRP78/BiP expression was significantly increased in the tunicamycin-treated cells compared with the untreated cells (E).

Mentions: The expression of the ER stress molecular chaperone GRP78/BiP was analyzed to investigate ER stress after transfection. Intracellular proteins were collected from HeLa cells at 12, 24, and 48 h after transfection with different plasmids. An equal amount of protein was subjected to electrophoresis. No significant differences were found between the mutant and wild-type groups at 12 and 48 h (p>0.05; Figure 6A, C,D). The intensity ratio of the T538P group was slightly lower than the wild-type group at 24 h (p=0.003; Figure 6B,D). However, this transient fluctuation returned to the same level as the wild-type group after 48 h. The expression of GRP78/BiP at 48 h was significantly higher than that at 12 and 24 h (p<0.05; Figure 6). In the positive control group, GRP78/BiP expression was significantly increased in the tunicamycin-treated cells compared with the untreated cells (Figure 6E).


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)

Western blot analysis of GRP78/BiP. HeLa cells were harvested at 12 (A), 24 (B), and 48 (C) h after transfection with the pcTGFBI-WT-myc (WT), pcTGFBI-T538P-myc (T538P), and pcTGFBI-R555W-myc (R555W) plasmids. The proteins were separated on 8% SDS-polyacrylamide gels, transferred, and probed with an anti-GRP 78 antibody. The amount of β-actin was analyzed with an anti-β-actin antibody to ensure that equal amounts of protein were loaded in each lane for electrophoresis. The intensity ratio was calculated by the intensity of the GRP78 band divided by the intensity of the β-actin band (D). No significant differences were found between the mutant and wild-type groups at 12 and 48 h (p>0.05). The intensity ratio of the T538P group was slightly lower than the wild-type group at 24 h (p=0.003). This transient fluctuation returned to the same level as the wild-type group after 48 h. The expression of GRP78/BiP at 48 h was significantly higher than that at 12 and 24 h (p<0.05). In the positive control group, GRP78/BiP expression was significantly increased in the tunicamycin-treated cells compared with the untreated cells (E).
© Copyright Policy - open-access
Related In: Results  -  Collection

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f6: Western blot analysis of GRP78/BiP. HeLa cells were harvested at 12 (A), 24 (B), and 48 (C) h after transfection with the pcTGFBI-WT-myc (WT), pcTGFBI-T538P-myc (T538P), and pcTGFBI-R555W-myc (R555W) plasmids. The proteins were separated on 8% SDS-polyacrylamide gels, transferred, and probed with an anti-GRP 78 antibody. The amount of β-actin was analyzed with an anti-β-actin antibody to ensure that equal amounts of protein were loaded in each lane for electrophoresis. The intensity ratio was calculated by the intensity of the GRP78 band divided by the intensity of the β-actin band (D). No significant differences were found between the mutant and wild-type groups at 12 and 48 h (p>0.05). The intensity ratio of the T538P group was slightly lower than the wild-type group at 24 h (p=0.003). This transient fluctuation returned to the same level as the wild-type group after 48 h. The expression of GRP78/BiP at 48 h was significantly higher than that at 12 and 24 h (p<0.05). In the positive control group, GRP78/BiP expression was significantly increased in the tunicamycin-treated cells compared with the untreated cells (E).
Mentions: The expression of the ER stress molecular chaperone GRP78/BiP was analyzed to investigate ER stress after transfection. Intracellular proteins were collected from HeLa cells at 12, 24, and 48 h after transfection with different plasmids. An equal amount of protein was subjected to electrophoresis. No significant differences were found between the mutant and wild-type groups at 12 and 48 h (p>0.05; Figure 6A, C,D). The intensity ratio of the T538P group was slightly lower than the wild-type group at 24 h (p=0.003; Figure 6B,D). However, this transient fluctuation returned to the same level as the wild-type group after 48 h. The expression of GRP78/BiP at 48 h was significantly higher than that at 12 and 24 h (p<0.05; Figure 6). In the positive control group, GRP78/BiP expression was significantly increased in the tunicamycin-treated cells compared with the untreated cells (Figure 6E).

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