Material and mechanical properties of bones deficient for fibrillin-1 or fibrillin-2 microfibrils

The contribution of non-collagenous components of the extracellular matrix to bone strength is largely undefined. Here we report that deficiency of fibrillin-1 or fibrillin-2 microfibrils causes distinct changes in bone material and mechanical properties. Morphometric examination of mice with hypomorphic or null mutations in fibrillin-1 or fibrillin-2, respectively, revealed appreciable differences in the postnatal shaping and growth of long bones. Fourier transform infrared imaging spectroscopy indicated that fibrillin-1 plays a predominantly greater role than fibrillin-2 in determining the material properties of bones. Biomechanical tests demonstrated that fibrillin-2 exerts a greater positive influence on the mechanical properties of bone than fibrillin-1 assemblies. Published evidence indirectly supports the notion that the above findings are mostly, if not exclusively, related to the differential control of TGFβ family signaling by fibrillin proteins. Our study therefore advances our understanding of the role that extracellular microfibrils play in bone physiology and implicitly, in the pathogenesis of bone loss in human diseases caused by mutations in fibrillin-1 or -2.     

Homo- and heterotypic fibrillin-1 and -2 interactions constitute the basis for the assembly of microfibrils

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.     

Homotypic fibrillin-1 interactions in microfibril assembly

We have defined the homotypic interactions of fibrillin-1 to obtain new insights into microfibril assembly. Dose-dependent saturable high affinity binding was demonstrated between N-terminal fragments, between furin processed C-terminal fragments, and between these N- and C-terminal fragments. The N terminus also interacted with a downstream fragment. A post-furin cleavage site C-terminal sequence also interacted with the N terminus, with itself and with the furin-processed fragment. No other homotypic fibrillin-1 interactions were detected. Some terminal homotypic interactions were inhibited by other terminal sequences, and were strongly calcium-dependent. Treatment of an N-terminal fragment with N-ethylmaleimide reduced homotypic binding. Microfibril-associated glycoprotein-1 inhibited N- to C-terminal interactions but not homotypic N-terminal interactions. These fibrillin-1 interactions are likely to regulate pericellular fibrillin-1 microfibril assembly.     

Versican interacts with fibrillin-1 and links extracellular microfibrils to other connective tissue networks.

Fibrillin-containing microfibrils are polymeric structures that are difficult to extract from connective tissues. Proteolytic digestion of tissues has been utilized to release microfibrils for study. Few of the molecules that connect microfibrils to other elements in the matrix have been identified. In this study, electron microscopic immunolocalization of anti-versican antibodies in tissues and in extracted microfibrils demonstrated that the C-terminal region of versican is found associated with fibrillin microfibrils. Extraction of microfibrils followed by treatment of microfibrils under dissociating conditions suggested that the versican C terminus is covalently bound to microfibrils. Binding assays using recombinant fibrillin-1 polypeptides and recombinant lectican lectin domains indicated that the versican lectin domain binds to specific fibrillin-1 polypeptides. The versican lectin domain also bound to molecules comigrating with authentic fibrillin-1 monomers in an assay using cell culture medium. In assays using microfibrils, the versican lectin domain demonstrated preferential binding compared with other lecticans. Binding was calcium-dependent. The binding site for versican in microfibrils is most likely within a region of fibrillin-1 between calcium-binding epidermal growth factor-like domains 11 and 21. Human mutations in this region can result in severe forms of the Marfan syndrome ("neonatal" Marfan syndrome). The connection between versican and fibrillin microfibrils may be functionally significant, particularly in cardiovascular tissues.     

Interactions of fibrillin-1 with heparin/heparan sulfate, implications for microfibrillar assembly.

