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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

Schematic representation of the recombinant mutant TGFBI proteins and the principal of the two-sequential PCR site-directed mutagenesis technique. A: S: signal sequence, EMI: cysteine-rich EMI domain, I to IV: four FAS1 domains, RGD: Arg-Gly-Asp domain, Myc: myc tag protein, TGFBI-T538P-I: bp 1 to 1613 (the 538 mutant site), TGFBI-R555W-I: bp 1 to 1663 (the 555 mutant site), TGFBI-T538P/R555W-II: from the mutant site to the 3′ end of the sequence. B: The rectangle represents the template DNA, the circle in the rectangle represents the mutant site, and the arrow represents the primer. In the first PCR, TGFBI-T538P/ R555W-I were amplified with the TGFBI-WT-F and TGFBI-T538P/ R555W-R primers. TGFBI-T538P/ R555W-II were amplified with the TGFBI-T538P/ R555W-F and TGFBI-WT-R primers. In the second PCR, full-length TGFBI-T538P/ R555W were amplified with the TGFBI-WT-F and TGFBI-WT-R primers.
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f2: Schematic representation of the recombinant mutant TGFBI proteins and the principal of the two-sequential PCR site-directed mutagenesis technique. A: S: signal sequence, EMI: cysteine-rich EMI domain, I to IV: four FAS1 domains, RGD: Arg-Gly-Asp domain, Myc: myc tag protein, TGFBI-T538P-I: bp 1 to 1613 (the 538 mutant site), TGFBI-R555W-I: bp 1 to 1663 (the 555 mutant site), TGFBI-T538P/R555W-II: from the mutant site to the 3′ end of the sequence. B: The rectangle represents the template DNA, the circle in the rectangle represents the mutant site, and the arrow represents the primer. In the first PCR, TGFBI-T538P/ R555W-I were amplified with the TGFBI-WT-F and TGFBI-T538P/ R555W-R primers. TGFBI-T538P/ R555W-II were amplified with the TGFBI-T538P/ R555W-F and TGFBI-WT-R primers. In the second PCR, full-length TGFBI-T538P/ R555W were amplified with the TGFBI-WT-F and TGFBI-WT-R primers.

Mentions: The mutations (T538P and R555W) were introduced with the two-sequential PCR site-directed mutagenesis technique (Figure 2). In the first PCR, the TGFBI-T538P/R555W-I and TGFBI-T538P/R555W-II fragments were amplified individually with pcTGFBI-WT-myc as a template. In the second PCR, full-length mutant TGFBI was amplified by primers TGFBI-WT-F and TGFBI-WT-R with purified TGFBI-T538P/R555W-I and II as the DNA templates. PCR was performed in a 50 μl reaction mixture containing 100 ng template DNA, 1 μM of each primer (Table 1), and 2.5 U DNA Polymerase (AccuPrime Taq DNA Polymerase High Fidelity, Invitrogen) in the 1× PCR buffer provided by the manufacturer. The thermocycling program was as follows: initial denaturation at 94 °C for 5 min; followed by 30 cycles of denaturation at 94 °C for 1 min, annealing at 65 °C for 1 min, and elongation at 68 °C for 3 min (for >1 kb PCR product) or 1.5 min (for <1 kb PCR product); and 5 min of final extension at 68 °C. The PCR products were gel-purified using the AxyPrep DNA Gel Extraction Kit (Axygen, Union City, CA) following the manufacturer’s instructions. After double digestion with Xho I/Hind III, the purified products were ligated into the pcDNA3.1(-)/myc-his vector to construct the pcTGFBI-T538P/R555W-myc plasmids. The plasmids were transformed into DH5а chemically competent cells and seeded onto 1% LB agar plates supplemented with ampicillin. After 18 h of incubation, 12 colonies were picked from each sample and cultured in LB medium with 200 rpm/min shaking overnight. The plasmid DNA was extracted using the Qiagen Plasmid Midi Kit (Qiagen, Hilden, Germany) following the manufacturer’s instructions. The sequences of all of the constructs were verified with direct sequencing.


