Abstract
Fibrillin-1 and fibrillin-2 constitute the backbone of extracellular filaments, called microfibrils. Fibrillin assembly involves complex multistep mechanisms to result in a periodical head-to-tail alignment in microfibrils. Impaired assembly potentially plays a role in the molecular pathogenesis of genetic disorders caused by mutations in fibrillin-1 (Marfan syndrome) and fibrillin-2 (congenital contractural arachnodactyly). Presently, the basic molecular interactions involved in fibrillin assembly are obscure. Here, we have generated recombinant full-length human fibrillin-1, and two overlapping recombinant polypeptides spanning the entire human fibrillin-2 in a mammalian expression system. Characterization by gel electrophoresis, electron microscopy after rotary shadowing, and reactivity with antibodies demonstrated correct folding of these recombinant polypeptides. Analyses of homotypic and heterotypic interaction repertoires showed N- to C-terminal binding of fibrillin-1, and of fibrillin-1 with fibrillin-2. The interactions were of high affinity with dissociation constants in the low nanomolar range. However, the N- and C-terminal fibrillin-2 polypeptides did not interact with each other. These results demonstrate that fibrillins can directly interact in an N- to C-terminal fashion to form homotypic fibrillin-1 or heterotypic fibrillin-1/fibrillin-2 microfibrils. This conclusion was further strengthened by double immunofluorescence labeling of microfibrils. In addition, the binding epitopes as well as the entire fibrillin molecules displayed very stable properties.
Highlights
Microfibrils are extracellular supramolecular aggregates found in many elastic and non-elastic tissues [1]
Microfibrils 10 –12 nm in diameter are supramolecular aggregates in the extracellular matrix consisting of fibrillins and other matrix proteins
Fibrillins are repetitively aligned within microfibrils and constitute their structural backbone
Summary
Human fibrillin-2 cDNA was synthesized by reverse transcription of total RNA isolated from MG-63 cells (American Type Culture Collection) using the antisense (as) primers specified below. The obtained cDNAs were used as templates for polymerase chain reactions (PCR) using the same antisense primers in combination with appropriate sense (s) primers (see below) and the high fidelity polymerase Pfx (Invitrogen). The 5Ј fragment rFBN2– 0s (nt 1–1782) was synthesized by PCR amplification using primers 0s (5Ј-ATTACCGCTAGCAACGGCTCGGCATCATGGGGAGAAGACGGAGGC-3Ј) and 0as (5Ј-ATCTGAGTTCACGCATCGACC-3Ј), which introduced a Kozak sequence and a NheI recognition site for cloning at the 5Ј end. The cDNAs from these constructs were combined to a full-length fibrillin-2 construct in several steps within the pBluescript II SK(ϩ) plasmid using appropriate restriction enzymes (New England Biolabs Inc.). The resulting plasmid, pBS-rFBN2full, contained fibrillin-2 cDNA positions 1– 8872.
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