Fibrillin-1 is a major constituent of the 10-12 nm extracellular microfibrils. Here we identify, characterize, and localize heparin/heparan sulfate-binding sites in fibrillin-1 and report on the role of such glycosaminoglycans in the assembly of fibrillin-1. By using different binding assays, we localize two calcium-independent heparin-binding sites to the N-terminal (Arg(45)-Thr(450)) and C-terminal (Asp(1528)-Arg(2731)) domains of fibrillin-1. A calcium-dependent-binding site was localized to the central (Asp(1028)-Thr(1486)) region of fibrillin-1. Heparin binding to these sites can be inhibited by a highly sulfated and iduronated form of heparan sulfate but not by chondroitin 4-sulfate, chondroitin 6-sulfate, and dermatan sulfate, demonstrating that the heparin binding regions represent binding domains for heparan sulfate. When heparin or heparan sulfate was added to cultures of skin fibroblasts, the assembly of fibrillin-1 into a microfibrillar network was significantly reduced. Western blot analysis demonstrated that this effect was not due to a reduced amount of fibrillin-1 secreted into the culture medium. Inhibition of the attachment of glycosaminoglycans to core proteins of proteoglycans by beta-d-xylosides resulted in a significant reduction of the fibrillin-1 network. These studies suggest that binding of fibrillin-1 to proteoglycan-associated heparan sulfate chains is an important step in the assembly of microfibrils.     

Microfibril-associated glycoprotein-2 interacts with fibrillin-1 and fibrillin-2 suggesting a role for MAGP-2 in elastic fiber assembly

Elastic fibers are composed of the protein elastin and a network of 10-12 nm microfibrils. The microfibrillar proteins include, among others, the fibrillins and microfibril-associated glycoproteins-1 and -2 (MAGP-1 and MAGP-2). Little is known about how microfibrillar proteins interact to support fiber assembly. We used the C-terminal half of MAGP-2 in a yeast two-hybrid library screen to identify relevant ligands. Six of 13 positive clones encoded known microfibrillar proteins, including fibrillin-1 and -2. Deletion analysis of partial fibrillin-1 and -2 clones revealed a calcium-binding epidermal growth factor repeat-containing region near the C terminus responsible for binding. This region is distinct from the region of fibrillin-1 reported by others to bind MAGP-1. The MAGP-2 bait was unable to interact productively with other epidermal growth factor repeats in fibrillin-1, demonstrating specificity of the interaction. Deletion analysis of the MAGP-2 bait demonstrated that binding occurred in a core region containing 48% identity and 7 conserved cysteine residues with MAGP-1. Immunoprecipitation of MAGP-2 from transfected COS-7 cells resulted in the coprecipitation of fibrillin. These results demonstrate that MAGP-2 specifically interacts with fibrillin-1 and -2 and suggest that MAGP-2 may help regulate microfibrillar assembly. The results also demonstrate the utility of the yeast two-hybrid system to study protein-protein interactions of the extracellular matrix.     

Cell adhesion to fibrillin-1 molecules and microfibrils is mediated by alpha 5 beta 1 and alpha v beta 3 integrins

Fibrillins are the major glycoprotein components of microfibrils that form a template for tropoelastin during elastic fibrillogenesis. We have examined cell adhesion to assembled purified microfibrils, and its molecular basis. Human dermal fibroblasts exhibited Arg-Gly-Asp and cation-dependent adhesion to microfibrils and recombinant fibrillin-1 protein fragments. Strong integrin alpha 5 beta 1 interactions with fibrillin ligands were identified, but integrin alpha v beta 3 also contributed to cell adhesion. Fluorescence-activated cell sorting analysis confirmed the presence of abundant alpha 5 beta 1 and some alpha v beta 3 receptors on these cells. Adhesion to microfibrils and to Arg-Gly-Asp containing fibrillin-1 protein fragments induced signaling events that led to cell spreading, altered cytoskeletal organization, and enhanced extracellular fibrillin-1 deposition. Differences in cell shape when plated on fibrillin or fibronectin implied substrate-specific alpha 5 beta 1-mediated cellular responses. An Arg-Gly-Asp-independent cell adhesion sequence was also identified within fibrillin-1. Adhesion and spreading of smooth muscle cells on fibrillin ligands was enhanced by antibody-induced beta1 integrin activation. A375-SM melanoma cells bound Arg-Gly-Asp-containing fibrillin-1 protein fragments mainly through alpha v beta 3, whereas HT1080 cells used mainly alpha 5 beta 1. This study has shown that fibrillin microfibrils mediate cell adhesion, that alpha 5 beta 1 and alpha v beta 3 are both important but cell-specific fibrillin-1 receptors, and that cellular interactions with fibrillin-1 influence cell behavior.     

