Microfibril-associated MAGP-2 stimulates elastic fiber assembly

Elastic fibers are complex structures composed of a tropoelastin inner core and microfibril outer mantle guiding tropoelastin deposition. Microfibrillar proteins mainly include fibrillins and microfibril-associated glycoproteins (MAGPs). MAGP-2 exhibits developmental expression peaking at elastic fiber onset, suggesting that MAGP-2 mediates elastic fiber assembly. To determine whether MAGP-2 regulates elastic fiber assembly, we used an in vitro model featuring doxycycline-regulated cells conditionally overexpressing exogenous MAGP-2 and constitutively expressing enhanced green fluorescent protein-tagged tropoelastin. Analysis by immunofluorescent staining showed that MAGP-2 overexpression dramatically increased elastic fibers levels, independently of extracellular levels of soluble tropoelastin, indicating that MAGP-2 stimulates elastic fiber assembly. This was associated with increased levels of matrix-associated MAGP-2. Electron microscopy showed that MAGP-2 specifically associates with microfibrils and that elastin globules primarily colocalize with MAGP-2-associated microfibrils, suggesting that microfibril-associated MAGP-2 facilitates elastic fiber assembly. MAGP-2 overexpression did not change levels of matrix-associated fibrillin-1, MAGP-1, fibulin-2, fibulin-5, or emilin-1, suggesting that microfibrils and other elastic fiber-associated proteins known to regulate elastogenesis do not mediate MAGP-2-induced elastic fiber assembly. Moreover, mutation analysis showed that MAGP-2 does not stimulate elastic fiber assembly through its RGD motif, suggesting that integrin receptor binding does not mediate MAGP-2-induced elastic fiber assembly. Because MAGP-2 interacts with Jagged-1 that controls cell-matrix interaction and cell motility, two key factors in elastic fiber macroassembly, microfibril-associated MAGP-2 may stimulate elastic fiber macroassembly by targeting the release of elastin globules from the cell membrane onto developing elastic fibers.     

Microfibril-associated glycoprotein-1 and fibrillin-2 are associated with tropoelastin deposition in vitro

Elastic system fibers consist of microfibrils and tropoelastin. During development, microfibrils act as a template on which tropoelastin is deposited. Microfibril-associated glycoprotein-1 (MAGP-1) and fibrillin-2, the major components of microfibrils, provide the likely template for tropoelastin deposition. In this study, we used the RNA interference (RNAi) technique to establish MAGP-1 and fibrillin-2 gene-specific knock-downs individually in elastin-producing cells (human gingival fibroblasts). We then examined the extracellular deposition of tropoelastin by western blotting. These two genes were specifically suppressed to < 30% of the control level, and this was responsible for the diminution of tropoelastin deposition. An immunofluorescence study also confirmed that RNAi-mediated down-regulation of MAGP-1 or fibrillin-2 led to the loss of tropoelastin immunoreactivity. These results suggest that MAGP-1 and fibrillin-2 are, directly or indirectly, associated with the extracellular deposition of tropoelastin during elastic fiber formation in human gingival fibroblasts in vitro.     

Differential gene expression of 36-kDa microfibril-associated glycoprotein (MAGP-36/MFAP4) in rat organs

By using quantitative Western blot analysis and the real time polymerase chain reaction technique, we investigated the differential gene expression of microfibril-associated glycoprotein (MAGP-36) in rat organs. The gene was expressed highly in sites rich in elastic fibers, such as aorta, skin, and esophagus. However, MAGP-36 was also expressed highly in some other sites containing no elastic fibers. In lung and trachea, the expression levels of MAGP-36 mRNA were about seven times higher than those in other elastic tissues, although the protein abundances were almost at the same levels as other elastic tissues. MAGP-36 seemed to be secreted outside these organs. In brain, kidney, and spleen, although the expression levels of MAGP-36 mRNA were low, substantial amounts of MAGP-36 protein were detected. An immunohistochemical study revealed that MAGP-36 was present at the brush border of the S3 segment of proximal tubules in kidney. Since MAGP-36 is known to bind to mannan, MAGP-36 might be involved in mannose transport in the S3 segment. Thus, MAGP-36 might be multifunctional and present in a wide variety of sites in various organs.     

