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Biomolecular analysis of elastic fibre molecules
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Methods 45 (2008) 42–52 www.elsevier.com/locate/ymeth
Biomolecular analysis of elastic fibre molecules Stuart A. Cain, Bertrand Raynal, Nigel Hodson, Adrian Shuttleworth, Cay M. Kielty * Wellcome Trust Centre for Cell-Matrix Research, Faculty of Life Sciences, University of Manchester, Michael Smith Building, Oxford Road, Manchester M13 9PT, UK Accepted 30 January 2008
Abstract Elastic fibres are macromolecular extracellular matrix assemblies that endow dynamic connective tissues such as arteries, lungs and skin with the property of elastic recoil. Here, we describe how we have purified elastic fibre molecules and then analysed them using a range of biochemical and biomolecular approaches. Such approaches have provided powerful insights into the complex hierarchical processes of extracellular matrix assembly. We outline molecular interaction and kinetics assays using Biacore, biophysical approaches such as multi-angle laser light scattering and analytical ultracentrifugation which provide information on molecular and macromolecular shape and mass in solution, the visualisation of molecules and assemblies using microscopy approaches such as atomic force microscopy and environmental scanning electron microscopy, and compositional analysis of macromolecular complexes using mass spectrometry. Data from these in vitro analytical approaches can be combined to develop powerful new models of elastic fibre assembly. Ó 2008 Elsevier Inc. All rights reserved. Keywords: Elastic fibres; Multi-angle laser light scattering; Analytical ultracentrifugation; Biacore; Mass spectrometry; Atomic force microscopy
1. Introduction Elastic fibres are large insoluble extracellular matrix assemblies comprising a central cross-linked core of elastin surrounded by a mantle of fibrillin microfibrils [1]. They endow dynamic tissues such as arteries, lungs, skin and ligaments with elastic recoil. Their assembly, which is developmentally regulated, has proved difficult to resolve due to their multimolecular composition and complex tissuespecific organization. Fibrillin microfibrils, which have a complex beaded morphology, assemble pericellularly and interact with a number of microfibril-associated molecules such as MAGP-1. In tissues, they form loose microfibrillar bundles that provide long-range tissue elasticity and, during elastic fibre formation, act as a template for elastin deposition. This higher-order assembly process involves several other molecules including lysyl oxidase cross-linking enzymes, and fibulins -4 and -5. Before describing biomolecular analyses of microfibril and elastic fibre assembly, *
Corresponding author. Fax: +44 161 275 5082. E-mail address: [email protected] (C.M. Kielty).
1046-2023/$ - see front matter Ó 2008 Elsevier Inc. All rights reserved. doi:10.1016/j.ymeth.2008.01.005
we first provide a brief summary of the molecules of the elastic fibre system. 1.1. Elastic fibre molecules The major structural components of elastic fibres are elastin and fibrillin-1 (for recent review, see [1]). Heritable diseases and mouse models have shown that several other molecules are also important in the assembly of functional elastic fibres. They include fibulins -4 and -5 which may regulate the deposition of tropoelastin on the microfibril template, and lysyl oxidase enzymes that cross-link and stabilize assembled elastin. Other molecules associated with microfibrils and elastic fibres include MAGP-1 and the glycosaminoglycan heparan sulphate. Each of these molecules has a unique molecular chemistry and interaction signature. In this article, we focus on the major elastic fibre molecules, namely fibrillin-1 and elastin. 1.1.1. Fibrillin-1 Fibrillin-1 is a large multidomain glycoprotein which contains 47 epidermal growth factor-like domains (EGF)
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domains, 43 of which are calcium-binding (cbEGF). These domains are interspersed with 8-cysteine containing (TB) modules, and there is an N-terminal proline-rich region that may act as a ‘hinge’ region [2] (Fig. 1.) In the presence of calcium, contiguous cbEGF domains adopt a relatively rod-like, although non-linear, conformation [3,4]). Fibrillin-1 has 14 N-glycosylation sites and, upon secretion, is processed at N- and C-termini by furin proteases. We have used mass spectrometry to determine the molecular composition of microfibrils, and biophysical and molecular interaction analyses to determine how fibrillin-1 molecules assemble to form tissue microfibrils. 1.1.2. Tropoelastin Tropoelastin is the soluble, secreted form of elastin. It comprises alternating hydrophobic and lysine-rich crosslinking domains [1] (Fig. 2). It acts as a ‘block co-polymer’ in vitro, undergoing a unique form of self-assembly, termed coacervation, as temperature increases. The rate of selfassembly and its initial reversibility, and the macromolecular form of the resultant multimers are regulated by salt concentration and rate of temperature change. In vitro, elastic multimers can form porous ‘gels’ that can be stabilized by synthetic crosslinks [5]. In vivo, assembled elastin is stabilized by lysyl-derived crosslinks catalyzed by members of the lysyl oxidase family, especially the lysyl oxidase (LOX) isoform [6]. We have used environmental electron scanning microscopy (ESEM) and mass spectrometry to analyze elastin coacervates, and how fibrillin-1 influences the coacervation process, and biophysical and molecular interaction analyses to determine how fibrillin microfibrils
