Microfibrils at basement membrane zones interact with perlecan via fibrillin-1.

Mutational defects in fibrillin-rich microfibrils give rise to a number of heritable connective tissue disorders, generally termed microfibrillopathies. To understand the pathogenesis of these microfibrillopathies, it is important to elucidate the supramolecular composition of microfibrils and their interaction properties with extracellular matrix components. Here we demonstrate that the proteoglycan perlecan is an associated component of microfibrils typically close to basement membrane zones. Double immunofluorescence studies demonstrate colocalization of fibrillin-1, the major backbone component of microfibrils, with perlecan in fibroblast cultures as well as in dermal and ocular tissues. Double immunogold labeling further confirms colocalization of perlecan to microfibrils in various tissues at the ultrastructural level. Extraction studies revealed that perlecan is not covalently associated with microfibrils. High affinity interactions between fibrillin-1 and perlecan were found by kinetic binding studies with dissociation constants in the low nanomolar range. A detailed mapping study of the interaction epitopes by solid phase binding assays primarily revealed interactions of perlecan domains I and II with a central region of fibrillin-1. Analysis of perlecan null embryos showed less microfibrils at the dermal-epidermal junction as compared with wild-type littermates. The data presented indicate a functional significance for perlecan in anchoring microfibrils to basement membranes and in the biogenesis of microfibrils.

Microfibrils, 10 -12 nm in diameter, are supramolecular aggregates found in many extracellular matrices. These entities always cover the surface of elastic fibers, where they are thought to play a crucial role in the biogenesis of elastic fibers (1). Microfibrils are also found in the absence of elastin in many tissues either as individual entities or intersecting with basement membranes. In fact, attachment of microfibrils to basement membranes represents a universal principle of virtually all types of basement membranes in a broad variety of tissues such as blood vessels (2), eye (3), kidney (4), lung (5), muscle (6), fetal membranes (7), and skin (8).
It has been clearly established that a family of proteins, the fibrillins, constitute the structural backbone of microfibrils (9 -14). Fibrillins-1-3 are large (ϳ350 kDa), highly homologous glycoproteins, which are composed of several extracellular protein domains such as the calcium-binding epidermal growth factor domain (cbEGF) 1 or the 8-Cys/transforming growth factor-␤ binding (TB) domain. Besides fibrillins, a number of other components have been reported to be associated with microfibrils as integral or peripherally associated components (for review see Ref. 15). Although most ligands described are localized to a tissue-specific subset of microfibrils, the microfibril-associated glycoprotein-1 (MAGP-1) codistributes with microfibrils in most if not all tissues where they occur (16,17). Fibulin-2 strongly interacts with fibrillin-1 (K D ϭ 56 nM) in a tissue-specific manner and is localized to the microfibril-elastic fiber interface, indicating a stabilizing role for elastic fibers (18). Latent transforming growth factor-␤-binding proteins (LTBP) have been found to serve as a vehicle for secretion of latent transforming growth factor-␤ into the extracellular matrix (19,20). LTBP-1 and -2 have been immunolocalized to fibrillin-containing microfibrils in tissues such as the heart, skin, and bone (21)(22)(23)(24), and direct interactions between LTBP-1 or -4 and fibrillins have been demonstrated (25). Thus, microfibrils potentially play a role in storage, presentation, and activation of transforming growth factor-␤ in the extracellular matrix. Other ligands such as collagen XVI (26,27), matrilin-2 (28), and the proteoglycan versican (29) also colocalize with microfibrils and directly interact with fibrillins. In addition to these protein ligands, glycosaminoglycans have been localized to microfibrils or have been found to interact directly with fibrillin-1 (30,31).
Microfibrils are the fundamental structure defective in a number of disorders summarized as microfibrillopathies, including Marfan syndrome and congenital contractural arachnodactyly (32). Mutations in fibrillin-1 result in Marfan syn-drome and some related disorders (33), whereas mutations in fibrillin-2 have been shown to lead to congenital contractural arachnodactyly (34,35). The "prototype" of microfibrillar disorders, Marfan syndrome, is primarily characterized by symptoms in the cardiovascular, skeletal, and ocular systems. With the exception of a small number of recurrent mutations, the vast majority of the currently known mutations (ϳ600) are unique to families (33). Inter-and intrafamilial variability is a common feature of Marfan syndrome, suggesting that other gene products play a modifying role in the pathogenesis of the disease. Microfibril-associated components are candidates for potential modulators in microfibrillopathies.