Protein interaction studies of MAGP-1 with tropoelastin and fibrillin-1

Elastic fibers consist primarily of an amorphous elastin core associated with microfibrils, 10-12 nm in diameter, containing fibrillins and microfibril-associated glycoproteins (MAGPs). To investigate the interaction of MAGP-1 with tropoelastin and fibrillin-1, we expressed human MAGP-1 as a T7-tag fusion protein in Escherichia coli. Refolding of the purified protein produced a soluble form of MAGP-1 that displayed saturable binding to tropoelastin. Fragments of tropoelastin corresponding to the N-terminal, C-terminal, and central regions of the molecule were used to characterize the MAGP-1 binding site. Cleavage of tropoelastin with kallikrein, which cleaves after Arg(515) in the central region of the molecule, disrupted the interaction, suggesting that the separated N- and C-terminal fragments were insufficient to determine MAGP-1 binding to intact tropoelastin. In addition, no evidence of an interaction was observed between MAGP-1 and a tropoelastin construct consisting of domains 17-27 that brackets the kallikrein cleavage site, suggesting a complex mechanism of interaction between the two molecules. Binding of MAGP-1 was also tested with overlapping recombinant fibrillin-1 fragments. MAGP-1 bound to a region at the N terminus of fibrillin-1 in a calcium-dependent manner. In summary, these results suggest a model for the interaction of elastin with the microfibrillar scaffold.     

Molecular interactions of biglycan and decorin with elastic fiber components: biglycan forms a ternary complex with tropoelastin and microfibril-associated glycoprotein 1.

The interactions of the dermatan sulfate proteoglycans biglycan and decorin have been investigated with the elastic fiber components, tropoelastin, fibrillin-containing microfibrils, and microfibril-associated glycoproteins (MAGP) 1 and 2. Both proteoglycans were found to bind tropoelastin and fibrillin-containing microfibrils but not MAGPs 1 and 2 in solid phase binding assays. The specificity of the binding of biglycan and decorin to tropoelastin was confirmed by co-immunoprecipitation experiments and by the blocking of the interactions with elastin-derived peptides. Isolated core proteins from biglycan and decorin bound to tropoelastin more strongly than the intact proteoglycans, and there were no differences in the tropoelastin binding characteristics of distinct glucuronate-rich and iduronate-rich glycoforms of biglycan. These findings indicated that the binding sites were contained in the protein cores of the proteoglycans rather than the glycosaminoglycan side chains. Scatchard analysis showed that biglycan bound more avidly than decorin to tropoelastin with K(d) values estimated as 1.95 x 10(-7) m and 5.3 x 10(-7) m, respectively. In blocking experiments each proteoglycan showed extensive inhibition of binding of the other to tropoelastin but was most effective at blocking its own binding. This result suggested that biglycan and decorin had closely spaced but distinct binding sites on tropoelastin. Addition of the elastin-binding protein MAGP-1 to the assays enhanced the binding of biglycan to tropoelastin but had no effect on the decorin-tropoelastin interaction. Co-immunoprecipitation experiments showed that MAGP-1 interacted with biglycan but not decorin in the solution phase. The results indicated that biglycan specifically formed a ternary complex with tropoelastin and MAGP-1. Overall the study supports the concept that biglycan may have a specific role in the elastinogenic phase of elastic fiber formation.     

Characterization of an in vitro model of elastic fiber assembly

Elastic fibers consist of two morphologically distinct components: elastin and 10-nm fibrillin-containing microfibrils. During development, the microfibrils form bundles that appear to act as a scaffold for the deposition, orientation, and assembly of tropoelastin monomers into an insoluble elastic fiber. Although microfibrils can assemble independent of elastin, tropoelastin monomers do not assemble without the presence of microfibrils. In the present study, immortalized ciliary body pigmented epithelial (PE) cells were investigated for their potential to serve as a cell culture model for elastic fiber assembly. Northern analysis showed that the PE cells express microfibril proteins but do not express tropoelastin. Immunofluorescence staining and electron microscopy confirmed that the microfibril proteins produced by the PE cells assemble into intact microfibrils. When the PE cells were transfected with a mammalian expression vector containing a bovine tropoelastin cDNA, the cells were found to express and secrete tropoelastin. Immunofluorescence and electron microscopic examination of the transfected PE cells showed the presence of elastic fibers in the matrix. Biochemical analysis of this matrix showed the presence of cross-links that are unique to mature insoluble elastin. Together, these results indicate that the PE cells provide a unique, stable in vitro system in which to study elastic fiber assembly.     

