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.

FIGURE 1.

Fibrillin-1 protein fragments used in this study. A, domain organization of the fibrillin-1 protein and the rF20 fragment is shown. The neonatal region and the four heparin binding sites in fibrillin-1 are indicated. Individual domains are represented as ellipses and described. The position of each generated MFS point mutation within the rF20 polypeptide is indicated by arrows. Mutations N548I (NI), R627C (RC), C750G (CG) cause the cMFS, and mutations G1013R (GR), C1032Y (CY), I1048T (IT), E1073K (EK), C1182S (CS) cause the nMFS. B, shown is a demonstration of purity of the protein preparations. The mutant rF20 proteins were purified to homogeneity as described under “Experimental Procedures.” 7 μg per lane were analyzed by SDS-PAGE under reducing (DTT+) and non-reducing (DTT−) conditions and stained with Coomassie Brilliant Blue. C, to test for potential residual proteolytic activity in the purified proteins samples, the proteins were incubated for 24 h at 37 °C and analyzed as described in B under reducing conditions. Positions of globular marker proteins are indicated in kDa.

FIGURE 2.

Gel filtration chromatography of the mutant rF20 polypeptides. The rF20 polypeptides were subjected to gel filtration chromatography, as detailed under “Experimental Procedures,” to separate monomers from potentially present multimers. Elution profiles at A₂₈₀ nm are shown. Aliquots of representative peak fractions labeled by bars (a, b, and c) were separated by SDS-PAGE under non-reducing (DTT−) and reducing (DTT+) conditions as indicated and silver-stained. Note that the silver-staining procedure stains reduced proteins to a lesser degree than non-reduced proteins. The positions of globular marker proteins are indicated in kDa. Each fraction was analyzed on the same gel under both conditions except for G1013R (b), for which samples were loaded on two gels.

FIGURE 3.

Thermal denaturation of rF20 polypeptides assessed by circular dichroism spectroscopy. Denaturation profiles were recorded at 204 nm, and the proteins were considered as 100% denatured at 95 °C. For all proteins analyzed, 50% denaturation was observed at similar temperatures between 71 and 77.5 °C (crossing point of the horizontal and vertical lines), indicating that the mutations did not have a profound effect on the overall structural stability of the polypeptide.

FIGURE 4.

Proteolysis of rF20 polypeptides with various non-physiological and physiological proteases. 7 μg of each polypeptide was digested with trypsin (A), chymotrypsin (B), thrombin (C), and plasmin (D) as detailed under “Experimental Procedures.” Proteolytic degradation products were separated by SDS-PAGE under reducing conditions and stained with Coomassie Brilliant Blue (upper panels). The numbered arrows correlate with the determined N-terminal sequences of the degradation products summarized in Table 3. The abbreviations of mutations are used according to Table 1. Mutations leading to classical Marfan syndrome are indicated as cMFS, and mutations leading to neonatal Marfan syndrome are indicated as nMFS. Fragments starting with the “APLADYCQ” amino acid sequence (N terminus of rF20) are labeled with an asterisk (*). Cleavage sites marked with an open circle were also found in the wild-type rF20 (WT). The lanes for each of the degradation analyses were analyzed by densitometry and plotted as a three-dimensional graph (lower panels). The profile of the WT is depicted in blue, the cMFS mutations is in shades of red, and the nMFS mutations are in shades of beige. High molecular weight bands are positioned on the left.

FIGURE 5.

Cleavage of non-mutated and mutated fibrillin-1 fragments with cathepsin K and cathepsin V. A, the N- and C-terminal half of non-mutated fibrillin-1 (rFBN1-N, rFBN1-C, respectively) was cleaved with cathepsin K (Cat K) and cathepsin V (Cat V) as outlined under “Experimental Procedures.” The numbered arrows correlate with the determined N-terminal sequences of the degradation products summarized in Table 4. Fragments labeled with an asterisk (*) could not be identified by N-terminal sequencing. Bands 1 and 5 represent the N-terminal APLA sequence of the rFBN1-C fragment. B, shown is schematic mapping of the cathepsins K and V cleavage sites identified in A. The domains are annotated as in Fig. 1. C and D, wild-type and mutant rF20 was digested with cathepsins K and V as indicated. Proteolytic degradation products were separated by SDS-PAGE under reducing conditions and stained with Coomassie Brilliant Blue (upper panels). Mutations leading to classical Marfan syndrome are indicated as cMFS, and mutations leading to neonatal Marfan syndrome are indicated as nMFS. Note that attempts to obtain sequence information revealed nine degradation products as the N terminus of the respective proteins, and some sequences were not interpretable. Each lane of the degradation analyses was analyzed by densitometry and plotted as a three-dimensional graph (lower panels). The profile of the WT is depicted in black, the cMFS mutations are in light gray, and the nMFS mutations are in dark gray. High molecular weight bands are positioned on the left.

FIGURE 6.

Proteolysis of rF20 polypeptides by MMPs. 7 μg of each polypeptide was digested with MMP-3 (A), MMP-12 (B), MMP-1 (C), MMP-2 (D), and MMP-9 (E). Degradation products were separated by SDS-PAGE under reducing conditions and stained with Coomassie Brilliant Blue (upper panels). The numbered arrows correlate with the determined N-terminal sequences of the degradation products summarized in Table 3. The abbreviations of mutations are used according to Table 1. Mutations leading to classical Marfan syndrome are indicated as cMFS, and mutations leading to neonatal Marfan syndrome are indicated as nMFS. Fragments with an N-terminal amino acid sequence of APLADYCQ (N terminus of rF20) are labeled with an asterisk (*). Each lane of the degradation analyses was analyzed by densitometry and plotted as a three-dimensional graph (lower panels). The profile of the WT is depicted in blue, the cMFS mutations is in shades of red, and the nMFS mutations are in shades of beige. High molecular weight bands are positioned on the left.

FIGURE 7.

Interaction of mutant fibrillin-1 polypeptides with heparin. The mutant polypeptides were subjected to heparin affinity chromatography and eluted by a linear NaCl gradient as described under “Experimental Procedures.” A, binding and elution profiles at 280 nm are shown. Non-bound protein is detected in the flow-through (FT), and the bound protein (BP) is eluted by a NaCl gradient. The insets confirm the presence of the protein in the peaks after SDS-PAGE under reducing (DTT+) and non-reducing (DTT−) conditions and silver staining. Note that the silver-staining procedure stains reduced proteins to a lesser degree than non-reduced proteins. B, shown is quantification of the amount of bound and non-bound protein of the affinity chromatographies shown in A. The bars show the amount of monomers and multimers as indicated.

FIGURE 8.

Schematic representation of the position of neo-cleavage sites in rF20 mutant proteins. The figure depicts graphically the data presented in Table 3 for each mutation separately. Only the domains for which neo-cleavage sites were identified are numbered. The domains are coded as described in Fig. 1. The position of the mutation is indicated with an asterisk. T, trypsin; C, chymotrypsin; P, plasmin; Th, thrombin; MMP, matrix metalloproteinase; Cat, cathepsin.