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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

Secretion profiles of GFP-Fbn constructs with disease-associated substitutions. Mutations associated with MFS, SSS, geleophysic dysplasia (GD) or acromicric dysplasia (AD) in domains TB4 and TB5 were engineered into a GFP-tagged fibrillin-1 construct (36) and the resulting constructs used to transiently transfect HEK293T cells. After 3 days in culture, samples of the medium and cell lysates were analysed by western blotting following separation on a reducing 6% SDS-PAGE gel, using an anti-GFP antibody. SSS, GD and AD mutants were detected in the culture medium, in contrast to the MFS mutants. Empty vector (pcDNA) and the wild-type construct (GFP-Fbn) were used as negative and positive controls. Cell lysate samples showed that the lack of recombinant material in the media of the MFS mutants was not due to a loss of protein expression.
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DDV181F2: Secretion profiles of GFP-Fbn constructs with disease-associated substitutions. Mutations associated with MFS, SSS, geleophysic dysplasia (GD) or acromicric dysplasia (AD) in domains TB4 and TB5 were engineered into a GFP-tagged fibrillin-1 construct (36) and the resulting constructs used to transiently transfect HEK293T cells. After 3 days in culture, samples of the medium and cell lysates were analysed by western blotting following separation on a reducing 6% SDS-PAGE gel, using an anti-GFP antibody. SSS, GD and AD mutants were detected in the culture medium, in contrast to the MFS mutants. Empty vector (pcDNA) and the wild-type construct (GFP-Fbn) were used as negative and positive controls. Cell lysate samples showed that the lack of recombinant material in the media of the MFS mutants was not due to a loss of protein expression.

Mentions: In a previous study using a fibrillin-1 mini-gene construct, we showed that the SSS-associated substitutions C1564S and W1570C in domain TB4 do not abrogate secretion of fibrillin-1 from fibroblasts (15). In this study, we compared the cell trafficking profiles of SSS substitutions C1564S and W1570C with a C1564Y substitution in domain TB4 associated with MFS (Fig. 1), in the context of a full-length GFP-tagged fibrillin-1 construct. Consistent with the fibrillin-1 mini-gene data, the GFP-Fbn constructs containing the SSS-associated substitutions C1564S and W1570C were detected in medium samples from cultures of transiently transfected HEK293T cells (Fig. 2). In contrast, the GFP-Fbn construct harbouring the MFS-associated substitution C1564Y was not detected in medium samples despite the recombinant protein being detected in cell fractions, suggesting either intracellular retention or a rapid turnover of the recombinant protein in the culture medium. Semi-quantitative RT–PCR analysis showed that the levels of recombinant transcript were similar in cells transfected with either the wild-type GFP-Fbn or C1564Y mutant (Supplementary Material, Fig. S1), indicating that the lack of recombinant protein observed in the medium in the C1564Y samples was not due to a lack of transcript.Figure 2.


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)

Secretion profiles of GFP-Fbn constructs with disease-associated substitutions. Mutations associated with MFS, SSS, geleophysic dysplasia (GD) or acromicric dysplasia (AD) in domains TB4 and TB5 were engineered into a GFP-tagged fibrillin-1 construct (36) and the resulting constructs used to transiently transfect HEK293T cells. After 3 days in culture, samples of the medium and cell lysates were analysed by western blotting following separation on a reducing 6% SDS-PAGE gel, using an anti-GFP antibody. SSS, GD and AD mutants were detected in the culture medium, in contrast to the MFS mutants. Empty vector (pcDNA) and the wild-type construct (GFP-Fbn) were used as negative and positive controls. Cell lysate samples showed that the lack of recombinant material in the media of the MFS mutants was not due to a loss of protein expression.
© Copyright Policy - creative-commons
Related In: Results  -  Collection

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getmorefigures.php?uid=PMC4492404&req=5

DDV181F2: Secretion profiles of GFP-Fbn constructs with disease-associated substitutions. Mutations associated with MFS, SSS, geleophysic dysplasia (GD) or acromicric dysplasia (AD) in domains TB4 and TB5 were engineered into a GFP-tagged fibrillin-1 construct (36) and the resulting constructs used to transiently transfect HEK293T cells. After 3 days in culture, samples of the medium and cell lysates were analysed by western blotting following separation on a reducing 6% SDS-PAGE gel, using an anti-GFP antibody. SSS, GD and AD mutants were detected in the culture medium, in contrast to the MFS mutants. Empty vector (pcDNA) and the wild-type construct (GFP-Fbn) were used as negative and positive controls. Cell lysate samples showed that the lack of recombinant material in the media of the MFS mutants was not due to a loss of protein expression.
Mentions: In a previous study using a fibrillin-1 mini-gene construct, we showed that the SSS-associated substitutions C1564S and W1570C in domain TB4 do not abrogate secretion of fibrillin-1 from fibroblasts (15). In this study, we compared the cell trafficking profiles of SSS substitutions C1564S and W1570C with a C1564Y substitution in domain TB4 associated with MFS (Fig. 1), in the context of a full-length GFP-tagged fibrillin-1 construct. Consistent with the fibrillin-1 mini-gene data, the GFP-Fbn constructs containing the SSS-associated substitutions C1564S and W1570C were detected in medium samples from cultures of transiently transfected HEK293T cells (Fig. 2). In contrast, the GFP-Fbn construct harbouring the MFS-associated substitution C1564Y was not detected in medium samples despite the recombinant protein being detected in cell fractions, suggesting either intracellular retention or a rapid turnover of the recombinant protein in the culture medium. Semi-quantitative RT–PCR analysis showed that the levels of recombinant transcript were similar in cells transfected with either the wild-type GFP-Fbn or C1564Y mutant (Supplementary Material, Fig. S1), indicating that the lack of recombinant protein observed in the medium in the C1564Y samples was not due to a lack of transcript.Figure 2.

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