Recently, we and others (31,36) have discovered that heparin/heparan sulfate is able to interact with several regions of the fibrillin-1 molecule. Addition of heparin/heparan sulfate to a cell culture microfibril assembly model effectively inhibited the formation of microfibrils in this system (31,36). These data resulted in a working hypothesis that heparan sulfate containing proteoglycans may be important components in the biogenesis of microfibrils. These results prompted us to analyze interactions of fibrillin-1 with heparan sulfate-containing proteoglycans including perlecan.
Perlecan consists of a 400 -480-kDa core protein substituted by heparan and/or chondroitin sulfate side chains. It is an intrinsic basement membrane constituent but is also expressed in other extracellular matrices (37,38). Perlecan has multiple functional roles, for example in the assembly and maintenance of basement membranes (39,40), as well as in skeletogenesis, and it plays a major role in the regulation of a wide variety of cellular processes (for review see Ref. 41). The cDNA sequence demonstrated the presence of distinct extracellular protein modules arranged in the five predicted domains I-V (42)(43)(44). The perlecan core protein is known to interact with a number of extracellular proteins including nidogen-1 and -2 (45,46), fibulin-2 (47), fibronectin (48), ␣-dystroglycan (49), and extracellular matrix protein 1 (50). It also binds cell surface molecules and growth factors such as the fibroblast growth factor-binding protein (51), the platelet-derived growth factor (52), fibroblast growth factor 7 (53), and progranulin (54). Additional interactions are mediated through the three heparan sulfate chains located in domain I, including interactions with laminin-1, collagen type IV, and fibronectin (45,55), as well as with fibroblast growth factor 2 (56,57). Targeting of the perlecan gene in mice leads to cardiovascular and cephalic abnormalities due to the loss of the structural integrity of basement membranes in regions with increased mechanical stress (39,40).
Here we report that perlecan interacts with fibrillin-containing microfibrils in the vicinity of basement membrane zones. We demonstrate direct and high affinity interaction between fibrillin-1 and perlecan, and we map the respective binding epitopes on both proteins. Analysis of microfibrils in perlecan null mice suggests a role for perlecan in the biogenesis of microfibrils. The interaction may also be involved in anchoring mechanisms of microfibrils to basement membranes.

Immunolabeling Experiments
Primary Cells-Indirect immunofluorescence double labeling experiments of cells were performed with primary human skin fibroblasts grown in Dulbecco's modified Eagle's medium (Invitrogen) supplemented with 2 mM glutamine, 100 units/ml penicillin, 100 g/ml streptomycin, and 10% fetal calf serum at 37°C in a 5% CO 2 atmosphere. Confluent cell layers were trypsinized and seeded at 7.5 ϫ 10 4 cells/well in an 8-well chamber slide (Permanox; Nalge Nunc International). After 4 -5 days, the cells were washed with phosphate-buffered saline (PBS), fixed with 70% methanol, 30% acetone, and rehydrated in PBS. Nonspecific binding sites were blocked with 10% normal goat serum in PBS for 30 min. The cells were incubated for 1 h with primary antibodies ␣-rF6H (1:250 diluted) or mAb69 (ϳ10 g/ml) against fibrillin-1, antiheparan sulfate (1:100 diluted), or a mixture of the antisera 1030ϩ and 1056ϩ (1:800 diluted) against perlecan. Antibody binding was detected by a 1-h incubation with goat anti-rabbit fluorescein conjugate and goat anti-mouse cyanine Cy3 conjugate (diluted 1:200 in PBS; Jackson Im-munoResearch). For visualization of the fluorescent signals, an Axio-plan2 microscope (Zeiss) was used. Digital images were recorded using a 3CCD color video camera (Sony) and the AxioVision version 3.0.6 software (Zeiss).
Human Tissue-Immunolabeling of human tissues was performed on three normal human donor eyes (age range 46 -72 years) obtained at autopsy and fixed less than 5 h after death. The eyes had no history or morphologic evidence of any known ocular disease. In addition, eyelid skin biopsies were obtained during eyelid resections from three patients (age range 63-78 years) with blepharochalasis.