Association of elastin with oxytalan fibers of the dermis and with extracellular microfibrils of cultured skin fibroblasts

The formation of a mature elastic fiber is thought to proceed by the deposition of elastin on pre-existing microfibrils (10-12 nm in diameter). Immunohistochemical evidence has suggested that in developing tissues such as aorta and ligamentum nuchae, small amounts of elastin are associated with microfibrils but are not detected at the light microscopic and ultrastructural levels. Dermal tissue contains a complex elastic fiber system consisting of three types of fibers--oxytalan, elaunin, and elastic--which are believed to differ in their relative contents of microfibrils and elastin. According to ultrastructural analysis, oxytalan fibers contain only microfibrils, elaunin fibers contain small quantities of amorphous elastin, and elastic fibers are predominantly elastin. Using indirect immunofluorescence techniques, we demonstrate in this study that nonamorphous elastin is associated with the oxytalan fibers. Frozen sections of normal skin were incubated with antibodies directed against human aortic alpha elastin and against microfibrillar proteins isolated from cultured calf aortic smooth muscle cells. The antibodies to the microfibrillar proteins and elastin reacted strongly with the oxytalan fibers of the upper dermis. Oxytalan fibers therefore are composed of both microfibrils and small amounts of elastin. Elastin was demonstrated extracellularly in human skin fibroblasts in vitro by indirect immunofluorescence. The extracellular association of nonamorphous elastin and microfibrils on similar fibrils was visualized by immunoelectron microscopy. Treatment of these cultures with sodium dodecyl sulfate/mercaptoethanol (SDS/ME) solubilized tropoelastin and other proteins that reacted with the antibodies to the microfibrillar proteins. It was concluded that the association of the microfibrils with nonamorphous elastin in intact dermis and cultured human skin fibroblasts may represent the initial step in elastogenesis.     

Ultrastructural immuno-localization of tropoelastin in the chick eye

Summary Immuno-gold labeling at the electron-microscopy level was used to investigate the distribution of tropoelastin in the chick eye. Intense staining was found in the amorphous part of mature elastic fibers in different regions of the organ. In elaunin fibers, both the amorphous core and the surrounding microfibrils were clearly labeled. In addition, reactive sites were detected in the oxitalan fibers of the stroma of the cornea and in Descemet's membrane, which showed a gradient of reactive sites increasing from the center toward the periphery. Oxitalan fibers of the stroma often fused with Descemet's membrane; the pattern of immunological staining suggested a continuity between the two structures. In the ciliary zonule, labeling for tropoelastin was observed in discrete areas on the bundles of microfibrils. The results show a complex structural organization of elastic tissue; this may be important in endowing the various parts of the eye with different mechanical properties.     

36-kDa microfibril-associated glycoprotein (MAGP-36) is an elastin-binding protein increased in chick aortae during development and growth

MAGP-36 was discovered in porcine aorta in 1989 and is thought to be one of the microfibril-associated proteins. MAGP-36 has been localized on the surface of elastic fibers or laminae in immunohistochemical studies. However, its functional role in the aorta is obscure. Herein, we report on the binding activity of MAGP-36 to components of the aortic wall and its accumulation pattern in the aorta during development and growth. In vitro, MAGP-36 bound to elastin and collagen in a Ca(2+)-dependent manner, and mediated the adhesion of human aortic smooth muscle cells. This cell adhesion mostly depended on the RGD-containing domain of MAGP-36. We examined the accumulation of MAGP-36 with quantitative Western blot analysis and immunoelectron microscopy in chick aortae during development and growth. The amount of MAGP-36 increased on the surface of elastic fibers or laminae between days 14 and 34 after the start of incubation, and reached a plateau at about 53 days. This accumulation of MAGP-36 roughly correlated with an increase in blood pressure for this period. Thus, MAGP-36 might be a bridging protein that connects elastin to other components of the aortic wall and might play a role in maintaining the integrity of the aortic structure under arterial pressure.     

