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Metal ion dependency of microfibrils supports a rod-like conformation for fibrillin-1 calcium-binding epidermal growth factor-like domains1

https://doi.org/10.1006/jmbi.1997.1593Get rights and content

Abstract

The effects of the removal and replacement of divalent cations on the ultrastructure of 10 to 12 nm fibrillin-1-containing microfibrils have been studied, in order to investigate the conformation of fibrillin-1 calcium-binding epidermal growth factor-like (cbEGF-like) domains within the microfibril. The NMR structure of a covalently linked pair of cbEGF-like domains from fibrillin-1 recently identified a rigid rod-like conformation for the domain pair stabilised by interdomain calcium binding. This suggested that tandem arrays of fibrillin-1 cbEGF-like domains may adopt an extended conformation within a microfibril. If correct, then removal of bound calcium from fibrillin-1 would be expected to increase the flexibility of each cbEGF-like interdomain linkage, resulting in a decrease in the length of the interbead region of the microfibril (and thus a decrease in bead to bead periodicity), a concomitant increase in its diameter, and an overall increase in the flexibility of the microfibril. Our results show that removal of calcium by treatment with EGTA causes a large alteration of the microfibril structure, resulting in microfibrils with a reduced beaded periodicity, a disrupted interbead region and an increased overall flexibility. These effects are readily reversible by the re-addition of calcium (in the form of CaCl2), but not by the addition of magnesium (MgCl2). This is consistent with conformational changes in cbEGF-like domains causing the major structural effects on the microfibril. These results provide the first direct experimental evidence to support an extended rod-like conformation for multiple tandem repeats of fibrillin-1 cbEGF-like domains within the microfibril, as predicted by the NMR structure of an isolated fibrillin-1 cbEGF-like domain pair.

Introduction

Fibrillin-1 is a ∼350 kDa extracellular glycoprotein. It has a highly modular organisation (Pereira et al., 1993), and the predominant motif of the protein is a domain with close homology to epidermal growth factor (EGF). Fibrillin-1 contains a total of 47 such domains, of which 43 contain a consensus sequence associated with calcium binding Dahlback et al 1990, Glanville et al 1994, Handford et al 1990, Handford et al 1991, Handford et al 1995, Mayhew et al 1992, Persson et al 1989, Selander et al 1990 and are therefore referred to as calcium-binding EGF-like (cbEGF-like) domains. The remainder of the fibrillin-1 molecule is composed of seven transforming growth factor β1-binding protein (TGFβ1-bp)-like domains (Kanzaki et al., 1990), which have a consensus sequence of eight cysteine residues, two hybrid domains sharing features of both the EGF-like and TGFβ1-bp-like motifs, a proline rich region, and two regions of sequence unique to fibrillin-1 at the extreme amino and carboxyl termini (Pereira et al., 1993).

Fibrillin-1 is major component of a distinct subset of microfibrils of the extracellular matrix (Sakai et al., 1986) which possess a diameter of 10 to 12 nm. Immunohistochemical data have revealed that fibrillin-1 monomers are arranged in a repetitive manner along the length of the microfibrils (Sakai et al., 1991). These structures have a “beads on a string” appearance when viewed by rotary shadowing electron microscopy, with an average, although variable, periodicity of 50 to 55 nm (Kielty & Shuttleworth, 1995). Tissue-extracted microfibrils possessing periodicities ranging from 30 nm to greater than 100 nm have been observed and the periodicity of tissue microfibrils can be extended from 50 nm to 80 nm if stretching forces are applied to the tissue (Keene et al., 1991).

Defects in fibrillin-1 are responsible for the manifestation of the connective tissue disorder, the Marfan syndrome Collod-Beroud et al 1997, Dietz et al 1991, Peltonen and Kainulainen 1992, which is characterised by abnormalities in the skeletal, cardiovascular and ocular systems. The Marfan phenotype exhibits striking intra- and interfamilial variability and mutations in the gene encoding fibrillin-1 have more recently been identified in patients with other related, but less common, connective tissue disorders (Dietz & Pyeritz, 1995).

As suggested by the high content of cbEGF-like repeats in fibrillin-1, calcium is essential for maintaining the structure of 10 to 12 nm microfibrils. Kielty & Shuttleworth (1993) reported that the removal of bound calcium from isolated microfibrils extracted from normal primary dermal fibroblast cultures caused a reversible disruption in the structure of the interbead region, whilst the beads remained intact. No change in microfibril periodicity was observed. Although the ultrastructural effects of calcium chelation suggest that multiple fibrillin-1 cbEGF domains are a major component of the interbead region, the precise arrangement of fibrillin-1 monomers within the assembled microfibril structure is still unknown and is currently the centre of some debate. It is also not known how the “bead” of the microfibril is formed, although the immunolocalisation of other proteins such as microfibril-associated glycoprotein (MAGP) to the microfibril (Gibson et al., 1986) suggest that other proteins may contribute to the bead structure. A number of possible models for the microfibrillar arrangement of fibrillin-1 molecules have been proposed based upon immunological and structural data Reinhardt et al 1996, Downing et al 1996. However, additional experimental analysis is still required to substantiate these models. Only when the microfibrillar organisation of fibrillin-1 is resolved can predictions regarding the structural and functional effects of particular fibrillin-1 mutations be made.

