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Madame Curie Bioscience Database [Internet]. Austin (TX): Landes Bioscience; 2000-.

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Microfibril-Associated Glycoprotein-1 (MAGP-1) and Other Non-fibrillin Macromolecules Which May Possess a Functional Association with the 10 nm Microfibrils

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Introduction

There is growing evidence that fibrillin-containing microfibrils are not just fibrillin polymers but that a variety of additional macromolecules may be associated with these structures. The functions of these molecules may be envisioned to include a) structural support to stabilize the interaction of fibrillin molecules within the microfibril; b) mediation of the interaction of adjacent microfibrils within bundles; c) assembly of elastin on the surface of the microfibrils; d) interfacing between the microfibrils and other structural elements of different matrices; e) modulation of the interaction of the microfibrils with cells to influence the deposition, orientation and organization of microfibrils and elastic fibers in different tissue environments; f ) provision and modulation of nonstructural functions of the microfibrils e.g., TGF-beta storage; g) enzymatic activity e.g., lysyl oxidase; and h) specific interactions with fibrillin-2-containing microfibrils. In recent years many candidate microfibril-associated macromolecules have been identified. Some of these molecules appear to be found in extracellular matrices associated exclusively with the fibrillin-containing microfibrils indicating that their function(s) are likely to be specific to some aspect of microfibril biology. Other candidates clearly have association with additional structural matrix components and thus they must possess some microfibril-independent functions. The best-characterized matrix macromolecule which is exclusively associated with fibrillin-containing microfibrils is Microfibril-Associated Glycoprotein-1, or MAGP-1, a small glycoprotein with an apparent molecular weight of 31 kDa.1,2 MAGP-1 was first identified as a component of the elastic-fiber rich tissue, developing bovine nuchal ligament. MAGP-1 was shown to be resistant to chaotropic extraction from tissue homogenates, unless a reducing agent was included in the extraction buffer. Such reductive treatment had been shown to selectively solubilize the microfibrillar component of developing elastic fibers, later to become known as fibrillin-containing microfibrils.1 MAGP-1 is the strongest candidate for a ubiquitous component of fibrillin-1-containing microfibrils since it extensively and specifically codistributes with these structures in tissues,36 and is localized periodically along these microfibrils in association with the bead regions.7 A structurally related molecule, MAGP-2 (apparent molecular weight 25 kDa) was later identified in fibrillin-1 enriched extracts of the same tissue along with MAGP-1 and two polypeptides of 78 and 70 kDa.8,9 MAGP-2 was also found to be exclusively associated with the microfibrils but it exhibited a more restricted distribution than MAGP-1.5 The 78 kDa and 70 kDa polypeptides, later referred to as MP78 and MP70, were identified as forms of βig-h39, a protein which appears to be associated with type VI collagen microfibrils rather than the fibrillin-containing structures,10 and thus these two polypeptides are not discussed further in this chapter.

The ultrastructural distribution, tissue expression, structure and function of MAGPs are discussed in more detail below. A number of other microfibril-associated components have been identified including MFAPs,1114 several proteoglycans,1519 fibulins,20,21 LTBPs2228 and EMILINs.29,30 Our knowledge of these macromolecules in relation to the fibrillin-containing microfibrils and to genetic disorders is also outlined below.

MAGPs

Ultrastuctural Localization and Developmental Tissue Expression Patterns of MAGPs and Their Relationships to Fibrillin-Containing Microfibrils

MAGP-1 has been shown, using immunofluorescence and immunoelectron microscopic techniques, with polyclonal and later monoclonal antibodies, to be specifically localized on fibrillin-containing microfibrils in a wide range of developing and mature elastic and nonelastic tissues. In all tissues examined the localization of MAGP-1 matched that of fibrillin-1.3,4,5 Interestingly, only one elastic tissue was identified which did not stain extensively with anti-MAGP-1 antibodies, the elastic ear cartilage, which is considered to contain predominantly fibrillin-2 containing microfibrils.5,31 The evidence suggests that MAGP-1 is specifically associated with fibrillin-1-containing microfibrils in most if not all situations where they occur.

Using pre-embedding labeling techniques, monoclonal anti-MAGP-1 antibodies were found to localize to fibrillin-containing microfibrils in ocular zonule and vitreous tissues in a cross-banding pattern at intervals of about 50 nm, the periodicity of the beaded filament structure of the microfibrils. Rotary shadowing of isolated microfibrils which had been immunogold-labeled with the anti-MAGP-1 antibody showed specific, apparently symmetrical, localization to the beads rather than the interbead regions (Fig. 1). Occasionally two gold particles were observed to be attached to opposite sides of the same bead, suggesting that multiple MAGP-1 molecules were present in the structure. The study concluded that MAGP-1 is intimately and regularly associated with the bead regions of fibrillin-containing microfibrils and that the findings are consistent with a major role for MAGP-1 in microfibril biology.7

Figure 1. Rotary shadowing and immunolabeling of isolated zonular microfibrils for MAGP-1.

