A standard control (glyceraldehyde-3-phosphate dehydrogenase) was included, which resulted in a 1173-bp DNA fragment. Polymerase chain reaction was performed using Taq polymerase (Roche Applied Science) and primer pair 5′-AAGTGTCAGTGTCCCAGTGG-3′ and 5′-TAGAAGGCACAGTCGAGGC-3′, which produced a 350-bp fragment specific for the rF45 constructs. After isolation of total RNA with Trizol following the supplier's instructions (Invitrogen), the mRNA was reverse transcribed into cDNA with Superscript II and oligo(dT) primer according to the manufacturer's protocol (Invitrogen). MRNA Synthesis and Secretion of Recombinant Proteins-Nontransfected and transfected Flp-In-293 cell clones were grown to confluence in 75-cm 2 flasks and used for RNA purification and secretion analysis. These results expand and further strengthen the concept that proteolytic degradation of mutated fibrillin-1 might be an important potential mechanism in the pathogenesis of Marfan syndrome and other disorders caused by mutations in fibrillin-1. Enhanced proteolytic susceptibility was observed for R627C and C750G to a variety of proteases. These changes occurred in the vicinity of the mutations either as short range effects (R627C) or both short and long range effects (C750G). Subtle structural changes caused by R627C and C750G, however, were monitored by proteolysis and heat denaturation experiments. The overall folds of the mutant polypeptides were indistinguishable from the wild-type as judged by the ultrastructural shape, CD analysis, and reactivity with a specific antibody sensitive for intact disulfide bonds. Purification was readily feasible for mutants R627C and C750G, but not for C926R, which restricted the availability of this mutant polypeptide to selected analyses. All three mutated polypeptides were secreted by embryonic kidney cells (293) into the culture medium. The mRNA levels for the mutation constructs were similar to wild-type levels. The mutations have been analyzed by means of recombinant polypeptides produced in mammalian expression systems. Here we report structural and functional consequences of three selected cysteine mutations (R627C, C750G, and C926R) in fibrillin-1. Many of the more than 600 mutations currently known in fibrillin-1 eliminate or introduce cysteine residues in epidermal growth factor-like modules. Mutations in fibrillin-1 lead to Marfan syndrome and some related genetic disorders. Glycobiology and Extracellular Matrices.
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