For indirect immunofluorescence double labeling experiments, ocular and dermal tissues were embedded in optimal cutting temperature compound and frozen in isopentane cooled with liquid nitrogen. Cryostat-cut sections (6 m) were fixed in cold acetone, blocked with 10% normal goat serum, and incubated in a mixture of primary antibodies (1:50 diluted mAb1948 and 1:2000 diluted ␣-rF6H) in PBS overnight at 4°C. Antibody binding was detected by Cy2-and Cy3-conjugated secondary antibodies diluted 1:300 (Alexa 488; Molecular Probes).
For postembedding immunogold labeling, human tissue specimens were fixed in 4% paraformaldehyde and 0.1% glutaraldehyde in 0.1 M cacodylate buffer, pH 7.4, for 2-5 h at 4°C. Specimens were dehydrated serially to 70% ethanol at Ϫ20°C and embedded in resin (LR White; Electron Microscopy Sciences). Ultrathin sections were pretreated with 1% hyaluronidase in Tris-buffered saline (TBS) for 30 min at 37°C. The sections were successively incubated in TBS, 0.05 M glycine in TBS, 0.5% ovalbumin, and 0.5% fish gelatin in TBS, a mixture of primary antibodies (1:20 diluted mAb1948 and 1:1500 diluted ␣-rF6H) in TBS/ ovalbumin overnight at 4°C, and finally in 10-and 18-nm gold-conjugated secondary antibodies (Biocell Laboratories) diluted 1:30 in TBS/ ovalbumin for 1 h. After rinsing, the sections were stained with uranyl acetate and examined with a transmission electron microscope (906E, Leo). In negative controls, the primary antibody was replaced by PBS or equimolar concentrations of nonimmune rabbit or mouse IgG or an irrelevant primary antibody.
Mouse Tissue-Perlecan null mice were generated by gene targeting and have been described previously (39). Embryos derived from perlecan heterozygous (ϩ/Ϫ) intercrosses were dissected at embryonic day 13.5 and 17.5. The yolk sac of each embryo was used for genomic DNA preparation and genotyping by PCR. Back skin from homozygous (Ϫ/Ϫ) perlecan null mice (n ϭ 3) and wild-type (ϩ/ϩ) littermates (n ϭ 3) was analyzed by pre-embedding immunogold labeling using the "en bloc" method as described by Sakai and Keene (69). Small tissue blocks (0.5-1-mm border length) were cut out and washed in PBS for 30 min at 4°C. The samples were incubated for 14 h with the primary antifibrillin-1 antibody (␣-rF6H, 1:5 diluted), washed again with PBS (6 h, five buffer changes), and incubated for 14 h with the gold-conjugated (5-nm gold particles) secondary antibodies (1:3 diluted; Amersham Bio-sciences) in 20 mM Tris-HCl, pH 8.2, in PBS including 0.1% bovine serum albumin. The samples were washed in PBS for 2 h and then fixed by immersion in 0.1 M cacodylate buffer, pH 7.4, containing 2.5% glutaraldehyde and 2% paraformaldehyde for 24 h, postfixed in 1% OsO 4 and stained en bloc with 2% uranyl acetate. After dehydration in graded alcohols, the specimens were embedded in Araldite. Semi-thin sections were stained with methylene blue and azure II to visualize the regions of interest containing the epidermis and the underlying dermal connective tissue. Ultrathin sections were cut from the regions defined previously, stained with lead citrate, and examined using a transmission electron microscope (EM 109, Phillips). In negative controls, the primary antibody was omitted and replaced by PBS.

Purification and Analysis of Microfibrils from Human Fibroblasts
Basically, purification of microfibrils from cell cultures followed the procedure by Kielty et al. (70). Primary normal human skin fibroblasts (passage 2) were grown to postconfluence for 6 weeks on a total culture area of 600 cm 2 under identical conditions as described under "Immunolabeling Experiments." The cells were washed with 50 mM Tris-HCl, pH 7.4, 400 mM NaCl, scraped from the culture flask, and incubated for 3 h with 1 mg/ml crude collagenase (from Clostridium histolyticum; Sigma) in the same buffer including 2 mM phenylmethylsulfonyl fluoride and 5 mM N-ethylmaleimide. The cell extract was centrifuged at 7000 ϫ g for 5 min and fractionated on a Sepharose CL-2B column (90 ml column volume; Amersham Biosciences) equilibrated in 50 mM Tris-HCl, pH 7.4, 400 mM NaCl, 5 mM CaCl 2 at a flow rate of 0.5 ml/min. From the eluted fractions, 10 l were spotted onto nitrocellulose for further analysis by standard immunoblotting. Fibrillin-1 and perlecan were detected using antibodies mAb69 and 1030ϩ, respectively. Fractions of the void volume, which were strongly positive for the presence of fibrillin-1, were dialyzed against distilled H 2 O and further analyzed by rotary shadowing and electron microscopy as described (71).