Figure 1

Rotary shadowing and immunolabeling of isolated zonular microfibrils for MAGP-1. (A-C) Isolated microfibrils were reacted with anti-MAGP-1 antibodies, followed by 10-nm immunogold particles (visualized as circles with black centers). Small arrows denote (more...)

MAGP-2 also shows specific ultrastructural localization to fibrillin-containing microfibrils in a variety of developing tissues.5 However, MAGP-2 has a more limited tissue pattern of expression than MAGP-1 and fibrillin-1 in late-fetal and adult bovine tissues. For instance, in late-trimester nuchal ligament, skeletal muscle and spleen the distribution of MAGP-2 appears to be indistinguishable from that of MAGP-1, but MAGP-2 is not associated with microfibrils of the medial layer of thoracic aorta or peritubular matrix of the kidney. MAGP-2 also appears not to be associated with the microfibrils of the ocular zonule. These observations are consistent with analysis of steady-state mRNA levels in developing bovine tissues. MAGP-2 expression appears to be higher in nuchal ligament, heart and skeletal muscle, but lower in aorta and kidney than that of MAGP-1.5 In nuchal ligament, MAGP-2 expression appears to peak at around 180 days of fetal development, which correlates with the period of onset of elastinogenesis and maximum expression of fibrillins in this tissue.5,32 In contrast, MAGP-1 expression remains consistently high throughout nuchal ligament development.

MAGP-2 was also shown to have a periodic association with microfibrils in pre-embedding labeling experiments similar to those described above for MAGP-17 but using nuchal ligament in place of zonule which lacks MAGP-2. MAGP-2 was found to copurify on CsCl density gradients with the fibrillin-containing microfibrils even in the presence of the strong chaotrope, 6 M guanidinium chloride indicating that the MAGP-2 is strongly associated with these structures. Dual immunolabeling of the isolated microfibrils indicated that individual microfibrils contain both MAGPs. In addition MAGP-2 appeared to be attached at two locations within each period, on the bead and at a specific point between the beads (Hanssen E and Gibson MA, unpublished observations). These findings suggest that in MAGP-2- rich tissues, the glycoprotein is an integral structural component of microfibrils. The ultrastructural and tissue expression data are consistent with a specialized role for MAGP-2 in the biology of the microfibrils, modulating their function in certain tissue-specific matrices during development and differentiation.

The Structure of MAGPs

MAGP-1 and MAGP-2 share a characteristic region of structural similarity and are the only members of this MAGP family to be identified so far. The structural features of MAGP-1 and MAGP-2 are shown in Figure 2. MAGP-1 is synthesized as a 21 kDa polypeptide of 183 amino acids from a 1.1 kb mRNA. The first 17 amino acids comprise the secretion signal peptide which is cleaved from the molecule during intracellular processing.2,33 Sequence analysis of the MAGP-1 cDNAs has shown that MAGP-1 consists of two structurally dissimilar regions, an amino-terminal segment containing high levels of glutamine, proline and acidic amino acids and a carboxyl-terminal segment containing all 13 of the cysteine residues involved in intra- or inter- molecular disulfide bonding.2 The amino acid sequence is highly conserved between the human, bovine and murine forms of the protein.2,34,35 Computer analysis has suggested that MAGP-1 consists of three domains, a small folded amino-terminal domain connected by an extended proline- and glutamine-rich domain to a globular, cysteine-rich, carboxyl-terminal domain.33 The extended domain appears to be responsible for the anomalous migration of MAGP-1 on polyacrylamide electrophoresis where it exhibits an apparent molecular weight of 31 kDa.1,2 Post-translational modifications exhibited by MAGP-1 include O-linked glycosylation and tyrosine sulfation, and the molecule also contains a potential amine acceptor site for transglutaminase cross-linking.1,2,36 These modifications occur within the amino-terminal region of the molecule which also contains a polyglutamine motif possibly involved in self-aggregation of MAGP-137 and a putative binding region for tropoelastin and type VI collagen.38 Since reduction of disulfide bonding is necessary to solubilize MAGP-1 from microfibrils it is likely that the microfibril-binding sequence is present in the cysteine-containing carboxyl-terminal half of the molecule.2 Recent data suggests that the region containing the first 7 cysteine residues of MAGP-1 is important for its incorporation into extracellular matrix.39

Figure 2. Structural comparison of MAGP-1 and MAGP-2.

Figure 2

Structural comparison of MAGP-1 and MAGP-2. The structures of MAGP-1 and MAGP-2 are aligned schematically to highlight the region of sequence similarity between the proteins, and to align structural features with the encoding exons in MAGP-1 and MAGP-2 (more...)

