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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

Construction of mutant plasmids. A: PCR products of the recombinant mutant TGFBI-T538P and TGFBI-R555W genes. M: 1 kb marker (Fermentas, Burlington, Canada), lane 1: TGFBI-T538P, lane 2: TGFBI-R555W. B: Constructed pcTGFBI-WT/T538P/R555W-myc plasmids were double digested into two bands approximately 2 kb and 5.5 kb, respectively, using Xho I and Hind III. M: 1 kb marker, lane 1: pcTGFBI-WT-myc, lane 2–lane 3: pcTGFBI-T538P-myc, lane 4–lane 5: pcTGFBI-R555W-myc. C: Right: Partial sequence of the pcTGFBI-T538P plasmid showing the A to C change at position c.1613 (the forward strand is shown) resulting in the p.Thr538Pro mutation (underlined). Left: Partial sequence of the pcTGFBI-R555W plasmid showing the C to T mutation at position c.1663 (the forward strand is shown) resulting in the p.Arg555Trp mutation (underlined). The arrow points toward the position of the mutations.
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f3: Construction of mutant plasmids. A: PCR products of the recombinant mutant TGFBI-T538P and TGFBI-R555W genes. M: 1 kb marker (Fermentas, Burlington, Canada), lane 1: TGFBI-T538P, lane 2: TGFBI-R555W. B: Constructed pcTGFBI-WT/T538P/R555W-myc plasmids were double digested into two bands approximately 2 kb and 5.5 kb, respectively, using Xho I and Hind III. M: 1 kb marker, lane 1: pcTGFBI-WT-myc, lane 2–lane 3: pcTGFBI-T538P-myc, lane 4–lane 5: pcTGFBI-R555W-myc. C: Right: Partial sequence of the pcTGFBI-T538P plasmid showing the A to C change at position c.1613 (the forward strand is shown) resulting in the p.Thr538Pro mutation (underlined). Left: Partial sequence of the pcTGFBI-R555W plasmid showing the C to T mutation at position c.1663 (the forward strand is shown) resulting in the p.Arg555Trp mutation (underlined). The arrow points toward the position of the mutations.

Mentions: To construct the mutant pcTGFBI-T538P/R555W-myc plasmid, the two-sequential PCR site-directed mutagenesis technique was used. Agarose gel electrophoresis showed a clear band with a size corresponding to approximately 2 kb after the second PCR (Figure 3A; lanes 1, 2). This corresponded with the size of the full-length TGFBI cDNA. The wild-type and mutant pcTGFBI plasmids were double digested with Xho I/Hind III. As expected, each plasmid was cut into 2-kb and 5.5-kb fragments (Figure 3B). The sequences of all the constructs were also verified with direct sequencing. The results clearly showed that A was replaced with C at position c.1613 of the T538P plasmid, while T substituted C at c.1663 of the R555W plasmid (Figure 3C). Aside from the induced point mutation, there were no other mutations throughout the entire sequence.


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)

Construction of mutant plasmids. A: PCR products of the recombinant mutant TGFBI-T538P and TGFBI-R555W genes. M: 1 kb marker (Fermentas, Burlington, Canada), lane 1: TGFBI-T538P, lane 2: TGFBI-R555W. B: Constructed pcTGFBI-WT/T538P/R555W-myc plasmids were double digested into two bands approximately 2 kb and 5.5 kb, respectively, using Xho I and Hind III. M: 1 kb marker, lane 1: pcTGFBI-WT-myc, lane 2–lane 3: pcTGFBI-T538P-myc, lane 4–lane 5: pcTGFBI-R555W-myc. C: Right: Partial sequence of the pcTGFBI-T538P plasmid showing the A to C change at position c.1613 (the forward strand is shown) resulting in the p.Thr538Pro mutation (underlined). Left: Partial sequence of the pcTGFBI-R555W plasmid showing the C to T mutation at position c.1663 (the forward strand is shown) resulting in the p.Arg555Trp mutation (underlined). The arrow points toward the position of the mutations.
© Copyright Policy - open-access
Related In: Results  -  Collection

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

f3: Construction of mutant plasmids. A: PCR products of the recombinant mutant TGFBI-T538P and TGFBI-R555W genes. M: 1 kb marker (Fermentas, Burlington, Canada), lane 1: TGFBI-T538P, lane 2: TGFBI-R555W. B: Constructed pcTGFBI-WT/T538P/R555W-myc plasmids were double digested into two bands approximately 2 kb and 5.5 kb, respectively, using Xho I and Hind III. M: 1 kb marker, lane 1: pcTGFBI-WT-myc, lane 2–lane 3: pcTGFBI-T538P-myc, lane 4–lane 5: pcTGFBI-R555W-myc. C: Right: Partial sequence of the pcTGFBI-T538P plasmid showing the A to C change at position c.1613 (the forward strand is shown) resulting in the p.Thr538Pro mutation (underlined). Left: Partial sequence of the pcTGFBI-R555W plasmid showing the C to T mutation at position c.1663 (the forward strand is shown) resulting in the p.Arg555Trp mutation (underlined). The arrow points toward the position of the mutations.
Mentions: To construct the mutant pcTGFBI-T538P/R555W-myc plasmid, the two-sequential PCR site-directed mutagenesis technique was used. Agarose gel electrophoresis showed a clear band with a size corresponding to approximately 2 kb after the second PCR (Figure 3A; lanes 1, 2). This corresponded with the size of the full-length TGFBI cDNA. The wild-type and mutant pcTGFBI plasmids were double digested with Xho I/Hind III. As expected, each plasmid was cut into 2-kb and 5.5-kb fragments (Figure 3B). The sequences of all the constructs were also verified with direct sequencing. The results clearly showed that A was replaced with C at position c.1613 of the T538P plasmid, while T substituted C at c.1663 of the R555W plasmid (Figure 3C). Aside from the induced point mutation, there were no other mutations throughout the entire sequence.

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