Consequences of Cysteine Mutations in Calcium-binding Epidermal Growth Factor Modules of Fibrillin-1

Abstract

Mutations in fibrillin-1 lead to Marfan syndrome and some related genetic disorders. Many of the more than 600 mutations currently known in fibrillin-1 eliminate or introduce cysteine residues in epidermal growth factor-like modules. Here we report structural and functional consequences of three selected cysteine mutations (R627C, C750G, and C926R) in fibrillin-1. The mutations have been analyzed by means of recombinant polypeptides produced in mammalian expression systems. The mRNA levels for the mutation constructs were similar to wild-type levels. All three mutated polypeptides were secreted by embryonic kidney cells (293) into the culture medium. Purification was readily feasible for mutants R627C and C750G, but not for C926R, which restricted the availability of this mutant polypeptide to selected analyses. 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. Subtle structural changes caused by R627C and C750G, however, were monitored by proteolysis and heat denaturation experiments. These changes occurred in the vicinity of the mutations either as short range effects (R627C) or both short and long range effects (C750G). Enhanced proteolytic susceptibility was observed for R627C and C750G to a variety of proteases. 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.

Fibrillin-1 directly regulates osteoclast formation and function by a dual mechanism

“…In contrast, most of the tested mutations resulted in enhanced degradation within disulfide-bonded loops of individual cbEGF or TB domains. Mutation-mediated cleavage events may also modulate interdomain processing by long-range structural effects that have been reported previously (Kirschner et al, 2011;Vollbrandt et al, 2004). In this regard it is important to note that the rF1F fragment tested in our study fully contains rF23, but lacks osteoclast inhibitory properties, which could be potentially explained by different folding of various length fibrillin-1 fragments.…”

“…This in turn would release the negative feedback controlled by N-terminal fibrillin-1 fragments resulting in enhanced osteoclast formation and activity. MFS mutations typically render fibrillin-1 more susceptible to proteolysis (Kirschner et al, 2011;McGettrick et al, 2000;Reinhardt et al, 2000;Vollbrandt et al, 2004). Cathepsin K cleaves rFBN1-N exclusively in the linker regions between domains producing N-terminal fragments almost identical to the inhibitory rF23 through cleavage sites at Val 444 and Leu 445 (Kirschner et al, 2011).…”

Potential Phenotype–Genotype Correlation in Marfan Syndrome

“…2,3 There is also evidence, however, that matrix incorporation of mutant fibrillin-1 (unique to DN-type mutations) can promote proteolytic clearance of microfibrils over time, perhaps further exacerbating the functional deficit and contributing to phenotypic progression. 4,5 Thus, although theoretical mechanistic distinctions between mutations with and without DN potential (DN mutations and haploinsufficiencyinducing (HI) mutations, respectively) are blurred, they are not necessarily irrelevant.…”

“…2,3 There is also evidence, however, that matrix incorporation of mutant fibrillin-1 (unique to DN-type mutations) can promote proteolytic clearance of microfibrils over time, perhaps further exacerbating the functional deficit and contributing to phenotypic progression. 4,5 Thus, although theoretical mechanistic distinctions between mutations with and without DN potential (DN mutations and haploinsufficiencyinducing (HI) mutations, respectively) are blurred, they are not necessarily irrelevant.Microfibrils serve diverse functions, including a direct contribution to the biomechanical properties of tissues, coordination of elastic fiber biogenesis and homeostasis, and regulation of the bioavailability and activity of growth factors that influence the performance of neighboring cells. The relative contribution of each of these functions to physiological and hence pathological tissue performance probably shows both spatial and temporal heterogeneity.…”

The N1317H Substitution Associated with Leber Congenital Amaurosis Results in Impaired Interdomain Packing in Human CRB1 Epidermal Growth Factor-like (EGF) Domains

“…1) (15,17,18). Substitutions that involve the replacement of a cysteine are likely to lead to misfolding, changes in calcium binding, and an increase in proteolytic susceptibility (36,37). The second category includes conservative and non-conservative substitutions (with respect to charge and size) distributed throughout the cbEGF module.…”