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 representation of the recombinant mutant TGFBI proteins and the principal of the two-sequential PCR site-directed mutagenesis technique. A: S: signal sequence, EMI: cysteine-rich EMI domain, I to IV: four FAS1 domains, RGD: Arg-Gly-Asp domain, Myc: myc tag protein, TGFBI-T538P-I: bp 1 to 1613 (the 538 mutant site), TGFBI-R555W-I: bp 1 to 1663 (the 555 mutant site), TGFBI-T538P/R555W-II: from the mutant site to the 3′ end of the sequence. B: The rectangle represents the template DNA, the circle in the rectangle represents the mutant site, and the arrow represents the primer. In the first PCR, TGFBI-T538P/ R555W-I were amplified with the TGFBI-WT-F and TGFBI-T538P/ R555W-R primers. TGFBI-T538P/ R555W-II were amplified with the TGFBI-T538P/ R555W-F and TGFBI-WT-R primers. In the second PCR, full-length TGFBI-T538P/ R555W were amplified with the TGFBI-WT-F and TGFBI-WT-R primers.
© Copyright Policy - open-access
Related In: Results  -  Collection

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

f2: Schematic representation of the recombinant mutant TGFBI proteins and the principal of the two-sequential PCR site-directed mutagenesis technique. A: S: signal sequence, EMI: cysteine-rich EMI domain, I to IV: four FAS1 domains, RGD: Arg-Gly-Asp domain, Myc: myc tag protein, TGFBI-T538P-I: bp 1 to 1613 (the 538 mutant site), TGFBI-R555W-I: bp 1 to 1663 (the 555 mutant site), TGFBI-T538P/R555W-II: from the mutant site to the 3′ end of the sequence. B: The rectangle represents the template DNA, the circle in the rectangle represents the mutant site, and the arrow represents the primer. In the first PCR, TGFBI-T538P/ R555W-I were amplified with the TGFBI-WT-F and TGFBI-T538P/ R555W-R primers. TGFBI-T538P/ R555W-II were amplified with the TGFBI-T538P/ R555W-F and TGFBI-WT-R primers. In the second PCR, full-length TGFBI-T538P/ R555W were amplified with the TGFBI-WT-F and TGFBI-WT-R primers.
Mentions: The mutations (T538P and R555W) were introduced with the two-sequential PCR site-directed mutagenesis technique (Figure 2). In the first PCR, the TGFBI-T538P/R555W-I and TGFBI-T538P/R555W-II fragments were amplified individually with pcTGFBI-WT-myc as a template. In the second PCR, full-length mutant TGFBI was amplified by primers TGFBI-WT-F and TGFBI-WT-R with purified TGFBI-T538P/R555W-I and II as the DNA templates. PCR was performed in a 50 μl reaction mixture containing 100 ng template DNA, 1 μM of each primer (Table 1), and 2.5 U DNA Polymerase (AccuPrime Taq DNA Polymerase High Fidelity, Invitrogen) in the 1× PCR buffer provided by the manufacturer. The thermocycling program was as follows: initial denaturation at 94 °C for 5 min; followed by 30 cycles of denaturation at 94 °C for 1 min, annealing at 65 °C for 1 min, and elongation at 68 °C for 3 min (for >1 kb PCR product) or 1.5 min (for <1 kb PCR product); and 5 min of final extension at 68 °C. The PCR products were gel-purified using the AxyPrep DNA Gel Extraction Kit (Axygen, Union City, CA) following the manufacturer’s instructions. After double digestion with Xho I/Hind III, the purified products were ligated into the pcDNA3.1(-)/myc-his vector to construct the pcTGFBI-T538P/R555W-myc plasmids. The plasmids were transformed into DH5а chemically competent cells and seeded onto 1% LB agar plates supplemented with ampicillin. After 18 h of incubation, 12 colonies were picked from each sample and cultured in LB medium with 200 rpm/min shaking overnight. The plasmid DNA was extracted using the Qiagen Plasmid Midi Kit (Qiagen, Hilden, Germany) following the manufacturer’s instructions. The sequences of all of the constructs were verified with direct sequencing.

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