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A microfibril assembly assay identifies different mechanisms of dominance underlying Marfan syndrome, stiff skin syndrome and acromelic dysplasias.

Jensen SA, Iqbal S, Bulsiewicz A, Handford PA - Hum. Mol. Genet. (2015)

Bottom Line: The majority of mutations affecting the human fibrillin-1 gene, FBN1, result in Marfan syndrome (MFS), a common connective tissue disorder characterised by tall stature, ocular and cardiovascular defects.Despite their apparent phenotypic differences, dysregulation of transforming growth factor β (TGFβ) is a common factor in all of these disorders.We show that substitutions in fibrillin-1 domains TB4 and TB5 that cause SSS and the acromelic dysplasias do not prevent fibrillin-1 from being secreted or assembled into microfibrils, whereas MFS-associated substitutions in these domains result in a loss of recombinant protein in the culture medium and no association with microfibrils.

View Article: PubMed Central - PubMed

Affiliation: Department of Biochemistry, University of Oxford, South Parks Rd, Oxford OX1 3QU, UK sacha.jensen@bioch.ox.ac.uk.

No MeSH data available.


Related in: MedlinePlus

Microfibril incorporation of mutant GFP-Fbn constructs. FS2 fibroblasts were co-cultured for 5 days with HEK293T cells transiently transfected to express GFP-Fbn (WT; Panel A) or GFP-Fbn variants associated with SSS (Panels B and C), geleophysic dysplasia (GD; Panels D–F), acromicric dysplasia (AD; Panels G–I) or MFS (Panels J–L). Co-cultures were then fixed and stained using an anti-GFP antibody as described previously (36). SSS-associated mutants C1564S and W1570C, as well as the geleophysic dysplasia and acromicric dysplasia mutants, produced recombinant microfibril networks that were indistinguishable from the wild type. Co-cultures expressing the MFS mutants showed no recombinant microfibril staining, consistent with the lack of recombinant material observed in medium samples by western blotting (Fig. 2). Bar = 100 μm. Staining for endogenous fibrillin-1, expressed by the FS2 cells, is shown in Supplementary Material, Figure S2.
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DDV181F3: Microfibril incorporation of mutant GFP-Fbn constructs. FS2 fibroblasts were co-cultured for 5 days with HEK293T cells transiently transfected to express GFP-Fbn (WT; Panel A) or GFP-Fbn variants associated with SSS (Panels B and C), geleophysic dysplasia (GD; Panels D–F), acromicric dysplasia (AD; Panels G–I) or MFS (Panels J–L). Co-cultures were then fixed and stained using an anti-GFP antibody as described previously (36). SSS-associated mutants C1564S and W1570C, as well as the geleophysic dysplasia and acromicric dysplasia mutants, produced recombinant microfibril networks that were indistinguishable from the wild type. Co-cultures expressing the MFS mutants showed no recombinant microfibril staining, consistent with the lack of recombinant material observed in medium samples by western blotting (Fig. 2). Bar = 100 μm. Staining for endogenous fibrillin-1, expressed by the FS2 cells, is shown in Supplementary Material, Figure S2.

Mentions: To assess the ability of fibrillin-1 molecules containing the SSS-associated substitutions to incorporate into microfibrils in culture, we used a microfibril incorporation assay (36) in which HEK293T cells transiently expressing wild-type GFP-Fbn (WT), GFP-FbnC1564S or GFP-FbnW1570C (Fig. 3, Panels A–C) were co-cultured with human dermal fibroblasts. As in the case of the wild-type recombinant protein, the GFP-Fbn variants containing the C1564S or W1570C substitutions were able to incorporate into the fibrillin-1 microfibril network. No GFP-tagged microfibrils were observed in cultures expressing the C1564Y construct (Fig. 3J), as expected by the lack of recombinant protein observed in medium samples from transiently transfected HEK293T cells. These data suggest that the C1564S- and W1570C-substituted fibrillin-1 mutants are not defective in their capacity to incorporate into microfibrils.Figure 3.