As a first step to determining fibrillin-1 organisation, the conformation of fibrillin-1 cbEGF-like domains within the microfibril was investigated. Experiments were performed to extensively analyse the effects of chelation and replacement of divalent cations on microfibril ultrastructure. The recent NMR structure of a covalently linked pair of fibrillin-1 cbEGF domains showed the pair to be in a near linear arrangement, stabilised by interdomain calcium ligation and hydrophobic interactions (Downing et al., 1996). In this orientation the two domains are in the most extended conformation possible with respect to each other. Hence if all tandem cbEGF-like domains in human fibrillin-1 adopt this structure within the microfibril, then removal of calcium would be predicted to shorten and not lengthen the interbead distance, as the linkages between cbEGF domains become more flexible. A transformed fibroblast cell line, MSU-1.1, was used as a source of microfibrils because of its long-term stability in culture and microfibrils produced by this line were morphologically similar to those extracted from low passage primary dermal fibroblasts when examined by rotary shadowing electron microscopy. Tissue-extracted microfibrils were not studied, since a population of microfibrils was required that had not been subjected to tension. Microfibrils were extracted and purified in the presence of CaCl2 and prepared for rotary shadowing (see the legend to Figure 1). The morphological appearance of a representative microfibril produced by the fibroblast cell line MSU-1.1 is shown in Figure 1(a).

The treatment of microfibrils with either EGTA or EDTA resulted in a substantial alteration of the microfibril structure. The periodicity (measured from mid-bead to mid-bead) of treated microfibrils decreased significantly with increasing concentration of chelating agent, and for EGTA appeared to reach a minimum at a concentration of 10 mM. Incubation at concentrations above 10 mM EGTA resulted in no further significant decrease in periodicity. Electron micrographs showing the decreased periodicity of EGTA and EDTA-treated microfibrils compared with a normal control microfibril are shown in Figure 1(a) to (f). Despite the marked reduction in periodicity, the bead domains of the microfibrils remained intact. No major structural change in the beads was observed. In contrast, the interbead regions became less well defined with either EGTA (Figure 1(d), arrowed) or EDTA treatment (Figure 1(e) and (f)), as has previously been reported by Kielty & Shuttleworth (1993). An increase in overall microfibril diameter was visible in many but not all preparations (Figure 1(b), arrowed). The morphological variation in interbead structure observed may reflect the flexibility of multiple repeats of fibrillin-1 cbEGF-like domains in the absence of calcium. These cbEGF-like domains would be expected to adopt a range of conformations that would be “fixed” by the freeze-drying process used prior to rotary shadowing. The conformational heterogeneity may reflect different dissociation constants for calcium within the microfibril. In addition to exhibiting a disrupted interbead domain, treated microfibrils displayed a marked increase in flexibility over the extended structures typically observed, frequently appearing curled or folded (Figure 1(c)). A comparison of a normal control and EGTA-treated microfibril is shown at lower magnification in Figure 2 to illustrate the fact that the effects of calcium chelation on ultrastructure were uniform along the microfibril. The effects of calcium chelation on microfibril architecture correspond with the recently reported calcium-dependent structural changes in fibrillin-1 as determined by velocity sedimentation and rotary shadowing of recombinant fibrillin-1 fragments (Reinhardt et al., 1997).

To confirm that the morphological changes in periodicity could be attributed to treatment with EGTA rather than inherent variability in all microfibrils studied, we compared the periodicity measurements of a population of microfibrils extracted and purified in the presence of 10 mM CaCl2 with those treated with EGTA. The mean periodicity (±SD) of a group of microfibrils was calculated at each concentration of EGTA (see Figure 1). The EGTA sample group employed for statistical analysis comprised microfibrils treated with 10 to 50 mM EGTA, since these showed the same reduction in periodicity. The mean periodicity of microfibrils was 56.0 (±1.6) nm (n = 19) for the normal control group and 39.97 (±3.3) nm (n = 21) for the EGTA group (difference between means 16.1 nm, 95% CI 14.4 to 17.8;p<0.0001, unpaired t-test). Similar results were obtained for EDTA-treated (10 to 50 mM) microfibrils, when compared with the normal control group (mean periodicity 40.5 (±4.64) nm, n = 27; difference between means 15.5 nm, 95% CI 13.3 to 17.0;p<0.0001). A comparison of the mean periodicity values of individual microfibrils from the normal control or EGTA-treated sample groups used for the above statistical comparison is shown in Figure 3(a). The distribution of individual bead to bead measurements from these sample groups (Figure 3(b)) shows that most cluster around the mean value obtained when the periodicity is averaged over the complete microfibril. This indicates that the reduction in bead to bead measurement was observed over the total length of the microfibril, not just in one region.