Kinetic Binding Studies
For kinetic binding studies of recombinant fibrillin-1 fragments with mouse perlecan by surface plasmon resonance, a BIAcore 1000 biosen-sor was used (BIAcoreAB). Purified recombinant fibrillin-1 fragments rF16 and rF6H were coupled to a CM5 sensor chip (BIAcore AB), which resulted in 6,000 -12,000 response units. Binding studies were performed with soluble perlecan in concentrations of 150 -300 nM in 50 mM Tris-HCl, pH 7.4, 150 mM NaCl including 1 mM CaCl 2 and 0.05% P20 (BIAcore AB) at flow rates of 20 l/min. The binding sites of the immobilized ligands were regenerated by washing with TBS after each cycle. After subtraction of the blank curves, the association and dissociation rate constants were determined by separate k a /k d fitting of all curves with the 1:1 Langmuir association/dissociation model (BIAevaluation software version 3.1, Biacore AB). Mass transfer limitations were not apparent.

Microtiter Solid Phase Binding Assay
Multiwell plates (96 wells, MaxiSorp, Nalge Nunc International) were coated with purified recombinant proteins (5 g/ml, 50 l/well) in TBS at 4°C. Nonspecific binding sites were blocked for 1 h with 5% nonfat milk in 20 mM Tris-HCl, pH 7.4, including 50 or 150 mM NaCl. Each of the following incubations was performed in 20 mM Tris-HCl, pH 7.4, 50 or 150 mM NaCl, 5 mM CaCl 2 with 2% nonfat milk at room temperature (ϳ20°C) and was followed by three washes with 20 mM Tris-HCl, pH 7.4, 50 or 150 mM NaCl, 5 mM CaCl 2 including 0.05% Tween 20. Coated proteins were incubated either with serial dilutions microtiter plate reader (Lucy 3, Anthos Labtec Instruments). Nonspecific binding representing interaction with the plastic surface was subtracted from each individual binding profile. Experiments were typically performed three to eight times.

Heparan Sulfate and Perlecan Are Colocalized with Fibrillin-
containing Microfibrils-Indirect double immunofluorescence experiments were performed by using primary human dermal fibroblasts (Fig. 1). Labeling with a monoclonal antibody specific for heparan sulfate and the specific polyclonal antiserum ␣-rF6H against fibrillin-1 resulted in a partially overlapping pattern on a light microscopic level (Fig. 1A). Double immunofluorescence labeling of perlecan and fibrillin-1 resulted in similar labeling patterns demonstrating significant colocalization of both proteins on microfibrillar networks (Fig. 1B). Double immunofluorescence studies on a light microscopic level were further extended to human tissues (Fig. 2). In general, fibrillin-1 and perlecan were found colocalized close to basement membrane zones, such as the epidermal-dermal junction in skin ( Fig. 2A), the vascular endothelial basement membrane in veins (Fig. 2B), or the basement membranes of the ciliary epithelium (Fig. 2C). Because the light microscopic level does not provide sufficient resolution to demonstrate colocalization on the molecular level, we performed double immunogold labeling of fibrillin-1 and perlecan in human tissues on a ultrastructural level (Fig. 3). Perlecan was clearly localized to fibrillin-1-containing microfibrils of the ciliary zonules, which attach to the lenticular basement membrane (Fig. 3A). In skin, perlecan was found colocalized with fibrillin-1 on microfibrils at the dermal-epidermal junction (Fig. 3B) or within perivascular microfibrils connecting endothelial cells with elastic fibers (Fig.  3C). In addition to the colocalization close to basement membrane zones, perlecan was also found occasionally colocalized with fibrillin-1 on isolated microfibrillar bundles, e.g. in the stroma of the ciliary body (Fig. 3D).