Confocal microscopy demonstrates association of LTBP-2 in fibrillin-1 microfibrils and colocalisation with perlecan in the disc cell pericellular matrix

Comparative immunolocalisations of latent transforming growth factor-beta-1 binding protein (LTBP)-2, fibrillin-1, versican and perlecan were undertaken in foetal human and wild type C57BL/6 mouse and Hspg2 exon 3 null HS deficient mouse intervertebral discs (IVDs). LTBP-2 was a prominent pericellular component of annular fibrochondrocytes in the posterior annulus fibrosus (AF), interstitial matrix adjacent to nucleus pulposus (NP) cells and to fibrillar and cell associated material in the anterior AF of the human foetal IVD and also displayed a pericellular localisation pattern in murine IVDs. Perlecan and LTBP-2 displayed strong pericellular colocalisation patterns in the posterior AF and to fibrillar material in the outer anterior AF in the foetal human IVD. Versican was a prominent fibril-associated component in the posterior and anterior AF, localised in close proximity to fibrillin-1 in fibrillar arrangements in the cartilaginous vertebral rudiments around paraspinal blood vessels, to major collagen fibre bundles in the anterior and posterior AF and shorter fibres in the NP. Fibrillin-1 was prominent in the outer anterior AF of the human foetal IVD and in fibres extending from the AF into the cartilaginous vertebral rudiments. LTBP-2 was prominently associated with annular fibrils containing fibrillin-1, versican was localised in close proximity to these but not specifically with LTBP-2. The similar deposition levels of LTBP-2 observed in the AF of the Hspg2 exon 3 null and wild type murine IVDs indicated that perlecan HS was not essential for LTBP-2 deposition but colocalisation of LTBP-2 with perlecan in the foetal human IVD was consistent with HS mediated interactions which have already been demonstrated in-vitro.     

Interaction of tropoelastin with the amino-terminal domains of fibrillin-1 and fibrillin-2 suggests a role for the fibrillins in elastic fiber assembly

Alignment of tropoelastin molecules during the process of elastogenesis is thought to require fibrillin-containing microfibrils. In this study, we have demonstrated that amino-terminal domains of two microfibrillar proteins, fibrillin-1 and fibrillin-2, interact with tropoelastin in solid phase binding assays. The tropoelastin-binding site was localized to a region beginning at the glycine-rich and proline-rich regions of fibrillin-2 and fibrillin-1, respectively, and continuing through the second 8-cysteine domain. Characterization of the binding requirements using the fibrillin-2 construct found that a folded, secondary structure was necessary for binding. Furthermore, binding between tropoelastin and fibrillin was mediated by ionic interactions involving the lysine side chains of tropoelastin. The importance of the lysine side chains was corroborated by the finding that the fibrillin-2 construct did not bind to mature elastin, whose lysine side chains have been modified to form cross-links. Interestingly, there was no interaction between the fibrillin constructs and tropoelastin in solution phase, suggesting that binding of tropoelastin to a solid substrate exposes a cryptic binding site. These results suggest that fibrillin plays an important role in elastic fiber assembly by binding tropoelastin and perhaps facilitating side chain alignment for efficient cross-linking.     

Initial steps in the degradation of n-alkane-1-sulphonates by Pseudomonas

The primary reaction in the degradation of n-alkane-1-sulfonates by Pseudomonas is the hydroxylation of the carbon atom bearing the sulfonate group. The 1-hydroxy-n-alkane-1-sulfonate (aldehyde-bisulfite adduct) formed easily hydrolyses to give the corresponding aldehyde and bisulfite. The enzyme catalysing the hydroxylation reaction depends for its action on the presence of molecular oxygen and NADH. The kinetics of this reaction and the substrate specificity of the enzyme were studied using a crude enzyme extract and a spectrophotometric assay method based on co-oxidation of NADH with the sulfonate.     