Distinct steps of cross-linking, self-association, and maturation of tropoelastin are necessary for elastic fiber formation

Elastic fibers play an important role in the characteristic resilience of many tissues. The assembly of tropoelastin into a fibrillar matrix is a complex stepwise process and the deposition and cross-linking of tropoelastin are believed to be key steps of elastic fiber formation. However, the detailed mechanisms of elastic fiber assembly have not been defined yet. Here, we demonstrate the relationship between deposition and the cross-linking/maturation of tropoelastin. Our data show that a C-terminal half-fragment of tropoelastin encoded by exons 16-36 (BH) is deposited onto microfibrils, yet we detect very limited amounts of the cross-linking amino acid, desmosine, an indicator of maturation, whereas the N-terminal half-fragment encoded by exons 2-15 (FH) was deficient for both deposition and cross-linking, suggesting that elastic fiber formation requires full-length tropoelastin molecules. A series of experiments using mutant BH fragments, lacking either exon 16 or 30, or a deletion of both exons showed that self-association of tropoelastin polypeptides was an early step in elastic fiber assembly. Immunofluorescence and Western blot assay showed that the treatment of cell culture medium or conditioned medium with beta-aminopropionitrile to inhibit cross-linking, prevented both the deposition and polymerization of tropoelastin. In conclusion, our present results support the view that self-association and oxidation by lysyl oxidase precedes tropoelastin deposition onto microfibrils and the entire molecule of tropoelastin is required for this following maturation process.     

Effect of monoclonal antibodies to defined regions of tropoelastin on elastogenesis in vitro.

Primary cultures of chick embryo aorta cells were grown for one week in the presence of mouse monoclonal antibodies directed against defined regions of chick tropoelastin. This treatment did not significantly alter cell proliferation, cell viability and incorporation of labeled amino acids into total protein or tropoelastin compared with control cultures in which antibodies were either omitted or substituted with an unrelated monoclonal antibody. Tropoelastin-reactive material in the cell layer was revealed by immunologic staining with rabbit antibodies against the chick protein both at the optical and ultrastructural level. Immunofluorescence of control cultures showed that tropoelastin was incorporated into thin and straight fibrils which were sometimes associated with spot-like elements. In the electron microscope tropoelastin-reactive sites were found mainly on the amorphous core of typical, small elastic fibers. The morphological picture of tropoelastin deposits in cultures exposed to anti-tropoelastin monoclonal antibodies depended on the molecular form (whole antibody or Fab fragments) and the binding specificity of the antibody used. Although alterations common to different antibodies were observed, the main structural features were peculiar for each antibody. Two antibodies which bound epitopes present in two regions of tropoelastin grossly altered the formation of amorphous elastin. Moreover, two antibodies directed against the region of tropoelastin containing the polypentapeptide-repeat (VPGVG)n stimulated the deposition of the protein into the amorphous core of normal-looking elastic fibers and disorganized the compact bundles of parallel microfibrils seen in controls. Finally, one antibody which recognized a unique epitope close to the carboxy-terminal end of tropoelastin and Fab fragments from all antibodies apparently inhibited the formation of the amorphous nuclei of elastic fibers, but not the association of tropoelastin with microfibrils. The data suggest that the association of tropoelastin molecules during fiber assembly is not random, but follows an ordered alignment process which the antibodies alter by imposing a different molecular packing.     

Microfibril-associated glycoprotein-1 binding to tropoelastin: multiple binding sites and the role of divalent cations.