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act as a template for tropoelastin deposition during elastic fibre formation. 2. Fibrillin isolation and purification 2.1. Cesium chloride density gradient centrifugation One of the problems encountered during the purification of cell matrix molecules is their large size. Therefore, we used a combination of density gradient and size exclusion chromatography strategies to purify fibrillin from cell media. Typically, fibrillin-1 molecules were purified on a density gradient, as previously achieved for microfibrils [7]. Caesium chloride was added to medium to give a starting density of 1.35 g/ml, and centrifuged in a Beckman Ti45 rotor at 125,000g for 70 h at 15 °C in 6 100 ml tubes. Tubes were emptied from the top and 20 fractions (5 ml) were collected. This strategy used the characteristic density of fibrillin-1 molecules (1.35 g/ml) which is larger than most of the globular proteins (1.20 g/ml). Fibrillin-1 containing fractions, detected by immuno-blotting, were pooled and concentrated by dialysis against Aquacide II (CalbiochemÒ) which decreased the volume from 125 ml to 5 ml. This 5 ml fraction was dialyzed against 0.1 M NaCl, 1 mM CaCl2, 50 mM Tris pH 8.0, and separated on a Superdex 200 size exclusion column eluted in 0.1 M NaCl, 1 mM CaCl2, 50 mM Tris pH 8.0 at a flow rate of 0.5 ml/min. Due to the characteristic large size of fibrillin-1 (350 kDa), these molecules elute in the dead volume of the column, whereas any smaller molecules (<200 kDa) would be included within the column volume.
Fig. 1. Schematic of the domain structure of fibrillin-1, including a key showing the different domains, N-linked glycosylation sites and C-terminal furin cleavage site.
Fig. 2. Schematic of the domain structure of human tropoelastin, indicating the corresponding exon numbers. Human tropoelastin consists of alternating hydrophobic (oval) and crosslinking domains (rectangle); there are two types of cross-linking domain, lysine–proline rich (KP) and lysine–alanine rich (KA).
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These combined approaches permit a pure fraction of fibrillin-1 molecules to be obtained. Having purified the protein of interest, its relative molecular mass (Mr) and N-glycosylation status can be assessed by denaturing SDS–PAGE in reducing and non-reducing conditions. The identity of the purified molecules can be confirmed using immunoblotting, using monoclonal antibodies directed to the protein of interest, for example fibrillin-1. 3. Monomer and multimer biophysical analysis For biomolecular analysis of purified molecules and multimers, it is important to establish whether the proteins of interest are monomeric, multimeric or non-specifically aggregated in native state. The size distribution of purified native proteins can readily be determined using non-denaturing agarose gel electrophoresis, or by size fractionation in native conditions. We routinely use Sephadex 200 columns for proteins in the size range 25–100 kDa. For structural and molecular interaction studies, high quality uniform monomer preparations are usually required. Extracellular matrix molecules are usually large, with a capacity to oligomerize, making it difficult to measure
molecular mass accurately. Due to their size, they are often also difficult to characterize in solution using techniques such as small angle X-ray scattering (SAXS). Furthermore, oligomers are difficult to separate and purify, making it essential to be able to analyze complex mixtures. Studies of molecular assembly intermediates and large multimers require determination of the shape and mass of a range of assemblies within a single sample. Size fractionation, laser light scattering and analytical ultracentrifugation are highly effective protocols, singly and in combination, for analyzing these parameters. For a detailed strategy for the biophysical analysis of molecules or multimers of interest, see Fig. 3. As an example, we have gained new insights into the process of fibrillin-1 microfibril assembly by defining the size distribution, molecular mass and shape of assembly intermediates of fibrillin-1 that were purified from cell culture medium, exploiting the powerful combination of analytical ultracentrifugation and multi-angle laser light scattering. The data were analysed using Sedfit software [8] which provides a means of accurately calculating the mass of individual molecular species within a complex velocity sedimentation profile. Below, we use this example
Fig. 3. Flow diagram showing potential strategies for biophysical analysis of oligomeric and non-oligomeric molecules using analytical ultracentrifugation, multi-angle laser light scattering (MALLS) and quasi-elastic light-scattering (QELS). These combined approaches allow the determination of molecular mass (Mr), size and shape of molecules and multimers in solution.
