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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

Construction of the NterPro-cbEGF18-26 fibrillin-1 fragments and fibroblast secretion assays. (A) NterPro-cbEGF18-26 is a fusion of the N-terminal region of fibrillin-1, up to the proline-rich domain (orange), with the cbEGF18-26 region, which encompasses domains TB4 and TB5. The smaller size of this construct allows it to be distinguished from full-length fibrillin-1, following reducing SDS-PAGE, in western blots of medium from stably transfected fibroblasts when developed with an antibody raised again the proline-rich domain. (B) Secretion profiles of TB4 and TB5 domain-mutant constructs associated with MFS, SSS, acromicric dysplasia (AD) or geleophysic dysplasia (GD). Medium and cell lysate samples from untransfected fibroblasts (MSU-1.1) and fibroblasts transfected with the wild-type control construct (WT) were run as controls, allowing the identification of the recombinant construct (fusion). The full-length fibrillin-1 band (Fbn-1) acts as an endogenously expressed loading control. In cell lysates, a band migrating at ∼230 kDa (*) is likely to be due to a degradation product of the endogenously expressed fibrillin-1.
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DDV181F4: Construction of the NterPro-cbEGF18-26 fibrillin-1 fragments and fibroblast secretion assays. (A) NterPro-cbEGF18-26 is a fusion of the N-terminal region of fibrillin-1, up to the proline-rich domain (orange), with the cbEGF18-26 region, which encompasses domains TB4 and TB5. The smaller size of this construct allows it to be distinguished from full-length fibrillin-1, following reducing SDS-PAGE, in western blots of medium from stably transfected fibroblasts when developed with an antibody raised again the proline-rich domain. (B) Secretion profiles of TB4 and TB5 domain-mutant constructs associated with MFS, SSS, acromicric dysplasia (AD) or geleophysic dysplasia (GD). Medium and cell lysate samples from untransfected fibroblasts (MSU-1.1) and fibroblasts transfected with the wild-type control construct (WT) were run as controls, allowing the identification of the recombinant construct (fusion). The full-length fibrillin-1 band (Fbn-1) acts as an endogenously expressed loading control. In cell lysates, a band migrating at ∼230 kDa (*) is likely to be due to a degradation product of the endogenously expressed fibrillin-1.

Mentions: To assess the effects of these substitutions on protein secretion from a microfibril-assembling cell line rather than HEK293T cells, which are epithelial in origin and do not assemble microfibrils, we used a previously described assay (32) in which pools of stably transfected clones of the human fibroblast cell line, MSU-1.1, are created to express a fibrillin-1 mini-gene (Fig. 4). MSU-1.1 fibroblasts are able to assemble extracellular microfibrils and contain all the cellular factors required for fibrillin-1 folding, processing, secretion and assembly (37). Pools of clones expressing the NPro-cbEGF18–26 fibrillin-1 mini-gene (Fig. 4A) were assayed to average out any variation in transgene expression levels caused by random genomic integration. Conditioned medium from pools of MSU-1.1 clones obtained after transfection with NPro-cbEGF18-26 wild-type, W1570C, C1564S or C1564Y constructs were analysed by western blotting using an antibody raised against the proline-rich region of fibrillin-1 (Fig. 4B). In medium samples from wild type, W1570C and C1564S pools, the recombinant NPro-cbEGF18-26 fragment, which migrates at ∼150 kD, was always detected in the medium. This band was absent in medium samples from untransfected MSU-1.1 cells, confirming that it is derived from the recombinant construct. In contrast, medium samples of pools expressing the C1564Y-substituted protein showed the presence of endogenous fibrillin-1 but greatly reduced the levels of the recombinant protein compared with the wild type and SSS forms. In addition, little C1564Y-substituted fusion was detected in the cell fraction, suggesting either a lack of expression or increased rate of turnover. Semi-quantitative RT–PCR analysis showed that the recombinant constructs were expressed at similar levels at the mRNA level (Supplementary Material, Fig. S3). The results suggest that the MFS-causing mutation C1564Y in domain TB4 results in a reduction in the amount of fibrillin-1 secreted by fibroblasts, consistent with the HEK293T data and in contrast to the SSS-causing mutants in domain TB4, which are secreted at levels comparable to the wild type.Figure 4.


