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Abnormal trafficking of endogenously expressed BMPR2 mutant allelic products in patients with heritable pulmonary arterial hypertension.

Frump AL, Lowery JW, Hamid R, Austin ED, de Caestecker M - PLoS ONE (2013)

Bottom Line: However expression of endogenous NMD negative BMPR2 mutant products and their effect on protein trafficking and signaling function have never been described.The endogenous BMPR2ΔEx2 mutant product does not reach the cell surface and is retained in the endoplasmic reticulum.These data indicate that the endogenous NMD negative BMPRΔEx2 mutant product is expressed but has a folding defect resulting in ER retention.

View Article: PubMed Central - PubMed

Affiliation: Department of Cell and Developmental Biology, Vanderbilt University Medical Center, Nashville, Tennessee, United States of America.

ABSTRACT
More than 200 heterozygous mutations in the type 2 BMP receptor gene, BMPR2, have been identified in patients with Heritable Pulmonary Arterial Hypertension (HPAH). More severe clinical outcomes occur in patients with BMPR2 mutations by-passing nonsense-mediated mRNA decay (NMD negative mutations). These comprise 40% of HPAH mutations and are predicted to express BMPR2 mutant products. However expression of endogenous NMD negative BMPR2 mutant products and their effect on protein trafficking and signaling function have never been described. Here, we characterize the expression and trafficking of an HPAH-associated NMD negative BMPR2 mutation that results in an in-frame deletion of BMPR2 EXON2 (BMPR2ΔEx2) in HPAH patient-derived lymphocytes and in pulmonary endothelial cells (PECs) from mice carrying the same in-frame deletion of Exon 2 (Bmpr2 (ΔEx2/+) mice). The endogenous BMPR2ΔEx2 mutant product does not reach the cell surface and is retained in the endoplasmic reticulum. Moreover, chemical chaperones 4-PBA and TUDCA partially restore cell surface expression of Bmpr2ΔEx2 in PECs, suggesting that the mutant product is mis-folded. We also show that PECs from Bmpr2 (ΔEx2/+) mice have defects in the BMP-induced Smad1/5/8 and Id1 signaling axis, and that addition of chemical chaperones restores expression of the Smad1/5/8 target Id1. These data indicate that the endogenous NMD negative BMPRΔEx2 mutant product is expressed but has a folding defect resulting in ER retention. Partial correction of this folding defect and restoration of defective BMP signaling using chemical chaperones suggests that protein-folding agents could be used therapeutically in patients with these NMD negative BMPR2 mutations.

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Differential N-linked glycosidase sensitivity of wild type Bmpr2 and Bmpr2ΔEx2 mutant products.Wild type control and Bmpr2ΔEx2/+ ciPEC lysates were immunoprecipitated and digested with N-linked glycosidases Endo-H or PNGase-F. The 150 kDa wild type Bmpr2 bands in both control and Bmpr2ΔEx2/+ ciPECs were sensitive to PNGase-F digestion, determined by a 15 kDa mobility shift (WT>WT1), but were insensitive to Endo-H digestion. Unlike wild type Bmpr2, the 130 kDa Bmpr2ΔEx2 mutant product in Bmpr2ΔEx2/+ ciPECs was sensitive to Endo-H digestion (ΔEx2>ΔEx21).
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pone-0080319-g003: Differential N-linked glycosidase sensitivity of wild type Bmpr2 and Bmpr2ΔEx2 mutant products.Wild type control and Bmpr2ΔEx2/+ ciPEC lysates were immunoprecipitated and digested with N-linked glycosidases Endo-H or PNGase-F. The 150 kDa wild type Bmpr2 bands in both control and Bmpr2ΔEx2/+ ciPECs were sensitive to PNGase-F digestion, determined by a 15 kDa mobility shift (WT>WT1), but were insensitive to Endo-H digestion. Unlike wild type Bmpr2, the 130 kDa Bmpr2ΔEx2 mutant product in Bmpr2ΔEx2/+ ciPECs was sensitive to Endo-H digestion (ΔEx2>ΔEx21).

Mentions: We used N-linked glycosidases to determine if the Bmpr2ΔEx2 mutant product was able to traffic correctly through the endoplasmic reticulum (ER) and Golgi apparatus. N-linked glycosylated proteins that are processed in the ER are sensitive to Endo-H glycosidase, but become resistant to Endo-H once the glycoprotein has passed through to the trans-Golgi [36]. In contrast, PNGase-F de-glycosylates all N-linked glycosylated proteins irrespective of their maturation state. This sensitivity to glycosidase digestion can be detected by a change in protein mobility resulting from cleavage of glycan moieties. As expected, the 150 kDa wild type Bmpr2 product in control and Bmpr2ΔEx2/+ ciPECs underwent a mobility shift after treatment with PNGase-F, but was resistant to Endo-H digestion (Figure 3). In contrast, the 130 kDa Bmpr2ΔEx2 mutant product underwent a mobility shift after digestion with PNGase-F and Endo-H (ΔEx2>ΔEx21). This indicates that wild type Bmpr2 is correctly processed through the ER and Golgi but that the 130 kDa Bmpr2ΔEx2 mutant product is likely to be retained in the ER.