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to highlight the potential of these combined approaches to dissect the assembly pathways of other extracellular matrix multimers.
3.1. Multi-angle laser light scattering Multi-angle laser light scattering (MALLS) allows the measurement of the average molecular mass (Mr), as well as the radius of gyration (Rg) which gives size information. This can be coupled with Quasi-elastic Light Scattering (QELS) to measure hydrodynamic radius (Rh) giving further information about the size of the molecule of interest. Prior to measurement, samples are separated by size exclusion chromatography, and measurements are performed on line, allowing the determination of Mr, Rg and Rh over the peak where polydisperse sample will show the range of Mr, Rg and Rh over the peak (Fig. 4). This enabled the separation of monomers from aggregates and assemblies. Interestingly, analysis of the relationship between Rg and the Mr of the polydisperse sample provides
Fig. 4. Characterisation of purified fibrillin-1 monomers and multimers by MALLS/QELS. (A) Size exclusion chromatography of purified fibrillin-1 in 0.1 M NaCl, 1 mM CaCl2, 50 mM Tris/HCl pH 8.0, using a G5000PW size fractionation column. The column effluent was monitored on-line with MALLS, a refractometer to measure refractive index, and with QELS. Line the refractive index signal, squares show measured Mr (blue). (B) Rg (blue) and Rh (red) dependence on elution volume. (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)
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details of the size and shape of the molecule, and how these parameters are affected by an increase in total mass. Rg ¼ KM ar
ð1Þ
where a is a parameter sensitive to the shape of the macromolecules, and K is an empirical constant. For a single molecule, a rod-like conformation gives an approximated value of 1 for a, whereas a compact symmetrical structure gives an approximated value of 0.33. Analysis of the fibrillin-1 assembly intermediates presented in Fig. 4 shows the shape change towards rod-like conformation with oligomerisation. 3.2. Analytical ultracentrifugation A second approach to measure the molecular mass as well as the size (Rh) and shape of the molecule of interest is to use analytical ultracentrifugation. Sedimentation equilibrium experiments allow the measurement of molecular mass (Mr), and provide a means to analyze association. Sedimentation velocity experiment allow the measurements of sedimentation value that permit the determination of size (Rh) and shape through the calculation of the frictional ratio (f/fO). These values can then be used to model the overall shape of the molecule or multimer, using different
Fig. 5. Sedimentation characteristics and model of fibrillin-1 monomers and multimers. (A) Sedimentation values of purified fibrillin-1 in 0.1 M NaCl, 1 mM CaCl2, 50 mM Tris/HCl pH 8.0 Arrows highlight the detected species (Table 1). (B) Shell modeling (Beads model) of some of the detected species, which predicts how a fibrillin microfibril may assemble.
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Table 1 Characterisation, identification and modelling of detected fibrillin-1 species detected by MALLS/QELS and analytical ultracentrifugation Identified number of monomer 1* 1 2 4 6 10 18 22
Theorotical Mr (kDa)
Measured S
S20,w
f/f0H
Calculated S20,w from modelling
340 340 680 1360 2040 3400 6120 7480
6.3 12.3 20.4 26.4 32.5 43.5 56.7 67.7
6.5 12.7 21.1 27.3 33.6 45.0 58.7 70.0
2.94 1.49 1.43 1.75 1.87 1.96 2.23 2.13
6.2 11.8 20.8 25.3 35.1 42.8 60.6 69.6
The number of fibrillin-1 monomers was calculated using MALLS/QELS, apart from * which were calculated by Sedfit analysis. The frictional ratios (f/f0H) were calculated, taking into account hydration of 0.328 g H2O/g for fibrillin-1, as calculated directly from the amino acid sequence.