Heparin/heparan sulfate controls fibrillin-1, -2 and -3 self-interactions in microfibril assembly

Fibrillins form multifunctional microfibrils in most connective tissues. Deficiencies in fibrillin assembly can result in fibrillinopathies, such as Marfan syndrome. We demonstrate the presence of heparin/heparan sulfate binding sites in fibrillin-2 and -3. Multimerization of all three fibrillins drastically increased the apparent affinity of their interaction with heparin/heparan sulfate. Surprisingly, contrary to other reports heparin/heparan sulfate strongly inhibited homo- and heterotypic N-to-C-terminal fibrillin interactions. These data suggest that heparin/heparan sulfate controls the formation of microfibrils at the bead interaction stage.     

Initial steps of photosystem II de novo assembly and preloading with manganese take place in biogenesis centers in Synechocystis

In the cyanobacterium Synechocystis sp PCC 6803, early steps in thylakoid membrane (TM) biogenesis are considered to take place in specialized membrane fractions resembling an interface between the plasma membrane (PM) and TM. This region (the PratA-defined membrane) is defined by the presence of the photosystem II (PSII) assembly factor PratA (for processing-associated TPR protein) and the precursor of the D1 protein (pD1). Here, we show that PratA is a Mn(2+) binding protein that contains a high affinity Mn(2+) binding site (K(d) = 73 μM) and that PratA is required for efficient delivery of Mn(2+) to PSII in vivo, as Mn(2+) transport is retarded in pratA(-). Furthermore, ultrastructural analyses of pratA(-) depict changes in membrane organization in comparison to the wild type, especially a semicircle-shaped structure, which appears to connect PM and TM, is lacking in pratA(-). Immunogold labeling located PratA and pD1 to these distinct regions at the cell periphery. Thus, PratA is necessary for efficient delivery of Mn(2+) to PSII, leading to Mn(2+) preloading of PSII in the periplasm. We propose an extended model for the spatial organization of Mn(2+) transport to PSII, which is suggested to take place concomitantly with early steps of PSII assembly in biogenesis centers at the cell periphery.     

Formation of fibrillar multimers of oat beta-glucosidase isoenzymes is mediated by the As-Glu1 monomer

Oat beta-glucosidase (EC 3.2.1.21) exists in two isomeric forms of homomultimer (type I) and heteromultimer (type II), which are comprised of two 60 kDa monomers of As-Glu1 and As-Glu2. The cDNA of As-Glu2 was cloned in this study, whereas As-Glu1 was previously cloned as As-P60. The As-Glu2 cDNA encodes a plastid-directing transit peptide of 57 amino acid residues and a mature protein of 521 amino acid residues. The amino acid sequence of As-Glu2 is highly homologous to that of As-Glu1, except for their C-terminal portions. When the two cDNAs of the mature proteins were expressed as T7.Tag-fused proteins in Escherichia coli, they produced soluble and enzymatically active T7.Tag-As-Glu1 and T7.Tag-As-Glu2 proteins. The T7.Tag-As-Glu1 was assembled into a donut-shaped hexamer ring which was in turn stacked in integer numbers to form long fibrillar homomultimers of different lengths with a molecular mass of up to several million daltons. On the other hand, the T7.Tag-As-Glu2 primarily formed a dimer rather than a multimer. When both cDNAs of As-Glu1 and As-Glu2 were co-expressed as T7.Tag-fused mature proteins, they were also assembled into a hexamer ring comprised of the two monomers in a 1:1 stoichiometry. The heteromeric hexamer was stacked in smaller numbers to form the heteromultimer of T7. Tag-As-Glu1 and -As-Glu2. The results indicate that the As-Glu1 monomer plays a crucial role in the formation of both the As-Glu1 homomultimer and the As-Glu1 and As-Glu2 heteromultimer. We describe here a unique structure for the oat beta-glucosidase fibrillar multimer that is formed by stacking the hexamer rings composed of As-Glu1 and/or As-Glu2.     

Early steps in assembly of the yeast vacuolar H+-ATPase.