Perlecan Is Not an Integral Component of Microfibrils-One important question was whether perlecan constitutes an integral or peripherally associated component of microfibrils. To answer this question, extracts from long term cultures of dermal fibroblasts were treated with collagenase to remove collagen fibers and separated by gel filtration chromatography. A typical elution profile is shown in Fig. 4A. The presence of fibrillin-1 and perlecan was determined by immunoblotting with specific antibodies (Fig. 4A, lower panels). A major peak of fibrillin-1 was present in the void volume (33-45 ml), whereas a smaller and wider peak eluted between 53 and 81 ml. In addition, immunoblotting revealed the presence of the ϳ31-kDa microfibrillar component MAGP-1 within the major peak (36 -42 ml; data not shown). This major fibrillin-1 and MAGP-1-containing peak was correlated with the presence of intact beaded microfibrils by rotary shadowing and electron microscopy (Fig. 4, A, MF, and B). The minor fibrillin-1 peak likely represents smaller microfibrillar aggregates or fibrillin-1 monomers and multimers. Perlecan was detected as one major peak eluting between 69 and 97 ml, which overlapped partially with the minor fibrillin-1 peak between 69 and 81 ml (Fig. 4A). These data indicate that perlecan dissociates from microfibrils during the extraction process. An alternative interpretation is that perlecan becomes degraded rather than dissociated during the extraction procedure. However, this interpretation is unlikely because (i) no residual perlecan signal was observed in the microfibril-containing fractions and (ii) perlecan always eluted in a relatively sharp peak as compared with a broad peak expected to result from proteolytically degraded perlecan. Together with the other data presented in this study (Figs.  1-3), we conclude that perlecan is a component that is peripherally and noncovalently associated with fibrillin-containing beaded microfibrils.
Fibrillin-1 Interacts with Perlecan on the Molecular Level-To analyze whether the major constituent of microfibrils, fibrillin-1, interacts with perlecan on the molecular level, the binding properties of authentic tissue perlecan with established recombinant halves of fibrillin-1 (see Fig. 5A) were studied in real time by surface plasmon resonance spectroscopy under physiological buffer conditions (Table I). Soluble perlecan in the concentration range of 150 -300 nM bound to the N-terminal half of fibrillin-1 (rF16) immobilized to a sensor chip with dissociation constants in the range of K D ϭ 9.6 -42.9 nM. No binding interaction was observed to the C-terminal half of fibrillin-1 (rF6H). These data demonstrate a high affinity interaction between the N-terminal half of fibrillin-1 and fulllength perlecan.
To study molecular interactions between fibrillin-1 and perlecan by an alternative method and to identify interacting subdomains, rF16 and rF6H were analyzed for interaction properties with overlapping recombinant fragments of perlecan spanning the entire length of this proteoglycan (Fig. 5A). Saturable binding curves were always observed between rF16 and perlecan domains I and II using relatively low salt concentrations of 50 mM NaCl (Fig. 5B, left panel). With this salt concentration, rF6H interacted moderately with perlecan domain I (Fig. 5B, right panel). At physiological salt concentrations (150 mM NaCl), rF16 demonstrated similar interaction properties with perlecan domain II as compared with binding in low ionic strength, whereas typically the interaction with perlecan domain I was significantly lower (Fig. 5B, left panel). Fragment rF6H did not show significant interactions with perlecan fragments at physiological salt concentrations. These data demonstrate that the interactions between fibrillin-1 and perlecan are mediated by the N-terminal half of fibrillin-1, which interacts with epitopes on perlecan domains I and II. Different sensitivities to salt further suggest that the interaction of fibrillin-1 with perlecan domain I is of relatively low affinity, whereas the interaction with perlecan domain II likely is of higher affinity. Both types of interactions contribute to the high affinity interactions observed between full-length perlecan and rF16 by surface plasmon resonance spectroscopy (Table I).
To test whether or not the heparan sulfate chains in perlecan domain I are involved in the interaction with fibrillin-1, an established mutant perlecan domain I (Mut-I) was employed, which does not contain heparan sulfate chains (55) (Fig. 5C). This mutant domain I did not interact with rF16 or with rF6H, indicating that the heparan sulfate chains of perlecan domain I mediate the low affinity interaction with fibrillin-1.