MAGP-2 is slightly smaller than MAGP-1, consisting of 173, 170 and 164 amino acids in the human, bovine and murine forms, respectively, including a signal peptide of 18 amino acids. Homology between each of the three species is around 75% and in each case the mature MAGP-2 polypeptide is predicted to be around 17 kDa in size.9,40 MAGP-2 has little similarity with other known proteins with the exception of MAGP-1. Like MAGP-1, MAGP-2 is a highly hydrophilic protein containing two distinct regions, an acidic cysteine-free amino terminal half and a basic cysteine-rich carboxyl-terminal half. However, there are many differences from MAGP-1. The amino-terminal region of MAGP-2 is rich in serine and threonine residues, and contains a RGD integrin binding motif and a consensus sequence for N-glycosylation.9 MAGP-2 lacks the proline, glutamine and tyrosine-rich sequences found in MAGP-1 including the putative elastin / collagen VI-binding region and the motifs for tyrosine sulfation and transglutamination.36,38 The carboxyl-terminal region of MAGP-2 contains 8 cysteines in contrast to the 13 found in MAGP-1, and MAGP-2 lacks a hydrophobic region at the extreme carboxyl-terminus. Close sequence similarity to MAGP-1 is confined to a central 60 amino acid region of MAGP-2 where there is precise alignment between the first seven cysteines of MAGP-2 and seven of the first eight cysteines of MAGP-1, with C3 the unaligned cysteine in MAGP-1.9 The spacing between the aligning cysteines (CX6CX12CX4CX14CX4CX10C) is conserved throughout the three species examined to date.2,34,35

The regions of similarity and dissimilarity in the two proteins are reflected in the exon structures of the MAGP-1 and MAGP-2 genes. The human and murine MAGP-1 and MAGP-2 genes exhibit exact size, sequence and junction alignment of the two penultimate exons (7 and 8 in MAGP-1 and 8 and 9 in MAGP-2) which encode the first six of the seven aligned cysteine residues. The seventh cysteine is encoded close to the 5' end of the final exon in both genes. Few other similarities are evident in the exon and promoter structures of the MAGP-1 and MAGP-2 genes.40,41 The structural differences between MAGP-1 and MAGP-2 proteins and their genes are consistent with the concept that the two proteins are structurally and functionally diverse proteins with distinct patterns of tissue and developmental expression. However, MAGP-1 and MAGP-2 share one characteristic cysteine-rich motif which is likely to confer important functionalities common to both proteins. This region is a strong candidate to contain the microfibril-binding sequence in both proteins.9,39,42

Function of MAGPs

MAGP-1

The functions of MAGP-1 remain unclear although several roles have been proposed for MAGP-1 in the biology of fibrillin-containing microfibrils. Possible roles include an integral structural component, an elastin-binding protein on the surface of the microfibrils and a link molecule mediating the interaction of the microfibrils with structural elements of the surrounding matrix. The evidence for each possible function is summarized below.

The widespread tissue codistribution of MAGP-1 with fibrillin-1-containing microfibrils and its periodic localization to the beads along the microfibrils suggest that the protein may be an integral structural element.4,7 This is supported by evidence that MAGP-1 is covalently attached to the microfibrils by disulfide bonding and that solubilization of the glycoprotein requires disruption of these structures with strong reducing agents.1,8 It has been suggested that MAGP-1 may stabilize by disulfide bonding the head to tail interaction of overlapping fibrillin-1 molecules within the “bead” regions of the microfibrils.7 MAGP-1 has been shown to interact with fibrillin in coimmunoprecipitation experiments. However, only one MAGP-1 binding site has so far been identified on fibrillin-1, close to the amino-terminus of the molecule.43 In the absence of evidence for a carboxyl-terminal MAGP-1 binding site on fibrillin-1, it would seem unlikely that MAGP-1 fulfills the stabilization role as proposed by Henderson et al.7 However, it remains possible that MAGP-1 may be involved in the stabilization of lateral interactions of fibrillin-1 molecules which appear to form parallel nonoverlapping dimers as an early step in microfibril assembly.4345

MAGP-1 may be involved in the lateral interactions between adjacent microfibrils. The cross-banding pattern obtained when tissues are pre-embedding labeled with anti-MAGP-1 monoclonal antibodies indicates that MAGP-1 is present on the surface of each microfibril and that there is lateral alignment of the “bead” regions of individual microfibrils within each bundle of microfibrils.7 Since MAGP-1 readily self-aggregates,36 it is possible that MAGP-1 forms bridges between the beads of adjacent microfibrils.7 MAGP-1 is a substrate for tissue transglutaminase and transglutamine cross-links may contribute to any structural role for MAGP-1, although evidence for such cross-links involving MAGP-1 is yet to be obtained.36,37