Abnormal trafficking of endogenously expressed BMPR2 mutant allelic products in patients with heritable pulmonary arterial hypertension.

Frump AL, Lowery JW, Hamid R, Austin ED, de Caestecker M - PLoS ONE (2013)

Differential N-linked glycosidase sensitivity of wild type Bmpr2 and Bmpr2ΔEx2 mutant products.Wild type control and Bmpr2ΔEx2/+ ciPEC lysates were immunoprecipitated and digested with N-linked glycosidases Endo-H or PNGase-F. The 150 kDa wild type Bmpr2 bands in both control and Bmpr2ΔEx2/+ ciPECs were sensitive to PNGase-F digestion, determined by a 15 kDa mobility shift (WT>WT1), but were insensitive to Endo-H digestion. Unlike wild type Bmpr2, the 130 kDa Bmpr2ΔEx2 mutant product in Bmpr2ΔEx2/+ ciPECs was sensitive to Endo-H digestion (ΔEx2>ΔEx21).
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC3818254&req=5

pone-0080319-g003: Differential N-linked glycosidase sensitivity of wild type Bmpr2 and Bmpr2ΔEx2 mutant products.Wild type control and Bmpr2ΔEx2/+ ciPEC lysates were immunoprecipitated and digested with N-linked glycosidases Endo-H or PNGase-F. The 150 kDa wild type Bmpr2 bands in both control and Bmpr2ΔEx2/+ ciPECs were sensitive to PNGase-F digestion, determined by a 15 kDa mobility shift (WT>WT1), but were insensitive to Endo-H digestion. Unlike wild type Bmpr2, the 130 kDa Bmpr2ΔEx2 mutant product in Bmpr2ΔEx2/+ ciPECs was sensitive to Endo-H digestion (ΔEx2>ΔEx21).
Mentions: We used N-linked glycosidases to determine if the Bmpr2ΔEx2 mutant product was able to traffic correctly through the endoplasmic reticulum (ER) and Golgi apparatus. N-linked glycosylated proteins that are processed in the ER are sensitive to Endo-H glycosidase, but become resistant to Endo-H once the glycoprotein has passed through to the trans-Golgi [36]. In contrast, PNGase-F de-glycosylates all N-linked glycosylated proteins irrespective of their maturation state. This sensitivity to glycosidase digestion can be detected by a change in protein mobility resulting from cleavage of glycan moieties. As expected, the 150 kDa wild type Bmpr2 product in control and Bmpr2ΔEx2/+ ciPECs underwent a mobility shift after treatment with PNGase-F, but was resistant to Endo-H digestion (Figure 3). In contrast, the 130 kDa Bmpr2ΔEx2 mutant product underwent a mobility shift after digestion with PNGase-F and Endo-H (ΔEx2>ΔEx21). This indicates that wild type Bmpr2 is correctly processed through the ER and Golgi but that the 130 kDa Bmpr2ΔEx2 mutant product is likely to be retained in the ER.

Bottom Line: However expression of endogenous NMD negative BMPR2 mutant products and their effect on protein trafficking and signaling function have never been described.The endogenous BMPR2ΔEx2 mutant product does not reach the cell surface and is retained in the endoplasmic reticulum.These data indicate that the endogenous NMD negative BMPRΔEx2 mutant product is expressed but has a folding defect resulting in ER retention.

View Article: PubMed Central - PubMed

Affiliation: Department of Cell and Developmental Biology, Vanderbilt University Medical Center, Nashville, Tennessee, United States of America.

ABSTRACT
More than 200 heterozygous mutations in the type 2 BMP receptor gene, BMPR2, have been identified in patients with Heritable Pulmonary Arterial Hypertension (HPAH). More severe clinical outcomes occur in patients with BMPR2 mutations by-passing nonsense-mediated mRNA decay (NMD negative mutations). These comprise 40% of HPAH mutations and are predicted to express BMPR2 mutant products. However expression of endogenous NMD negative BMPR2 mutant products and their effect on protein trafficking and signaling function have never been described. Here, we characterize the expression and trafficking of an HPAH-associated NMD negative BMPR2 mutation that results in an in-frame deletion of BMPR2 EXON2 (BMPR2ΔEx2) in HPAH patient-derived lymphocytes and in pulmonary endothelial cells (PECs) from mice carrying the same in-frame deletion of Exon 2 (Bmpr2 (ΔEx2/+) mice). The endogenous BMPR2ΔEx2 mutant product does not reach the cell surface and is retained in the endoplasmic reticulum. Moreover, chemical chaperones 4-PBA and TUDCA partially restore cell surface expression of Bmpr2ΔEx2 in PECs, suggesting that the mutant product is mis-folded. We also show that PECs from Bmpr2 (ΔEx2/+) mice have defects in the BMP-induced Smad1/5/8 and Id1 signaling axis, and that addition of chemical chaperones restores expression of the Smad1/5/8 target Id1. These data indicate that the endogenous NMD negative BMPRΔEx2 mutant product is expressed but has a folding defect resulting in ER retention. Partial correction of this folding defect and restoration of defective BMP signaling using chemical chaperones suggests that protein-folding agents could be used therapeutically in patients with these NMD negative BMPR2 mutations.

Show MeSH
Related in: MedlinePlus