Microfibrils and elastin are major constituents of elastic fibers, the assembly of which is dictated by multimolecular interactions. Microfibril-associated glycoprotein-1 (MAGP-1) is a microfibrillar component that interacts with the soluble elastin precursor, tropoelastin. We describe here the adaptation of a solid-phase binding assay that defines the effect of divalent cations on the interactions between MAGP-1 and tropoelastin. Using this assay, a strong calcium-dependent interaction was demonstrated, with a dissociation constant of 2.8 +/- 0.3 nm, which fits a single-site binding model. Manganese and magnesium bestowed a weaker association, and copper did not facilitate the protein interactions. Three constructs spanning tropoelastin were used to quantify their relative contributions to calcium-dependent MAGP-1 binding. Binding to a construct spanning a region from the N-terminus to domain 18 followed a single-site binding model with a dissociation constant of 12.0 +/- 2.2 nm, which contrasted with the complex binding behavior observed for fragments spanning domains 17-27 and domain 27 to the C-terminus. To further elucidate binding sites around the kallikrein cleavage site of domains 25/26, MAGP-1 was presented with constructs containing C-terminal deletions within the region. Construct M1659, which spans a region from the N-terminus of tropoelastin to domain 26, inclusive, bound MAGP-1 with a dissociation constant of 9.7 +/- 2.0 nm, which decreased to 4.9 +/- 1.0 nm following the removal of domain 26 (M155n), thus displaying only half the total capacity to bind MAGP-1. These results demonstrate that MAGP-1 is capable of cumulative binding to distinct regions on tropoelastin, with different apparent dissociation constants and different amounts of bound protein.     

Deposition of tropoelastin into the extracellular matrix requires a competent elastic fiber scaffold but not live cells.

The initial steps of elastic fiber assembly were investigated using an in vitro assembly model in which purified recombinant tropoelastin (rbTE) was added to cultures of live or dead cells. The ability of tropoelastin to associate with preexisting elastic fibers or microfibrils in the extracellular matrix was then assessed by immunofluorescence microscopy using species-specific tropoelastin antibodies. Results show that rbTE can associate with elastic fiber components in the absence of live cells through a process that does not depend on crosslink formation. Time course studies show a transformation of the deposited protein from an initial globular appearance early in culture to a more fibrous structure as the matrix matures. Deposition required the C-terminal region of tropoelastin and correlated with the presence of preexisting elastic fibers or microfibrils. Association of exogenously added tropoelastin to the cellular extracellular matrix was inhibited by the addition of heparan sulfate but not chondroitin sulfate sugars. Together, these results suggest that the matrix elaborated by the cell is sufficient for the initial deposition of tropoelastin in the extracellular space and that elastin assembly may be influenced by the composition of sulfated proteoglycans in the matrix.     

MAGP-2 has multiple binding regions on fibrillins and has covalent periodic association with fibrillin-containing microfibrils

The interactions of microfibril-associated glycoprotein (MAGP)-2 have been investigated with fibrillins and fibrillin-containing microfibrils. Solid phase binding assays were conducted with recombinant fragments covering fibrillin-1 and most of fibrillin-2. MAGP-2, and its structure relative MAGP-1, were found to bind two fragments spanning the N-terminal half of fibrillin-1 and an N-terminal fragment of fibrillin-2. Blocking experiments indicated that MAGP-2 had a binding site(s) close to the N terminus of the fibrillin-1 molecule that was distinct from that for MAGP-1 and an additional, more central binding site(s) that may be shared by the two MAGPs. Immunogold labeling of developing nuchal ligament tissue showed that MAGP-2 had regular covalent and periodic (about 56 nm) association with fibrillin-containing microfibrils of elastic fibers in this tissue. Further analysis of isolated microfibrils indicated that MAGP-2 was attached at two points along the microfibril substructure, "site 1" on the "beads" and "site 2" at the "shoulder" of the interbead region close to where the two "arms" fuse. In contrast, MAGP-1 was located only on the beads. Comparison of the MAGP-2 binding data with known fibrillin epitope maps of the microfibrils showed that site 1 correlated with the N-terminal MAGP-2 binding region, and site 2 correlated with the second, more central, MAGP-2 binding region on the fibrillin-1 molecule. Of particular note, immunolabeling at site 2 was markedly decreased, relative to that at site 1, on extended microfibrils with bead-to-bead periods over 90 nm, suggesting that site 2 may move toward the beads when the microfibril is stretched. The study points to MAGP-2 being an integral component of some populations of fibrillin-containing microfibrils. Moreover, the identification of multiple MAGP-binding sequences on fibrillins supports the concept that MAGPs may function as molecular cross-linkers, stabilizing fibrillin monomers in folded conformation within or between the microfibrils, and thus MAGPs may be implicated in the modulation of the elasticity of these structures.     