To further narrow down perlecan interaction epitopes in fibrillin-1, full-length tissue perlecan (Fig. 6A) or recombinant perlecan domains I and II (Fig. 6B) were analyzed for binding to subfragments of rF16 (rF1F, rF18, rF51, see Fig. 5A) by solid phase binding assays. These experiments demonstrate that most of the binding property for full-length perlecan is con- tained within fragment rF18 representing the longest stretch of tandemly repeated cbEGF modules in the center of the fibrillin-1 molecule. Perlecan domains I and II both contributed to the interaction with rF18. In addition, fibrillin-1 fragment rF1F (N terminus to 8-Cys/TB domain 2) also contributed somewhat to binding of perlecan domain II.
Microfibrils Are Reduced in Perlecan Null Mice-In order to study the functional relevance of the perlecan interaction with fibrillin-1 and microfibrils, we have analyzed microfibrils at the dermal-epidermal junction in homozygous perlecan null and wild-type mouse embryos (Fig. 7). In wild-type mice (n ϭ 3) at E13.5, immunogold-labeled microfibrils with anti-fibrillin-1 antibodies (␣-rF6H) were present in the dermis close to the epithelial basement membrane as well as in deeper areas of the dermis (Fig. 7A). Analysis of perlecan null embryos (n ϭ 3) at E13.5 clearly revealed reduced amounts of immunostainable microfibrils in the vicinity of the epithelial basement membrane at the dermal-epidermal junction (Fig. 7B). The same tendency was observed at E17.5 but was less pronounced (data not shown). Homozygous perlecan null mice die either between E10 and E12 or perinatally, and thus it is not possible to analyze postnatal stages (39,40). These data suggest that perlecan plays a role either in the biogenesis of microfibrils close to basement membrane zones or, alternatively, in anchoring microfibrils to basement membranes. DISCUSSION Over the last years, it became evident that fibrillin-containing microfibrils are supramolecular and multifunctional aggregates in extracellular matrices. In a recent review (15), at least 15 different components associated with microfibrils have been summarized. It is very likely that the composition of microfibrils in various tissues does not always involve all of these components rather than a certain subset, which is specific for the functional and mechanical requirements of a given tissue. On the other hand, microfibrillopathies, resulting from genetic mutations in microfibrillar components such as fibrillin-1 (Marfan syndrome) or fibrillin-2 (congenital contractural arachnodactyly), are characterized by a wide intra-and interfamilial variability. In this regard, microfibril-associated components are certainly good candidates that may play modifying roles in the pathogenesis of these disorders. To understand the full functional spectrum of microfibrils and the pathogenetic   pathways of microfibrillopathies, information about components, which interact and associate with microfibrils, is urgently needed.
In an attempt to identify proteoglycan ligands of microfibrils, we found that perlecan interacts with microfibrils and fibrillin-1 as observed on several experimental levels. Double immunofluorescence labeling of the extracellular matrix produced by primary dermal fibroblasts showed significant colocalization of heparan sulfate and perlecan with the fibrillin-1 network representing probably an early stage of microfibrillar aggregates (72). These data are in agreement with observations by others describing the presence of perlecan (synonym: heparan sulfate proteoglycan) or the 10E4 epitope (representing heparan sulfate) on extracellular fibers produced by fibroblasts (73)(74)(75). Singer et al. (75) reported that the heparan sulfate proteoglycan colocalized to fibronectin-containing fibers produced by fibroblasts after short culture periods of up to 24 h. In the early stages of fibroblast cultures, fibrillin-1 also colocalizes with fibronectin fibers, 2 but fibrillin-1 cannot directly interact with fibronectin (18). In the light of these data, perlecan may function to connect fibrillin-1 to fibronectin fibers in the early stages of microfibril biogenesis.