models such as prolate, spherical, circular cylinder or Shell modelling [9]. Development of new analysis software such as SEDFIT permits the analysis of complex mixtures and determination of their sedimentation characteristics, Mr and f/fO. In the example presented in Fig. 5A, the oligomerisation of fibrillin-1 has been studied. Fibrillin-1 was purified by CsCl centrifugation and size exclusion chromatography on a Sephacryl S200 column. The data show the presence of multiple oligomers that can be characterized by the Rh, f/fO and sedimentation values (Fig. 5A; Table 1). As the number of fibrillin-1 monomers increases within a multimer, there is a corresponding increase in stiffness and length, as shown by the increase in f/fOH (Table 1). Every oligomer shape has then been modelled using Shell modeling [9] techniques, and an example of such modelling data is shown in Fig. 5B. 4. Monomer and multimer imaging
brils and collagen VI microfibrils [11,12] to individual protein subunits (e.g. collagen (VIII) alpha1 subunits [13]. A fine tip, usually with a radius of curvature of around 10 nm, which is located at the end of a flexible cantilever is scanned across the surface of the sample (Fig. 6). In intermittent or ‘‘tapping” mode, the cantilever is oscillated at its resonant frequency during the scanning, which results in minimal damage to the specimen due to little or no lateral force. Repulsive forces between the tip and the sample cause the cantilever to be deflected as it scans across surface features. Laser light reflected off the back of the cantilever is collected by photodiodes and deflections of the cantilever caused by these features changes the amount of light received by the photodetectors; it is this output that is used to produce the image [14,15]. Imaging of air-dried specimens requires little sample preparation. An aliquot of sample is deposited onto a substrate surface, often freshly cleaved mica, and then allowed to air-dry. However, both substrate surface charge and buffer conditions affect the physical properties of the sample, including shape and dimensions. For example, we showed that the characteristic double beads of type VI collagen (white arrows) were apparent when the microfibrils were deposited on to mica coated with poly-L-lysine (PLL) but absent from those on untreated mica (Fig. 7) [16]. Fibrillin microfibrils (the large beaded structures in Fig. 7) also exhibit different morphology on mica or mica-PLL. A later study showed that biological functions such as cell adhesion were markedly affected by choice of substrate [17]. Calcium-bound fibrillin microfibrils have very different dimensions when adsorbed on mica or in physiological solution [18]. Our studies employ a Multimode AFM with a Nanoscope IIIa controller and an ‘‘E” scanner (Veeco Instruments), giving a scan area of 12 12 lm, and a height range up to 10 lm. Routinely, the Nanoscope software is used to determine cantilever oscillation frequency and drive
Whilst biophysical approaches provide quantitative data on molecular shape and mass, complementary microscopy approaches provide the means to visualize directly extracellular matrix molecules and assemblies. 4.1. AFM Atomic force microscopy is a powerful technique that allows the direct imaging of purified recombinant monomers and multimers in hydrated or air-dried states, without metal or carbon coating. The protein is adsorbed onto freshly cleaved mica, or to other flat surfaces such as graphite. Visualisation allows direct measurements of characteristics such as shape, molecular length, assembly periodicities and molecular organization. Developed by Binnig and colleagues [10] as an alternative method to scanning tunnelling microscopy, AFM has been used in our group to visualize a range of isolated extracellular matrix components, from fibrillin-1 microfi-
Fig. 6. Operation of an atomic force microscope. The diagram shows the arrangement of the cantilever, tip, photodetector and sample.
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Fig. 7. Atomic force microscopy of a tissue isolated sample containing fibrillin microfibrils and type VI collagen microfibrils. Samples were adsorbed onto freshly cleaved mica, or onto mica that had been pretreated with poly-L-lyine (PLL). The thin filaments (white arrows) are type VI collagen microfibrils, and the wider diameter microfibrils with prominent beads are fibrillin microfibrils. Bar = 100 nm.