Significant decrease in tropoelastin gene expression in fibroblasts from a Japanese Costello syndrome patient with impaired elastogenesis and enhanced proliferation

Costello syndrome is a connective tissue disorder associated with sparse, thin, and fragmented elastic fibers in tissues. In this study we demonstrated a significant decrease in the expression of tropoelastin mRNA in fibroblasts derived from a Japanese Costello syndrome patient with impaired elastogenesis and enhanced proliferation. In contrast, there were no changes in expression of the Harvey ras (HRAS), fibrillin-1, fibulin-5, microfibril-associated glycoprotein-1 (MAGP-1), lysyl oxidase (LOX), or 67-kDa non-integrin elastin-binding protein (EBP) gene. The proliferative activity of the Costello fibroblasts was about 4-fold higher than that of the normal and pathological control ones. However, no mutations were detected in the coding region of HRAS mRNA. Transduction of the bovine tropoelastin (bTE) gene with the lentiviral vector restored the elastic fiber formation and decreased the growth rate in the Costello fibroblasts. These results strongly suggest that the defect of human tropoelastin (hTE) gene expression should induce the impaired elastogenesis and enhanced proliferation of Costello fibroblasts, and that a primary cause other than the HRAS gene mutation should contribute to the pathogenesis in the present Costello case.     

The alpha 1 chain of type VIII collagen is associated with many but not all microfibrils of elastic fiber system.

The antigen of monoclonal antibodies which had labeled the hexagonal lattice of Descemet's membrane in a specific manner was shown to be the alpha 1 chain of type VIII collagen by immunoblotting followed by amino acid sequence analysis. With this antibody, the localization of alpha 1 (VIII) in various tissues was studied by several immunocytochemical methods. Under light microscopy, the alpha 1 (VIII) was found in a fine fibrillar form in various capsular tissues such as capsules of the liver, kidneys, adrenals, lungs and so on. It was also present in dense connective tissues such as the Achilles tendon, and periodontal and perivertebral ligaments. When some dense connective tissues which had been negative to the label including the intima of aorta, perimysium and Glisson's sheath of the liver, were subjected to pepsin digestion, epitopes were revealed which showed a specific immunofluorescence pattern. In many locations the pattern of localization coincided with that of elastic fiber components, and full or partial colocalization with tropoelastin or costaining with resorcin-fuchsin staining was observed. In immunoelectron microscopy, the antigen (alpha 1 (VIII)) was localized on the surface of, but not inside, elastic fibers. However, some tissues which are rich in elastic fibers or microfibrils remained unlabeled. These included elastic fibers of the aortic media and ligamentum nuchae as well as ciliary zonules. Therefore it is suggested that alpha 1 (VIII) is a collagen associated with microfibrils of some elastic fiber systems, but is not an intrinsic component of either elastic fibers or of microfibrils.     

Emilin, a component of elastic fibers preferentially located at the elastin-microfibrils interface

The fine distribution of the extracellular matrix glycoprotein emilin (previously known as glycoprotein gp115) (Bressan, G. M., I. Castellani, A. Colombatti, and D. Volpin. 1983. J. Biol. Chem. 258: 13262-13267) has been studied at the ultrastructural level with specific antibodies. In newborn chick aorta the protein was exclusively found within elastic fibers. In both post- and pre-embedding immunolabeling emilin was mainly associated with regions where elastin and microfibrils are in close contact, such as the periphery of the fibers. This localization of emilin in aorta has been confirmed by quantitative evaluation of the distribution of gold particles within elastic fibers. In other tissues, besides being associated with typical elastic fibers, staining for emilin was found in structures lacking amorphous elastin, but where the presence of tropoelastin has been demonstrated by immunoelectron microscopy. This was particularly evident in the oxitalan fibers of the corneal stroma, in the Descemet's membrane, and in the ciliary zonule. Analysis of embryonic aorta revealed the presence of emilin at early stages of elastogenesis, before the appearance of amorphous elastin. Immunofluorescence studies have shown that emilin produced by chick embryo aorta cells in culture is strictly associated with elastin and that the process of elastin deposition is severely altered by the presence of antiemilin antibodies in the culture medium. The name of the protein was derived from its localization at sites where elastin and microfibrils are in proximity (emilin, elastin microfibril interface located protein).     

