Missense mutations impair intracellular processing of fibrillin and microfibril assembly in Marfan syndrome

Abstract

Dermal fibroblasts from nine Marfan syndrome patients with missense mutations in the fibrillin-1 gene (FBN1) produced nearly normal amounts of fibrillin as determined by quantitative pulse-chase experiments. However, six of the seven mutations involving substitutions of highly conserved cysteine residues exhibited lower rates of intracellular transport and secretion. This effect is likely due to improper folding, since intracellular fibrillin processing was also affected by the reducing agent dithiothreitol. Normal secretion patterns were seen in three mutations that either change the conformation of EGF-like domains or change consensus amino acids required for Ca(++)-binding. In all nine fibroblasts strains, however, the deposition of fibrillin in the extracellular matrix was reduced to 50% of normal in two and to less than 30% in seven of the nine samples studied. The protein alterations caused by these missense mutations are associated with moderate to severe features of Marfan syndrome and a dominant negative mechanism is suggested to play a major role in their pathogenesis.

Structural Consequences of Cysteine Substitutions C1977Y and C1977R in Calcium-binding Epidermal Growth Factor-like Domain 30 of Human Fibrillin-1

“…It was suggested that the delay in secretion might be due to targeted recognition of misfolded protein. In contrast, a smaller number of cases (24% of those studied) have been found in MFS patients with cysteine substitutions where normal secretion of fibrillin-1 has been observed (14,18). The C1977R substitution is one example where pulse-chase analysis of patient fibroblasts harboring this mutation showed normal synthesis, secretion, and deposition of fibrillin-1 compared with normal fibroblasts (Ref.…”

Physiology and pathology of notch signalling system

“…Otherwise, transport of the mutant receptor through the endoplasmic reticulum (ER) may be reduced or slowed, as occurs for other proteins, the mutations of which recall those of CADASIL and which consequently have folding or protein aggregation problems in ER. In Marfan syndrome, for example the altered number of cysteines in EGF-repeats of fibrillin-1 causes accumulation of fibrillin in ER (Godfrey et al, 1990;Aoyama et al, 1993;Schrijver et al, 1999). Karlström et al (2002) observed that in their experimental system, the mutant receptor was more likely to form cell aggregates than the normal receptor.…”

Expression of a mutant human fibrillin allele upon a normal human or murine genetic background recapitulates a Marfan cellular phenotype.

“…The disorder is inherited in an autosomal dominant fashion and is caused by mutations in a gene encoding a protein known to multimerize. In addition, a hallmark feature of classic MFS is severe deficiency of extracellular fibrillin, far below the amount predicted by a single functioning FBN1 allele ( 15,16). Finally, mutations causing very reduced levels of FBN1 mutant transcript have been associated with an extremely mild disease phenotype ( 17).…”

“…Polyacrylamide electrophoresis gels of cellular protein extracts after 35S-cysteine pulse and unlabeled-cysteine chase were evaluated by autoradiography and scanning densitometry as previously described (15). Calculated levels of fibrillin synthesis and deposition are presented in Table II.…”