MAGP-1 has also been shown to bind to the elastin precursor tropoelastin in vitro36,38,43 and it has been proposed that MAGP-1 may mediate the binding and alignment of tropoelastin onto a microfibril template during elastinogenesis.2 Further evidence for a role for MAGP-1 in elastinogenesis has come from cell culture experiments.46 In particular, an antibody raised to the amino-terminal region of MAGP-1 (amino acids 21–35), when added to the culture medium, was found to prevent elastic ear cartilage cells from organizing newly-synthesized tropoelastin into elastic fibers. The distribution of MAGP-1 in the matrix appeared to be normal when detected by an antibody raised to a different region of the molecule. These findings led the authors to suggest that the interaction between tropoelastin and fibrillin-containing microfibrils may be mediated by a domain involving the N-terminal half of the MAGP-1.46 In another study a peptide corresponding to amino acids 29–38 of MAGP-1was found specifically to inhibit the interaction of MAGP-1 and tropoelastin in an in vitro binding assay. This finding indicated that the sequence, close to the N-terminus of MAGP-1, contains a major elastin-binding site.38 The sequence(s) on the tropoelastin molecule important for binding MAGP-1 is less well characterized. Additional antibody inhibition studies have shown that the cysteine-containing, carboxyl terminal sequence of tropoelastin is important for incorporation into the extracellular matrix and binding to MAGP-1.46 However, recent in vitro interaction studies, using a comprehensive range of tropoelastin fragments, failed to identify regions important for interaction with MAGP-1. This result suggests that the conformation of the intact tropoelastin molecule may be important for the binding of MAGP-1.43 It should also be noted that tropoelastin can bind directly to fibrillin-1 suggesting that elastin deposition onto the microfibrils may occur independently from MAGP-1.47

MAGP-1 has been demonstrated to interact with the small dermatan sulfate proteoglycans, decorin16 and biglycan17 (see below), and with collagen VI via the pepsin-resistant domain of the alpha 3(VI) chain.38 These findings support the concept that MAGP-1 plays a role in mediating the interaction of the microfibrils with constituents of the surrounding matrix. Interestingly, inhibition studies have shown that the binding site for collagen VI on MAGP-1 appears to be very close to that for tropoelastin, near the N-terminus of the MAGP-1 molecule. It has been suggested that the interaction of MAGP-1 with collagen VI may be important for the anchorage of the elastin-associated, fibrillin-containing microfibrils to the surrounding matrix during elastic fiber stretching in tissues such as ligament, lung and aorta. It is also possible that the interaction provides an indirect structural link between elastic fibers and collagen fibers via collagen VI microfibrils.38

MAGP-2

As with MAGP-1, the functions of MAGP-2 remain to be determined. Major differences in the composition of the amino- and carboxyl- terminal regions of the two MAGPs, and the more restricted expression patterns of MAGP-2 indicate that its functions are likely to be different from those of MAGP-1.5,9 An early suggestion that MAGP-2 may be exclusively associated with fibrillin-2 containing microfibrils5, based on some correlation of expression patterns in tissues such as kidney, lung and zonule appears to be unfounded. MAGP-2 was later shown to be absent from several fibrillin-2-rich tissues including ear cartilage and the medial layer of elastic blood vessels.5,31 More recently yeast two hybrid studies have shown that MAGP-2 interacts with both fibrillin-1 and fibrillin-2. Interestingly, the binding site on each fibrillin is located within the final 7 EGF-like repeats in the carboxyl-terminal region which is at the opposite end of the molecule to the MAGP-1 binding site on fibrillin-1. The reciprocal binding site on MAGP-2 was identified within the central cysteine-rich region conserved between MAGP-1 and MAGP-2. The authors suggested that the interactions may be important for assembly of both fibrillin-1 and fibrillin-2 containing microfibrils.42 In nuchal ligament development, MAGP-2 expression does mimic the expression patterns for fibrillins -1 and -2, supporting a possible role for MAGP-2 in microfibril assembly and elastinogenesis in this tissue.5,32 However, the absence of MAGP-2 from microfibrils in several tissues has been documented, indicating that a ubiquitous structural role for MAGP-2 in the microfibrils is unlikely.

The amino acid sequence of MAGP-2 provides a clue to a possible function of the molecule. MAGP-2 lacks the putative tropoelastin and collagen VI-binding region of MAGP-1.38 However, the corresponding region of MAGP-2 contains an active RGD integrin recognition sequence. This sequence has been shown to mediate the attachment and spreading of a range of cell types on MAGP-2 substrate via specific interaction with alpha V beta 3 integrin.48 Thus MAGP-2 may modulate microfibril-cell interactions at specific stages during development and differentiation in particular tissue environments.48

Further insight into the functions of MAGP-1 and MAGP-2 should be provided when the results of gene knockout experiments in mice are published.

Other Small Microfibril-Associated Proteins (MFAPS)

In addition to MAGP-1 and MAGP-2, several other small proteins have been identified as potential components of fibrillin-containing microfibrils. As the human form of each protein was cloned it was given the name MFAP and the series currently includes four members, one of which, MFAP2, is MAGP-1. It should be noted that there are no structural similarities between any of the four proteins. Since the nomenclature can be confusing the data has been presented in table form (Table 1).

Table 1. Characteristics of small candidate microfibril-associated proteins.

Table 1

Characteristics of small candidate microfibril-associated proteins.

MFAP1 was originally identified as ‘associated microfibril protein’ or AMP, following expression screening of a whole chick embryo cDNA library with antiserum raised to the microfibril-rich bovine ocular zonule.11 Antiserum raised to a synthetic MFAP1 peptide localized specifically to fibrillin-containing microfibrils in several tissues, including the zonule fibers. MFAP1 was characterized as a 54 kDa protein which is processed to a 32 kDa protein, that had previously been identified in zonular extracts.49 The human gene for MFAP1 has been characterized and mapped, close to the fibrillin-1 gene locus on chromosome 15.12,50

MFAP-3 is a 41 kDa serine-rich protein also identified by cDNA library screening with a polyclonal antibody to zonular fibers.13 Antibodies raised to recombinant MFAP3 were found to localize to zonular microfibrils and identify the 41 kDa protein in extracts of developing nuchal ligament. Northern blotting indicated that the protein was also expressed in fetal aorta and lung. Genomic analysis revealed that the MAFP3 gene contained only two exons and mapped to chromosome 5q32-q33.2.