Vacuolar proton-translocating ATPases are composed of a complex of integral membrane proteins, the Vo sector, attached to a complex of peripheral membrane proteins, the V1 sector. We have examined the early steps in biosynthesis of the yeast vacuolar ATPase by biosynthetically labeling wild-type and mutant cells for varied pulse and chase times and immunoprecipitating fully and partially assembled complexes under nondenaturing conditions. In wild-type cells, several V1 subunits and the 100-kDa Vo subunit associate within 3-5 min, followed by addition of other Vo subunits with time. Deletion mutants lacking single subunits of the enzyme show a variety of partial complexes, including both complexes that resemble intermediates in the assembly pathway of wild-type cells and independent V1 and Vo sectors that form without any apparent V1Vo subunit interaction. Two yeast sec mutants that show a temperature-conditional block in export from the endoplasmic reticulum accumulate a complex containing several V1 subunits and the 100-kDa Vo subunit during incubation at elevated temperature. This complex can assemble with the 17-kDa Vo subunit when the temperature block is reversed. We propose that assembly of the yeast V-ATPase can occur by two different pathways: a concerted assembly pathway involving early interactions between V1 and Vo subunits and an independent assembly pathway requiring full assembly of V1 and Vo sectors before combination of the two sectors. The data suggest that in wild-type cells, assembly occurs predominantly by the concerted assembly pathway, and V-ATPase complexes acquire the full complement of Vo subunits during or after exit from the endoplasmic reticulum.     

Initial steps of phloroglucinol metabolism in Penicillium simplicissimum

The pathway for the aerobic catabolism of 1,3,5-trihydroxybenzene (phloroglucinol) by a new strain of Penicillium was investigated using both in vivo and in vitro cell-free systems. The fungal strain was isolated by enrichment on phloroglucinol and identified as P. simplicissimum (Oud) Thom. It grew optimally at pH 5.5 and 27°C with 119 mM (1.5%w/v) of phloroglucinol in a basal mineral salts medium. Vapours of the crystalline substrate placed in a Petri-plate lid supported the growth of the fungal colonies on the agar surface. Mycelia grown on phloroglucinol accumulated 1,2,4-trihydroxybenzene and resorcinol in the medium. Washed, resting mycelia grown on phloroglucinol, when resuspended in a buffer utilized oxygen in the presence of catechol, resorcinol, pyrogallol and phloroglucinol. A NADPH-dependent reductase in the cell-free extract reduced phloroglucinol to dihydrophloroglucinol. This electron donor could not be replaced by NADH. Resorcinol hydroxylase, phloroglucinol reductase, catechol-1,2-oxygenase, and catechol-2,3-oxygenase were detected in cell-free extracts of mycelia grown on phloroglucinol. The possible steps in the degradation of phloroglucinol are discussed.     

Influence of fibrillin-1 genotype on the aortic stiffness in men.

Aortic stiffness is a predictor of cardiovascular mortality. The mechanical properties of the arterial wall depend on the connective tissue framework, with variation in fibrillin-1 and collagen I genes being associated with aortic stiffness and/or pulse pressure elevation. The aim of this study was to investigate whether variation in fibrillin-1 genotype was associated with aortic stiffness in men. The mechanical properties of the abdominal aorta of 79 healthy men (range 28-81 yr) were investigated by ultrasonographic phase-locked echo tracking. Fibrillin-1 genotype, characterized by the variable tandem repeat in intron 28, and collagen type I alpha 1 genotype characterized by the 2,064 G>T polymorphism, were determined by using DNA from peripheral blood cells. Three common fibrillin-1 genotypes, 2-2, 2-3, and 2-4, were observed in 50 (64%), 10 (13%), and 11 (14%) of the men, respectively. Those of 2-3 genotype had higher pressure strain elastic modulus and aortic stiffness compared with men of 2-2 or 2-4 genotype (P = 0.005). Pulse pressure also was increased in the 2-3 genotype (P = 0.04). There was no significant association between type 1 collagen genotype and aortic stiffness in this cohort. In conclusion, the fibrillin-1 2-3 genotype in men was associated with increased aortic stiffness and pulse pressure, indicative of an increased risk for cardiovascular disease.