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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

Western blot analysis of the recombinant wild-type and mutant proteins. HCE and HeLa cells (A, B: HCE cell; C, D: HeLa cells) were transfected with wild-type (lane 2) and two mutant TGFBI plasmids (lanes 3 and 4) or were not transfected plasmid (lane 1). The culture medium was replaced with serum-free medium 48 h post-transfection. Another 48 h later, the serum-free media from overexpressed cells were collected and concentrated. A and C: 65 µg protein was loaded into each well. The TGFBI-T538P-myc and TGFBI-R555W-myc protein were detected with an anti-c-myc-tag antibody (lanes 3 and 4), while the wild-type TGFBI-myc protein was not (lane 2). B and D: 65 µg protein was loaded on to each well and detected with an anti-TGFBIp antibody. The wild-type and mutant TGFBI-myc fusion proteins were detected as two bands of different intensities (lanes 2–4). Endogenous TGFBIp is shown in the control lane (lane 1). The stronger bands in lanes 3 and 4 represent full-length mutant TGFBI-myc proteins, while the stronger band in lane 2 represents the wild-type TGFBI-myc protein that lacks its C-terminus. There was an approximate 10 kDa difference between the full-length and cleaved proteins.
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f4: Western blot analysis of the recombinant wild-type and mutant proteins. HCE and HeLa cells (A, B: HCE cell; C, D: HeLa cells) were transfected with wild-type (lane 2) and two mutant TGFBI plasmids (lanes 3 and 4) or were not transfected plasmid (lane 1). The culture medium was replaced with serum-free medium 48 h post-transfection. Another 48 h later, the serum-free media from overexpressed cells were collected and concentrated. A and C: 65 µg protein was loaded into each well. The TGFBI-T538P-myc and TGFBI-R555W-myc protein were detected with an anti-c-myc-tag antibody (lanes 3 and 4), while the wild-type TGFBI-myc protein was not (lane 2). B and D: 65 µg protein was loaded on to each well and detected with an anti-TGFBIp antibody. The wild-type and mutant TGFBI-myc fusion proteins were detected as two bands of different intensities (lanes 2–4). Endogenous TGFBIp is shown in the control lane (lane 1). The stronger bands in lanes 3 and 4 represent full-length mutant TGFBI-myc proteins, while the stronger band in lane 2 represents the wild-type TGFBI-myc protein that lacks its C-terminus. There was an approximate 10 kDa difference between the full-length and cleaved proteins.

Mentions: To investigate the expression of the wild-type and mutant TGFBI-myc fusion proteins, HCE and HeLa cells were used for transient plasmid transfection. Each protein expressed a Myc tag at its C-terminus. In Figure 4A, 65 µg protein from the media of HCE cells was loaded into each well. As expected, the wild-type TGFBI-myc protein was not detected by an anti-c-myc-tag antibody due to C-terminal cleavage (lane 2). This was consistent with Kim et al.’s study, which indicated that wild-type TGFBI undergoes C-terminal cleavage processing after secretion [11]. Surprisingly, TGFBI-T538P-myc (lane 3) and TGFBI-R555W-myc (lane 4) fusion proteins were detected by anti-c-myc-tag antibody at approximately 70 kDa.


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 the recombinant wild-type and mutant proteins. HCE and HeLa cells (A, B: HCE cell; C, D: HeLa cells) were transfected with wild-type (lane 2) and two mutant TGFBI plasmids (lanes 3 and 4) or were not transfected plasmid (lane 1). The culture medium was replaced with serum-free medium 48 h post-transfection. Another 48 h later, the serum-free media from overexpressed cells were collected and concentrated. A and C: 65 µg protein was loaded into each well. The TGFBI-T538P-myc and TGFBI-R555W-myc protein were detected with an anti-c-myc-tag antibody (lanes 3 and 4), while the wild-type TGFBI-myc protein was not (lane 2). B and D: 65 µg protein was loaded on to each well and detected with an anti-TGFBIp antibody. The wild-type and mutant TGFBI-myc fusion proteins were detected as two bands of different intensities (lanes 2–4). Endogenous TGFBIp is shown in the control lane (lane 1). The stronger bands in lanes 3 and 4 represent full-length mutant TGFBI-myc proteins, while the stronger band in lane 2 represents the wild-type TGFBI-myc protein that lacks its C-terminus. There was an approximate 10 kDa difference between the full-length and cleaved proteins.
© Copyright Policy - open-access
Related In: Results  -  Collection

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

f4: Western blot analysis of the recombinant wild-type and mutant proteins. HCE and HeLa cells (A, B: HCE cell; C, D: HeLa cells) were transfected with wild-type (lane 2) and two mutant TGFBI plasmids (lanes 3 and 4) or were not transfected plasmid (lane 1). The culture medium was replaced with serum-free medium 48 h post-transfection. Another 48 h later, the serum-free media from overexpressed cells were collected and concentrated. A and C: 65 µg protein was loaded into each well. The TGFBI-T538P-myc and TGFBI-R555W-myc protein were detected with an anti-c-myc-tag antibody (lanes 3 and 4), while the wild-type TGFBI-myc protein was not (lane 2). B and D: 65 µg protein was loaded on to each well and detected with an anti-TGFBIp antibody. The wild-type and mutant TGFBI-myc fusion proteins were detected as two bands of different intensities (lanes 2–4). Endogenous TGFBIp is shown in the control lane (lane 1). The stronger bands in lanes 3 and 4 represent full-length mutant TGFBI-myc proteins, while the stronger band in lane 2 represents the wild-type TGFBI-myc protein that lacks its C-terminus. There was an approximate 10 kDa difference between the full-length and cleaved proteins.
Mentions: To investigate the expression of the wild-type and mutant TGFBI-myc fusion proteins, HCE and HeLa cells were used for transient plasmid transfection. Each protein expressed a Myc tag at its C-terminus. In Figure 4A, 65 µg protein from the media of HCE cells was loaded into each well. As expected, the wild-type TGFBI-myc protein was not detected by an anti-c-myc-tag antibody due to C-terminal cleavage (lane 2). This was consistent with Kim et al.’s study, which indicated that wild-type TGFBI undergoes C-terminal cleavage processing after secretion [11]. Surprisingly, TGFBI-T538P-myc (lane 3) and TGFBI-R555W-myc (lane 4) fusion proteins were detected by anti-c-myc-tag antibody at approximately 70 kDa.

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