MFAP4 was identified from a novel cDNA mapping to human chromosome 17p11.2.14 The clone encoded a 29 kDa protein which was named MFAP4 due to its high level of sequence identity with a partially sequenced 36 kDa protein extracted from porcine and bovine aortas. The 36 kDa protein showed ultrastructural localization to fibrillin-containing microfibrils surrounding elastic fibers in aorta, skin and spleen. Interestingly, the protein appeared not to be associated with the elastin-free microfibrils in ocular zonule and kidney.5153 It has been suggested that the 36 kDa protein plays a role in elastinogenesis and it has been named MAGP-36.53 MFAP-4 also shares some sequence similarity with a 40 kDa protein identified using IgG from the aortic wall of patients with abdominal aortic aneurysms. The protein has been named aortic aneurysm associated protein (AAAP-40) and MAGP-3 due to its similarity to MAGP-36.54,55 It should be noted that MFAP4, MAGP-36 and AAAP-40 have no structural resemblance to the MAGP family of proteins (MAGP-1 and MAGP-2) described in the previous section. The functions of MFAP1, MFAP2 and MFAP4 in the biology of fibrillin-containing microfibrils remain to be elucidated.

Proteoglycans

A number of ultrastructural and histochemical studies have identified proteoglycans in close association with developing elastic fibers in tissues such as skin and aorta,5661 and with ocular zonule microfibrils.62,63 In addition, immunohistochemical studies on human skin have suggested that two small dermatan sulfate proteoglycans, decorin and biglycan, are closely associated with elastic fibers, decorin with the fibrillin-containing microfibrils and biglycan with the elastin component.60,61 More recent studies have supported these observations. Kielty et al15 demonstrated that chondroitinase AC treatment disrupts the bead component and increases the inter-bead periodicity of fibrillin-containing microfibrils. In addition they showed that a small chondroitinase AC sensitive proteoglycan could be coprecipitated with fibrillin from smooth muscle cell medium.15 The proteoglycan could be from either the chondroitin sulfate or dermatan sulfate family since chondroitinase AC specifically cleaves glycosaminoglycans at D-glucuronate residues which occur in the side-chains of chondroitin sulfate and also dermatan sulfate proteoglycans containing copolymeric side-chains.64 The authors concluded that a small chondroitin sulfate proteoglycan associates with fibrillin and contributes to microfibril assembly, and suggested that proteoglycan that could be decorin, biglycan or fibromodulin.

In other studies decorin has been shown to form a ternary complex with fibrillin-1 and MAGP-1 in chrondrocyte culture medium. Further experiments with recombinant fibrillin fragments indicated that the decorin binding site on fibrillin-1 was located in an amino-terminal region containing the proline-rich domain and the following five EGF-like repeats.16 This region of fibrillin-1 is considered to be in or close to the ‘beads’ within the microfibril and thus decorin may be the chondroitinase AC sensitive proteoglycan identified by Kielty et al15 to be associated with these structures. The corresponding fragment of fibrillin-2 did not bind decorin suggesting that the binding was specific for fibrillin-1. Interestingly, decorin and MAGP-1 were also found to coimmunoprecipitate from chondrocyte cultures which suggested that there was also a direct interaction between these two macromolecules. The authors speculated that decorin may be involved in regulating assembly of microfibrils or in cocoordinating individual microfibrils into bundles.16 Decorin has also been shown to bind tropoelastin in vitro suggesting that the proteoglycan may be involved in elastic fiber assembly.17

Biglycan may also be involved in microfibril biology. In studies on glycosaminoglycan and proteoglycan content of developing nuchal ligament, expression of a specific glycoform of biglycan was shown to correlate with the elastinogenic phase of elastic fiber formation in this tissue. In contrast, decorin expression was shown to peak early and was relatively low during the periods of maximum microfibril and elastin deposition.65 Subsequent in vitro binding studies showed that biglycan binds via its core protein to tropoelastin and to MAGP-1 and that the three macromolecules can form a ternary complex. Decorin was also found to bind to tropoelastin but less strongly than biglycan. However, in contrast to biglycan, decorin showed no binding affinity for MAGP-1 in both solid phase and immunoprecipitation assays.17 The authors suggested that the differential binding to tropoelastin and MAGP-1 points to decorin and biglycan possessing distinct functions in elastic fiber biology. They also suggested that biglycan may be involved with MAGP-1 in the deposition of tropoelastin onto the surface of the microfibrils during elastinogenesis or in the stabilization of the mature elastic fiber perhaps by mediating interactions with elements of the surrounding matrix.