The flexibility of microfibrils in the presence and absence of calcium was analysed by measuring the internal angle defined by each set of three adjacent beads along the microfibril (Figure 4). To confirm the qualitative observation of increased microfibril flexibility following calcium removal, the variances of the measured internal angle from the control and EGTA-treated microfibrils were compared and shown to be significantly different (F-statistic 4.78, p < 0.00001). The means of the two groups were also significantly different, confirming the increased curvature of microfibrils in the EGTA-treated groups (control group μ=165 (±13.1)°; EGTA group μ=138 (±28.7)°; difference between means 27°, 95% CI 21.9 to 32.1; p<0.0001, unpaired t-test for unequal variances). These data confirm that removal of Ca2+ increases the flexibility of the microfibril.

The effects of calcium chelation on microfibril ultrastructure were readily reversible. Microfibrils incubated initially with EGTA and subsequently with an equimolar concentration of calcium were generally morphologically indistinguishable from untreated controls (see Figure 1(g)), with the vast majority of microfibrils exhibiting intact bead and interbead domains, a normal periodicity (58.3±0.6, n = 7, Figure 3(a)) and an extended conformation. The effects of EGTA treatment were not reversed by the subsequent addition of magnesium (see Figure 1(h)), confirming the specificity of the microfibril structure for calcium. The periodicity of the microfibrils remained reduced (41.7±2.3, n = 6, Figure 3(a)), the structure of the interbead region remained disrupted and the increased overall flexibility of the microfibrils persisted. Taken together, these periodicity and flexibility changes are consistent with multiple tandem repeats of fibrillin-1 cbEGF-like domains existing in a rod-like conformation within the microfibril structure.

The results of these studies preclude an earlier model of microfibrillar fibrillin-1 organisation proposed by one of us in which the fibrillin-1 cbEGF-like domains adopted a calcium-dependent helical conformation, thus compacting each fibrillin-1 monomer to span one interbead region of the microfibril Handford et al 1995, Rao et al 1995. Chelation of calcium from these structures, although causing perturbations of the interbead region, might be expected to maintain or increase the interbead length, since the cbEGF-like domains would no longer be compacted (the presence of any cross-links may limit any increase in interbead length).

The effects of calcium chelation on microfibril ultrastructure reported here, demonstrate that calcium levels should be maintained during the extraction and purification of microfibrils to preserve structural integrity. One possible explanation for low periodicity measurements (<55 nm) of microfibrils previously reported may have been due to a failure to maintain calcium binding to these structures, either by inclusion of calcium chelators such as EDTA or PBS during extraction or immunolabelling procedures, or the use of denaturants. Many studies utilised such reagents for microfibril analyses (e.g. see Sakai et al 1986, Kielty et al 1991, Keene et al 1991), prior to the identification of the domain structure of fibrillin-1, which indicated that it was a calcium-binding protein Maslen et al 1991, Lee et al 1991. An extended rod-like conformation for fibrillin-1 cbEGF-like domains, however, does not account for the potential of the microfibril structure to be extended beyond an average periodicity of ∼55 nm when placed under tension. The periodicity of tissue microfibrils can be increased nearly twofold by applying stretching forces to the tissue (Keene et al., 1991), and indeed entangled intact rotary shadowed microfibrils were occasionally observed during these studies that displayed a beaded periodicity of ∼100 nm. Recently the microfibril network from Cucumaria frondosa has been shown to be reversibly extensible (Thurmond & Trotter, 1996). Our studies suggest that the elastic properties of microfibrils are conferred by fibrillin-1 domains other than the multiple cbEGF-like motifs, since the periodicity of isolated microfibrils is maximal in the presence of calcium and therefore already consistent with an extended conformation for contiguous cbEGF-like domains within the microfibril.

In summary the results of this study provide the first direct evidence to support a calcium-dependent rod-like arrangement of fibrillin-1 cbEGF-like domains within the microfibril. These data exclude models for fibrillin-1 organisation within the microfibril based on calcium-dependent compacted arrays of cbEGF-like domains and highlight the importance of calcium in the maintenance of microfibril structure.

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Acknowledgements

We thank Jeremy Sanderson for technical assistance with electron microscopy and production of electron micrographs, and Drs M. Hollinshead, R. J. O. Davies, S. Kettle and A. K. Downing for helpful discussions. The MSU-1.1 fibroblast cell-line was a kind gift from Dr J. McCormick, Carcinogenesis Laboratory, Michigan State University, USA. This work was supported by the MRC, grant numbers G9403796MA and G9435839MB. P.A.H. is a Royal Society University Research Fellow and a member of the Oxford

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