A microfibril assembly assay identifies different mechanisms of dominance underlying Marfan syndrome, stiff skin syndrome and acromelic dysplasias.

Jensen SA, Iqbal S, Bulsiewicz A, Handford PA - Hum. Mol. Genet. (2015)

Microfibril incorporation of mutant GFP-Fbn constructs. FS2 fibroblasts were co-cultured for 5 days with HEK293T cells transiently transfected to express GFP-Fbn (WT; Panel A) or GFP-Fbn variants associated with SSS (Panels B and C), geleophysic dysplasia (GD; Panels D–F), acromicric dysplasia (AD; Panels G–I) or MFS (Panels J–L). Co-cultures were then fixed and stained using an anti-GFP antibody as described previously (36). SSS-associated mutants C1564S and W1570C, as well as the geleophysic dysplasia and acromicric dysplasia mutants, produced recombinant microfibril networks that were indistinguishable from the wild type. Co-cultures expressing the MFS mutants showed no recombinant microfibril staining, consistent with the lack of recombinant material observed in medium samples by western blotting (Fig. 2). Bar = 100 μm. Staining for endogenous fibrillin-1, expressed by the FS2 cells, is shown in Supplementary Material, Figure S2.
© Copyright Policy - creative-commons
Related In: Results  -  Collection

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Show All Figures
getmorefigures.php?uid=PMC4492404&req=5

DDV181F3: Microfibril incorporation of mutant GFP-Fbn constructs. FS2 fibroblasts were co-cultured for 5 days with HEK293T cells transiently transfected to express GFP-Fbn (WT; Panel A) or GFP-Fbn variants associated with SSS (Panels B and C), geleophysic dysplasia (GD; Panels D–F), acromicric dysplasia (AD; Panels G–I) or MFS (Panels J–L). Co-cultures were then fixed and stained using an anti-GFP antibody as described previously (36). SSS-associated mutants C1564S and W1570C, as well as the geleophysic dysplasia and acromicric dysplasia mutants, produced recombinant microfibril networks that were indistinguishable from the wild type. Co-cultures expressing the MFS mutants showed no recombinant microfibril staining, consistent with the lack of recombinant material observed in medium samples by western blotting (Fig. 2). Bar = 100 μm. Staining for endogenous fibrillin-1, expressed by the FS2 cells, is shown in Supplementary Material, Figure S2.
Mentions: To assess the ability of fibrillin-1 molecules containing the SSS-associated substitutions to incorporate into microfibrils in culture, we used a microfibril incorporation assay (36) in which HEK293T cells transiently expressing wild-type GFP-Fbn (WT), GFP-FbnC1564S or GFP-FbnW1570C (Fig. 3, Panels A–C) were co-cultured with human dermal fibroblasts. As in the case of the wild-type recombinant protein, the GFP-Fbn variants containing the C1564S or W1570C substitutions were able to incorporate into the fibrillin-1 microfibril network. No GFP-tagged microfibrils were observed in cultures expressing the C1564Y construct (Fig. 3J), as expected by the lack of recombinant protein observed in medium samples from transiently transfected HEK293T cells. These data suggest that the C1564S- and W1570C-substituted fibrillin-1 mutants are not defective in their capacity to incorporate into microfibrils.Figure 3.

Bottom Line: The majority of mutations affecting the human fibrillin-1 gene, FBN1, result in Marfan syndrome (MFS), a common connective tissue disorder characterised by tall stature, ocular and cardiovascular defects.Despite their apparent phenotypic differences, dysregulation of transforming growth factor β (TGFβ) is a common factor in all of these disorders.We show that substitutions in fibrillin-1 domains TB4 and TB5 that cause SSS and the acromelic dysplasias do not prevent fibrillin-1 from being secreted or assembled into microfibrils, whereas MFS-associated substitutions in these domains result in a loss of recombinant protein in the culture medium and no association with microfibrils.

View Article: PubMed Central - PubMed

Affiliation: Department of Biochemistry, University of Oxford, South Parks Rd, Oxford OX1 3QU, UK sacha.jensen@bioch.ox.ac.uk.

No MeSH data available.


Related in: MedlinePlus