On the tissue level, fibrillin-1 and perlecan have been found by double immunofluorescence and immunogold labeling to typically, but not exclusively, colocalize in close vicinity of various dermal and ocular basement membrane zones. These tissues included the epithelial basement membrane in skin, the endothelial basement membrane of retinal veins, basement membranes of the ciliary body epithelium, the lenticular base-ment membrane, or the stroma of the ciliary body. These are tissue locations, where both individual components, fibrillin-1 and perlecan, are typically expressed (38,76,77). For ciliary zonules, labeling with perlecan-specific antibodies is in agreement with an earlier study by Inoue (78) using an immunoperoxidase staining method. Most of the microfibrils that showed positive immunogold labeling for both fibrillin-1 and perlecan intersected with the respective basement membrane. It is of interest that perlecan null mice demonstrated fewer microfibrils at the dermal epithelial basement membrane as compared with their wild-type littermates, suggesting a potential role of perlecan in the biogenesis of microfibrils during development in the vicinity of basement membranes. Perhaps perlecan acts as a nucleation molecule for the biogenesis of microfibrils. Previously, we and others (31,36) demonstrated that heparin/ heparan sulfate is able to inhibit the formation of fibrillin networks in cell culture systems. It is conceivable that soluble heparin/heparan sulfate used in these studies competitively withdraws fibrillin from important interaction epitopes with proteoglycan heparan sulfate chains that are potentially necessary for microfibril assembly. In this study, we have found 2 S. Dallas, personal communication. Transmission electron microscopy of immunogold-labeled (anti-fibrillin-1) microfibrils within the skin of wild-type and homozygous perlecan null mice. A, in wild-type animals, immunogold-labeled microfibrils are readily discernible (arrows) either in direct contact or adjacent to the dermal-epidermal basement membrane (BM) where they often form a dense network. Occasionally, microfibrils are entangled with collagen fibers (arrowheads). B, in contrast, homozygous perlecan null mice are characterized by a considerable reduction of immunogold-labeled microfibrils (arrows) both at the level of the basement membrane (BM) and within the adjacent dermis. In both micrographs, the undulated dermal-epidermal junction zone was cut in part tangentially, so that the basement membranes vary in width and contact the dermis from both sides. Bars represent 150 nm in each figure.
that fibrillin-1 interacts with the heparan sulfate chains of perlecan domain I. This individual interaction is very likely of relatively low affinity (see below) because it can be significantly inhibited at physiological salt concentrations of 150 mM as compared with sub-physiological salt levels of 50 mM. A low affinity interaction between fibrillin-1 and heparan sulfate chains, however, could be suitable for a "nucleation" mechanism of microfibrils, because such interactions may be of transient nature.
Although it is a general principle that microfibrils intersect with basement membranes in various tissues (2)(3)(4)(5)(6)(7)(8), virtually nothing is known about how microfibrils are physically connected to these basement membranes. In this respect, the functional significance of the perlecan presence on microfibrils may also include mechanical anchoring of microfibrils to basement membranes or to structures in close vicinity of basement membranes. By studying binding kinetics in real time, we have identified interaction of the N-terminal half of recombinant fibrillin-1 and authentic tissue perlecan with dissociation constants in the low nanomolar range. This high affinity interaction between fibrillin-1 as the major microfibrillar component and perlecan as a ubiquitously expressed basement membrane protein likely provides a solid basis for anchoring microfibrils to basement membranes. In addition, fibulin-2 may further stabilize these types of connections, because it is able to interact with both fibrillin-1 (18) and perlecan (65,66). Fibrillin-1 has also been described in the lamina densa of the dermalepidermal junction in the absence of microfibrillar structures (79), and thus, the interaction between fibrillin-1 and perlecan may also be relevant for stabilizing basement membranes.
It has been found by solid phase binding assays using recombinant fragments of fibrillin-1 and perlecan that the interaction epitopes are primarily located in the central region of fibrillin-1 represented by fragment rF18 (8-Cys/TB domain 3-cbEGF 21) and in perlecan domains I and II. Although it is not possible to calculate binding affinities based on such assays (80), rough estimates suggest lower affinities as compared with affinities observed by Biacore using full-length tissue perlecan. These observations suggest that individual interaction epitopes located in the adjacent perlecan domains I and II contribute to a cooperative binding mechanism with the central region of fibrillin-1, resulting in an overall high affinity interaction between full-length fibrillin-1 and perlecan. Most interestingly, a nearly identical region of fibrillin-1 (cbEGF [11][12][13][14][15][16][17][18][19][20][21] was described to interact with another proteoglycan, versican, an interaction that is thought to mediate anchoring of microfibrils to hyaluronan-rich matrices (29). Mutations in this central region of fibrillin-1 can lead to the very severe neonatal form of Marfan syndrome, which is often lethal within the first year of life. It remains to be elucidated whether or not fibrillin-1 mutations in this region lead to a destabilization of the fibrillin-1-perlecan and/or the fibrillin-1-versican interactions and whether these interactions modulate the pathogenesis of Marfan syndrome and perhaps other microfibrillopathies.