Abstract
Fibrillin-1 is the major component of the 10–12 nm diameter extracellular matrix microfibrils. 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. Recently, stiff skin syndrome (SSS) and a group of syndromes known collectively as the acromelic dysplasias, which typically result in short stature, skin thickening and joint stiffness, have been linked to FBN1 mutations that affect specific domains of the fibrillin-1 protein. Despite their apparent phenotypic differences, dysregulation of transforming growth factor β (TGFβ) is a common factor in all of these disorders. Using a newly developed assay to track the secretion and incorporation of full-length, GFP-tagged fibrillin-1 into the extracellular matrix, we investigated whether or not there were differences in the secretion and microfibril assembly profiles of fibrillin-1 variants containing substitutions associated with MFS, SSS or the acromelic dysplasias. 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. These results suggest fundamental differences in the dominant pathogenic mechanisms underlying MFS, SSS and the acromelic dysplasias, which give rise to TGFβ dysregulation associated with these diseases.
Highlights
Human fibrillin-1 is a 350 kDa glycoprotein that constitutes the major structural component of the 10-12 nm diameter extracellular microfibrils of metazoan species
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
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
Summary
Human fibrillin-1 is a 350 kDa glycoprotein that constitutes the major structural component of the 10-12 nm diameter extracellular microfibrils of metazoan species. In addition to conferring structural support to tissues and providing a scaffold for the deposition of elastin during elastogenesis [1,2,3], fibrillin microfibrils are involved in multiple interactions with cells and other connective tissue components that function to regulate the production of extracellular matrix These include the binding of cells to fibrillin-1 via the integrins αvβ, α5β1 and αvβ6 [4,5,6], and the sequestration on microfibrils of growth factors such as transforming growth factor-β (TGFβ) and the bone morphogenetic proteins (BMPs) [7,8,9]. The majority of mutations affecting the gene encoding human fibrillin-1, FBN1, result in Marfan syndrome (MFS), a common (1:5000 incidence), autosomal dominant disorder of the fibrous connective tissue with highly variable clinical manifestations characterised by skeletal, ocular and cardiovascular defects
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