title: Classical and neonatal Marfan syndrome mutations in fibrillin-1 cause differential protease susceptibilities and protein function.
abstract: Mutations in fibrillin-1 give rise to Marfan syndrome (MFS) characterized by vascular, skeletal, and ocular abnormalities. Fibrillins form the backbone of extracellular matrix microfibrils in tissues including blood vessels, bone, and skin. They are crucial for regulating elastic fiber biogenesis and growth factor bioavailability. To compare the molecular consequences of mutations causing the severe neonatal MFS with mutations causing the milder classical MFS, we introduced representative point mutations from each group in a recombinant human fibrillin-1 fragment. Structural effects were analyzed by circular dichroism spectroscopy and analytical gel filtration chromatography. Proteolytic susceptibility was probed with non-physiological and physiological proteases, including plasmin, thrombin, matrix metalloproteinases, and cathepsins. All mutant proteins showed a similar gross secondary structure and no differences in heat stability as compared with the wild-type protein. Proteins harboring neonatal mutations were typically more susceptible to proteolytic cleavage compared with those with classical mutations and the wild-type protein. Proteolytic neo-cleavage sites were found both in close proximity and distant to the mutations, indicating small but significant structural changes exposing cryptic cleavage sites. We also report for the first time that cathepsin K and V cleave non-mutated fibrillin-1 at several domain boundaries. Compared with the classical mutations and the wild type, the group of neonatal mutations more severely affected the ability of fibrillin-1 to interact with heparin/heparan sulfate, which plays a role in microfibril assembly. These results suggest differential molecular pathogenetic concepts for neonatal and classical MFS including enhanced proteolytic susceptibility for physiologically relevant enzymes and loss of function for heparin binding.
authors.affiliation: Faculty of Dentistry, Division of Biomedical Sciences, Faculty of Medicine, McGill University, Montreal H3A 2B2, Canada.
authors.forename: Ryan
authors.initials: R
authors.lastname: Kirschner
authors.forename: Dirk
authors.initials: D
authors.lastname: Hubmacher
authors.forename: Garud
authors.initials: G
authors.lastname: Iyengar
authors.forename: Jasvir
authors.initials: J
authors.lastname: Kaur
authors.forename: Christine
authors.initials: C
authors.lastname: Fagotto-Kaufmann
authors.forename: Dieter
authors.initials: D
authors.lastname: Brömme
authors.forename: Rainer
authors.initials: R
authors.lastname: Bartels
authors.forename: Dieter P
authors.initials: DP
authors.lastname: Reinhardt
mesh_terms: D002850:Chromatography, Gel; D002942:Circular Dichroism; D000071838:Fibrillin-1; D000071837:Fibrillins; D057809:HEK293 Cells; D006801:Humans; D007231:Infant, Newborn; D008382:Marfan Syndrome / Q000235:genetics / Q000378:metabolism*; D008840:Microfilament Proteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D010447:Peptide Hydrolases / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D017354:Point Mutation; D017434:Protein Structure, Tertiary; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D054392:Adipokines
chemical_list: D000071838:Fibrillin-1; D000071837:Fibrillins; D008840:Microfilament Proteins; D010447:Peptide Hydrolases; D011994:Recombinant Proteins; C000605722:FBN1 protein, human; D054392:Adipokines