Although evidence is mounting that decorin and biglycan are involved in microfibril and elastic fiber biology, it is interesting that decorin and biglycan null mice show no obvious elastic fiber abnormalities.66,67 These findings suggests that, in the absence of the other proteoglycan, decorin and biglycan may be able to substitute for each other in their elastic fiber-related functions.

While roles for decorin and biglycan remain uncertain, recent evidence has underlined the importance of proteoglycans for microfibril and elastic fiber assembly. Independent cell culture studies have clearly demonstrated that disruption of proteoglycan synthesis, by blocking sulfation or GAG side-chain assembly, prevents incorporation of fibrillin-1 into the extracellular matrix. 16,18 Other proteoglycan candidates for involvement in microfibril function include, the large chondroitin sulfate proteoglycan, versican,19 and an as yet unidentified heparan sulfate proteoglycan.18 A combined ultrastructural, immunolocalization and molecular binding study has shown that versican binds, apparently covalently, to microfibrils and to fibrillin-1 via its C-terminal domain. The binding site on fibrillin-1 is located centrally between calcium binding EGF-like domains 11 and 21, the region involved in the severe 'neo-natal' form of Marfan syndrome. Immunolabeling along the microfibrils was found to be relatively sparse and to lack a discernible periodicity. This finding suggested to the authors that versican does not serve an integral structural function but connects microfibrils to hyaluronan in the surrounding matrix. 19 In other recent studies it has been reported that over-expression of a variant of versican in cultured arterial smooth muscle cells significantly increased tropoelastin expression and formation of elastic fibers in the extracellular matrix. Moreover, the seeding of these transfected cells into rat carotid arteries, damaged by balloon catheter, induced the formation of a compact neointima which was rich in elastic lamellae. These results clearly implicate versican in elastic fiber assembly.68

Tiedemann et al18 have shown that heparin binds to fibrillin-1 at three sites along the molecule and that heparin and heparan sulfate inhibit, when added to the medium, the assembly of fibrillin-1 into the matrix of cultured skin fibroblasts. The authors suggest that the binding of proteoglycan-associated heparan sulfate chains to fibrillin-1 is an important step in microfibril assembly. The proteoglycan (s) involved remain to be identified. Evidently, research into proteoglycan involvement in the function of fibrillin-containing microfibrils is entering an exciting phase.

Fibulins

Recent evidence indicates that fibulins play an important role in elastic fiber biology. The fibulin family consists of five distinct rod-like proteins containing multiple EGF-like repeat motifs and a globular C-terminal domain exhibiting some sequence similarity with the C-terminus of fibrillins.6971 Fibulins have extensive but distinct tissue distributions. Fibulin-1 (100 kDa) is found in plasma and the extracellular matrix of a wide variety of tissues where it is associated with structures such as the elastin core of elastic fibers and several basement membranes. 20,72 Fibulin-2 (195 kDa) has been shown to be colocalized with fibrillin-1 in some but not all tissues.21 Ultrastructurally, fibulin-2 was found to be located at the junction between the microfibrils and the amorphous elastin core in elastic fibers of the skin.21 Fibulins 1 and 2 have been shown to bind an extensive range of matrix macromolecules including basement membrane and elastic fiber components. In particular, both fibulin-1 and 2 bind tropoelastin73 and fibulin-2, but not fibulin-1, binds to the N-terminal region of fibrillin-1.21 It has been proposed that fibulin-2 in particular may have a role in mediating the attachment of the microfibrils to elastin73 and to basement membranes.21

More recently, targeted disruption of the fibulin-5 gene in mice has demonstrated that this fibulin, at least, is profoundly involved in elastic fiber development. The phenotype exhibits severe disorganization of the elastic fiber systems throughout the body leading to the development of marked elastinopathies including cutis laxa, emphysema and tortuosity of the aorta.74,75 Fibulin-5 has been shown to interact with tropoelastin and several integrins suggesting it may have a critical role in the anchorage of elastic fibers to cells during elastic fiber development. Interestingly, fibulin 5 did not bind fibrillin-1 in in vitro binding experiments75 and its relationship with fibrillin-containing microfibrils remains to be elucidated. Overall the evidence suggests that several fibulins may be important binding partners for elastic fiber components rather than functioning as structural components of the microfibrils themselves.

LTBPs

Latent transforming growth factor-beta binding proteins (LTBPs) are a family of four proteins which share structural characteristics with fibrillins in that they are rod-like extracellular matrix molecules consisting predominantly of tandem EGF-like 6-cysteine repeats interspersed with 8-cysteine motifs. Since the 8-cysteine motifs have only been found in fibrillins and LTBPs, the two groups of proteins are now considered to comprise a superfamily.7678 A major functional characteristic of LTBPs is considered to be the intracellular covalent binding of latent forms of TGF-beta and the facilitation of subsequent secretion and targeting of the growth factor to sites in the extracellular matrix. Latent TGF beta has been shown to bind covalently to LTBPs 1, 3 and 4 via a site in the third 8-cysteine motif of each protein. However, LTBP-2, like fibrillins, does not appear to bind to TGF-beta.79 In addition, LTBP-1 and LTBP-2 can be secreted and deposited into extracellular matrix without attached TGF-beta.22,80,81 Thus the evidence indicates that LTBPs may have additional functions as structural components of the matrix.