Development of elastic fibers of nuchal ligament, aorta, and lung of fetal and postnatal sheep: an ultrastructural and electron microscopic immunohistochemical study

The morphogenesis of elastic fibers of the nuchal ligament, aorta, and lung of sheep was studied by light microscopy, transmission electron microscopy, and immunohistochemical methods for the detection of elastin. The degree of maturation of the amorphous materials of elastic fibers was assessed morphologically in preparations stained by the tannic acid and periodic acid methenamine-silver methods. With both of these methods, the amorphous components of mature fibers stained less intensely than did those of immature fibers. Elastic fibers in early stages of development consisted of many microfibrils and few, small, branching masses of immature amorphous material. Thicker fibers were formed by the coalescence of growing masses of amorphous materials. In late stages of formation of elastic fibers, the mature amorphous materials were associated with few microfibrils; and they were partially surrounded by immature amorphous materials associated with many microfibrils. Antielastin antibody reacted evenly with amorphous materials in very early stages of elastic-fiber development, but reacted only with the other zones of amorphous materials in later stages; it also reacted with the microfibrils in all stages. These findings were interpreted as indicating that the microfibrils were associated with small amounts of elastin on their surfaces. This conclusion is in agreement with ultrastructural observations showing 1) that development of microfibrils precedes that of the amorphous material and 2) that the microfibrils adjacent to the immature amorphous materials are covered with small amounts of tannic acid-positive amorphous materials. These observations suggest that microfibrils serve as sites for elastin deposition, both in early elastogenesis and in subsequent growth of elastic fibers. However, the nature of the interaction between elastin and microfibrils remains unknown.     

Coacervation is promoted by molecular interactions between the PF2 segment of fibrillin-1 and the domain 4 region of tropoelastin

In forming elastic fibers, microfibrils act as the scaffold sites for depositing the elastin precursor tropoelastin. We examined key binding interactions that promote massive tropoelastin association through coacervation. Using a segment of the microfibril protein fibrillin-1, PF2, known to bind full-length tropoelastin, we mapped its interaction site to the N-terminal region of tropoelastin bounded by domains 2 and 18. Precise contact residues between domain 4 of tropoelastin and domain 16 of fibrillin-1 were discovered through a novel combination of transglutaminase cross-linking and mass spectroscopy, with contact sites at residues K38 of tropoelastin and Q669 of fibrillin-1. This is the first report of a role for this region of tropoelastin in microfibril interactions. The addition of PF2 thermodynamically facilitated the coacervation of tropoelastin, resulting in smaller changes in entropy and enthalpy values for the coacervating system. A novel multicomponent in vitro tropoelastin assembly reaction system demonstrated that amassed tropoelastin was spatially and preferentially directed to surfaces coated with PF2 as expected for organized three-dimensional distribution during tissue elastogenesis. This study underscores the role of this part of fibrillin-1 as an anchor point for tropoelastin at the microfibril-elastin junction during the initial stages of elastic fiber assembly.     

Fibrillin-1 interactions with heparin. Implications for microfibril and elastic fiber assembly

Fibrillin-1 assembly into microfibrils and elastic fiber formation involves interactions with glycosaminoglycans. We have used BIAcore technology to investigate fibrillin-1 interactions with heparin and with heparin saccharides that are analogous to S-domains of heparan sulfate. We have identified four high affinity heparin-binding sites on fibrillin-1, localized three of these sites, and defined their binding kinetics. Heparin binding to the fibrillin-1 N terminus has particularly rapid kinetics. Hyaluronan and chondroitin sulfate did not interact significantly with fibrillin-1. Heparin saccharides with more than 12 monosaccharide units bound strongly to all four fibrillin-1 sites. Heparin did not inhibit fibrillin-1 N- and C-terminal interactions or RGD-dependent cell attachment, but heparin and MAGP-1 competed for binding to the fibrillin-1 N terminus, and heparin and tropoelastin competed for binding to a central fibrillin-1 sequence. By regulating these key interactions, heparin can profoundly influence microfibril and elastic fiber assembly.