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)

Construction of the NterPro-cbEGF18-26 fibrillin-1 fragments and fibroblast secretion assays. (A) NterPro-cbEGF18-26 is a fusion of the N-terminal region of fibrillin-1, up to the proline-rich domain (orange), with the cbEGF18-26 region, which encompasses domains TB4 and TB5. The smaller size of this construct allows it to be distinguished from full-length fibrillin-1, following reducing SDS-PAGE, in western blots of medium from stably transfected fibroblasts when developed with an antibody raised again the proline-rich domain. (B) Secretion profiles of TB4 and TB5 domain-mutant constructs associated with MFS, SSS, acromicric dysplasia (AD) or geleophysic dysplasia (GD). Medium and cell lysate samples from untransfected fibroblasts (MSU-1.1) and fibroblasts transfected with the wild-type control construct (WT) were run as controls, allowing the identification of the recombinant construct (fusion). The full-length fibrillin-1 band (Fbn-1) acts as an endogenously expressed loading control. In cell lysates, a band migrating at ∼230 kDa (*) is likely to be due to a degradation product of the endogenously expressed fibrillin-1.
© Copyright Policy - creative-commons
Related In: Results  -  Collection

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DDV181F4: Construction of the NterPro-cbEGF18-26 fibrillin-1 fragments and fibroblast secretion assays. (A) NterPro-cbEGF18-26 is a fusion of the N-terminal region of fibrillin-1, up to the proline-rich domain (orange), with the cbEGF18-26 region, which encompasses domains TB4 and TB5. The smaller size of this construct allows it to be distinguished from full-length fibrillin-1, following reducing SDS-PAGE, in western blots of medium from stably transfected fibroblasts when developed with an antibody raised again the proline-rich domain. (B) Secretion profiles of TB4 and TB5 domain-mutant constructs associated with MFS, SSS, acromicric dysplasia (AD) or geleophysic dysplasia (GD). Medium and cell lysate samples from untransfected fibroblasts (MSU-1.1) and fibroblasts transfected with the wild-type control construct (WT) were run as controls, allowing the identification of the recombinant construct (fusion). The full-length fibrillin-1 band (Fbn-1) acts as an endogenously expressed loading control. In cell lysates, a band migrating at ∼230 kDa (*) is likely to be due to a degradation product of the endogenously expressed fibrillin-1.
Mentions: To assess the effects of these substitutions on protein secretion from a microfibril-assembling cell line rather than HEK293T cells, which are epithelial in origin and do not assemble microfibrils, we used a previously described assay (32) in which pools of stably transfected clones of the human fibroblast cell line, MSU-1.1, are created to express a fibrillin-1 mini-gene (Fig. 4). MSU-1.1 fibroblasts are able to assemble extracellular microfibrils and contain all the cellular factors required for fibrillin-1 folding, processing, secretion and assembly (37). Pools of clones expressing the NPro-cbEGF18–26 fibrillin-1 mini-gene (Fig. 4A) were assayed to average out any variation in transgene expression levels caused by random genomic integration. Conditioned medium from pools of MSU-1.1 clones obtained after transfection with NPro-cbEGF18-26 wild-type, W1570C, C1564S or C1564Y constructs were analysed by western blotting using an antibody raised against the proline-rich region of fibrillin-1 (Fig. 4B). In medium samples from wild type, W1570C and C1564S pools, the recombinant NPro-cbEGF18-26 fragment, which migrates at ∼150 kD, was always detected in the medium. This band was absent in medium samples from untransfected MSU-1.1 cells, confirming that it is derived from the recombinant construct. In contrast, medium samples of pools expressing the C1564Y-substituted protein showed the presence of endogenous fibrillin-1 but greatly reduced the levels of the recombinant protein compared with the wild type and SSS forms. In addition, little C1564Y-substituted fusion was detected in the cell fraction, suggesting either a lack of expression or increased rate of turnover. Semi-quantitative RT–PCR analysis showed that the recombinant constructs were expressed at similar levels at the mRNA level (Supplementary Material, Fig. S3). The results suggest that the MFS-causing mutation C1564Y in domain TB4 results in a reduction in the amount of fibrillin-1 secreted by fibroblasts, consistent with the HEK293T data and in contrast to the SSS-causing mutants in domain TB4, which are secreted at levels comparable to the wild type.Figure 4.

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