Ultrastructural immunolocalization studies have identified fibrillin-containing microfibrils as the location of matrix-associated LTBPs in a range of tissues. LTBP-1 has been immunolocalized to microfibrils in skin,24,25 bone26 and kidney.27 In a very recent immunohistochemical study of human tissues, LTBP-1 was found to codistribute extensively with fibrillin-1 in tendon, perichondrium and blood vessels but to be absent or scarce in skeletal muscle and lung.28 LTBP-2 has been located on microfibrils in elastic nuchal ligament and aorta.22 In another study, both LTBP-1 and LTBP-2 showed partial coimmunolocalization with fibrillin-1 in the wall of coronary arteries. In addition, the intensity of staining increased following angioplasty-induced injury to the arteries.82

It is still unclear if LTBPs are integral structural components of the microfibrils or are associated with the surface of these structures. Both LTBP-1 and LTBP-2 have been demonstrated to bind strongly to the microfibrils22,23,80 and LTBP-1 has been found to contain three potential matrix binding domains.81 Very recently, LTBP-1 has been shown to bind to the N-terminal regions of fibrillins 1 and 2 via its carboxyl-terminal region.28 LTBP-4 also appears to bind fibrillins in a similar manner. However, LTBP-1 was not detected in association with microfibrils extracted from tissues using crude bacterial collagenase, suggesting that the protein is not an integral component of these structures.28 LTBP-1 and LTBP-2 can also be separated from fibrillin-containing microfibrils using density gradient centrifugation, indicating a lack of covalent association with these structural elements.83 This finding is partially supported by studies on extraction of LTBP-2 from developing nuchal ligament. Most of the LTBP-2 was shown to be solubilized from the tissue, in monomeric form, using the chaotrope, 6M guanidinium chloride. Since the microfibrils remain morphologically intact after the treatment, it would appear that the majority of the LTBP-2 was not covalently bound to these structures. However, a significant proportion of the LTBP-2 could only be solubilized using a reducing agent, suggesting that some of the protein was covalently attached to the microfibrils, presumably by reducible disulfide bonding.22

Overall, the balance of evidence points to LTBPs being located on the surface of fibrillin-containing microfibrils rather than occurring as integral components of these structures. The functions of LTBPs in microfibril biology are yet to be determined. Some clues have been forthcoming from LTBP gene knock-out mice. LTBP-2 null mice have proved to be uninformative as they die in early embryogenesis.84 The LTBP-3 null mice have bone abnormalities attributed to alteration of normal TGF-beta bioavailability.85 However, the disruption of the gene for LTBP-4 in mice resulted in a homozygous phenotype exhibiting severe tissue specific disruption of elastic fiber structure in lung and colon.86 Thus it appears that individual LTBPs may modulate microfibril function to suit particular tissue environments.

Emilins

Elastin-microfibril interface located proteins, or EMILINS, are a two-member family of matrix glycoproteins.30 EMILIN-1 is a 115 kDa glycoprotein which, as its name suggests, has been ultrastructurally immunolocalized to the interface between the microfibrils and the elastin core of developing elastic fibers.87 Molecular cloning has shown EMILIN-1 to contain several distinct domains including a short collagenous region and a C-terminal domain with close sequence similarity to C1q.88 The molecule appears to form homotrimers and larger disulfide-bonded multimers. EMILIN-2 is similar in structure to EMILIN-1 and the two proteins have overlapping but distinct tissue expression patterns. EMILIN-1 has been shown to interact with EMILIN-2 via the C-terminal domains and to possess cell adhesive properties. However, binding of EMILINS to elastin and microfibrillar components has not yet been reported and EMILIN function in microfibril biology remains unknown. It has been suggested that EMILIN-1 may be involved in elastic fiber formation, in anchoring smooth muscle cells to elastic fibers and in regulation of blood vessel assembly.30

Other Proteins

Other proteins which may be involved with the function of fibrillin-containing microfibrils include the cross-linking enzyme, lysyl oxidase89 and the 67 kDa cell surface elastin-binding protein.90 Lysyl oxidase (32 kDa) has been localized by immuno-electron microscopy on, or very close to, the elastin-associated microfibrils in developing elastic tissues. This observation raises the possibility that an interaction of lysyl oxidase with fibrillin or other microfibrillar component may be important in the early stages of elastinogenesis when tropoelastin is deposited onto the microfibrils.91

The cell surface-associated, 67 kDa elastin binding protein appears to play a major role in tropoelastin secretion and assembly into elastic fibers.90,92 Evidence suggests that the binding protein becomes bound to tropoelastin intracellularly and then accompanies the elastin precursor to the extracellular side of the plasma membrane. It is postulated that tropoelastin then remains bound to the 67 kDa protein until there is an interaction with a microfibril-associated galactoside sugar which induces the transfer of the tropoelastin onto an acceptor site on the microfibril.93 The identity of the microfibril component(s) involved is unknown but it may be fibrillin-1 or MAGP-1 as both have been shown to bind tropoelastin and a proteoglycan component rich in galactosamine.

Involvement in Human Genetic Diseases

It has been postulated that mutations in the genes for non-fibrillin microfibrillar components may result in phenotypes with some similarities to the physical manifestations of Marfan syndrome. To date no mutations in human MAGP genes have been identified, although several alternatively spliced isoforms of MAGP-1 have been characterized including one isoform in MG-63 cells which lacks an alanine residue in the signal peptide.94 A search for mutations in the MFAP-1 gene also proved unsuccessful.50 The MFAP-4 gene has been found to be commonly deleted in Smith-Magenis syndrome, the characteristics of which include skeletal abnormalities of the head and mental retardation. However, the contribution of the lack of the MFAP-4 gene to this phenotype is unclear.14

Fibulin gene defects have been linked to several genetic disorders. Haploinsufficiency of the fibulin-1 gene has been linked to a case of synpolydactyly95 and a mutation in the fibulin-3 gene has been shown to segregate with the autosomal dominant eye diseases, Malattia Leventinese and Doney honeycomb retinal dystrophy.96 It is unclear if either of these mutations affects the function of fibrillin-containing microfibrils or elastic fibers. However, a missense mutation in the fibulin-5 gene has been linked to a severe recessive form of cutis laxa, exhibiting reduced, disorganized elastic fibers in the skin, thickened aortic valve, supravalvular aortic stenosis and the development of pulmonary emphysema.97 These manifestations indicate disruption of normal elastin synthesis in these patients and are consistent with a proposed role for fibulin-5 in elastic fiber formation deduced from fibulin-5 gene knock out mice.74,75 Interestingly, heterozygotes for the mutation were clinically normal, indicating that the mutant fibulin-5 molecules did not interfere with the function of normal fibulin-5 molecules. This finding suggested that fibulin-5 functions as a monomer molecule in its role in elastinogenesis and thus it is unlikely to be a structural component of the fibrillin-containing microfibrils.97

In preliminary studies, several distinct missense point mutations in the LTBP-2 gene have been reported in patients exhibiting a range of Marfan-like manifestations including mitral valve prolapse, aortic dilatation and spinal scoliosis. However, it needs to be determined if the mutations have a causative link to the phenotypes of these patients.98 If such a link were to be established then it would point to a structural role for LTBP-2 in fibrillin-containing microfibrils in a variety of tissues.

It remains to be established if a mutation in the gene for any non-fibrillin microfibril-associated protein can result in physical manifestations attributable to the disruption of the normal function of fibrillin-containing microfibrils.

Concluding Remarks

In recent years a diverse range of macromolecules have been identified as candidates for nonfibrillin components of the 10 nm microfibrils. The evidence for an association can vary in each case from immunolocalization only, to binding interactions with fibrillins and biological effects of knock-out mice. Some of the macromolecules discussed above appear to be exclusively associated with the microfibrils suggesting that their functions relate specifically to the microfibrils. This group can be divided into macromolecules which have widespread codistribution with the microfibrils such as MAGP-1 and macromolecules which have limited codistribution with the microfibrils suggesting that they may modulate the function of these structures to suit particular tissue environments, for example MAGP-2 and some MFAPs. Another group of microfibril-associated macromolecules also have known association with other structural elements of extracellular matrices indicating that they also fulfill microfibril-independent functions. This group may include several proteoglycans, fibulins and certain LTBPs. As knowledge of each microfibril-associated protein grows, candidates are appearing for each category of function proposed in the introduction. Molecules providing structural and linkage functions within microfibrillar bundles would be expected to have widespread codistribution with the microfibrils and be difficult to extract without disruption of the microfibrils. From the evidence presented above, MAGP-1 would appear to be the chief candidate for such roles. In addition, there is evidence that an unidentified proteoglycan, possibly decorin, may have a structural role as a component of the bead substructures. Several nonfibrillin molecules, in close association with microfibrils, may be involved in the assembly of elastin onto these structures. Molecules in this category may include MAGP-1, emilin-1, fibulin-5, the 67 kDa elastin-binding protein, lysyl oxidase and biglycan. Molecules which may mediate interactions of microfibrils with the surrounding matrix include versican, fibulins 1 and 2, MAGP-1 and LTBP-2. MAGP-2 and fibulin-5 may be involved in specific microfibril-cell interactions. LTBPs may extend the function of the microfibrils by acting as stores for TGF-β on the surface of the microfibrils in particular tissue environments. However, despite the rapidly accumulating information about the above macromolecules, the role(s) of each protein in microfibril function remains unclear. Obviously, further research is needed to clarify which proteins are important in microfibril biology and to identify the full range of human genetic disorders linked to their genes.

Acknowledgments

The author would like to acknowledge the financial support the National Health and Medical Research Council of Australia for the contributions from his laboratory cited in this review. Dr. E.G. Cleary and Dr. E. Hanssen are also thanked for their advice and critical comment during the preparation of this article.

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