amplitudes, but we adjust the setpoint to just below the point where tip-sample interaction is lost. Scanning is usually performed at rates of 1.49 Hz with scan sizes of either 5 or 2 lm2. First order flattening of images is performed using the Nanoscope software prior to exporting data to other packages for analysis. In addition to direct imaging, molecular or multimer height data can be readily obtained by AFM. For example, Sherratt and coworkers have identified age-related deterioration of fibrillin microfibrils isolated from skin biopsies by measuring changes in interbead lengths following mechanical stretching. Height data can also be used to investigate specific points of interaction, for example, to determine the location of SPARC binding sites on collagen I and procollagen I [19]. When using data derived from height measurements, it should be noted that, due to the geometry of the tip, it is often necessary to correct for the inherent widening effect that is seen [17,20]. Tip correction is a straight-forward process achieved using a calibration specimen of known dimensions, such as colloidal gold. In addition to direct imaging of unshadowed, unstained molecules, AFM can also be used as a force spectrometer. By coating the tip with specific bioactive species (such as monoclonal antibodies), it is possible to determine the binding forces between a cell surface receptor and extracellular matrix ligand. Sun et al. used this approach to study mechanical interactions between fibronectin and a5b1-integrin on vascular smooth muscle cells [21]. Graham et al. used a similar approach to acquire force elongation/relaxation profiles for human type I collagen microfibrils [22]. 4.2. ESEM The environmental scanning electron microscope (ESEM) is a relatively recent development in electron microscopy that enables the imaging of hydrated biological material without pre-treatment [23], and with a high spatial
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resolution, in some cases in the order of 1–2 nm [24]. One of the main areas where the technique has made an impact is in the field of biomaterials and tissue engineering. In particular, the ability to study cellular material in situ on a scaffold material without subjection to elaborate fixation/ dehydration procedures whilst maintaining fully hydrated state, makes the ESEM a valuable tool (Fig. 8A) [25]. Our recent ESEM studies have focused on tissue engineered arterial grafts [26] and analysis of recombinant elastin coacervates in the presence or absence of fibrillin-1 or other elastic fibre molecules [5]. Other groups have used ESEM to study hydrogels [27,28], cell growth on bioactive matrix surfaces [29] and osteoblast attachment to polymer/ hydoxyapatite composites [30]. ESEM differs from conventional scanning electron microscopy in its use of pressure limiting apertures along the length of the microscope column, such that a high vacuum is maintained at the electron gun whilst the specimen chamber is under poor vacuum. The chamber is filled with a gas, usually water vapor, and it is this feature that maintains the hydrated state of the sample whilst also providing, via the cascade effect [31], the imaging medium. Briefly, when the primary electron beam is scanned across the surface of the sample, secondary electrons are released from the surface. These secondary electrons then collide with water vapor molecules which results in further secondary electrons being released from the water molecules. These electrons then collide with further water molecules, and a cascade amplification results. The resulting secondary electron signal is collected by a specialized type of detector known as gaseous secondary electron detector, which may even form the final aperture of the microscope column itself. Positive ions produced as a result of the collisions are drawn towards the specimen surface and neutralize the charging effects caused by the non-conductive nature of the sample. We use ESEM to image a variety of extracellular matrix and fibrin-based support materials used in tissue engineering and regeneration [13,32]. These range from electro-spun polycaprolactone to biomaterials such as tropoelastin aggregates. In the latter case, a solution of coacervated and crosslinked tropoelastin was deposited onto an ESEM specimen mount and then imaged using a FEI Quanta 200 with an accelerating voltage of 10– 30 keV (Fig. 8B). The sample was held at a temperature of 5 oC using a Peltier cooling stage and, at a pressure ca 3.5 Torr, highly ordered linear arrays were observed. It is important to note that the conditions used for ESEM are more likely to result in specimen damage from the electron beam than conventional scanning electron microscopy, and so care must be observed in image acquisition and interpretation. 5. Monomer and multimer interaction analysis The processes of fibrillin-1 microfibril assembly and elastic fibre formation have been extensively probed, in
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Fig. 8. Environmental scanning electron microscopy. (A) The arrangement of microscope column, electron beams and hydrated sample; (B) ESEM analysis of coacervated recombinant human tropoelastin (globules and linear arrays).
our laboratory and others, at the molecular level using in vitro molecular interaction studies. The ultimate aims are to develop a model based on high and low affinity interactions that predicts how, and to what extent, these macromolecular complexes can undergo self-assembly. These predictions can then be tested in cell culture models, e.g. using knock-down and knock-in approaches, or in mouse models, to establish the roles for individual components in this hierarchical process. Molecular binding assays can also be used to validate models derived from biophysical analyses, e.g. our fibrillin-1 microfibril assembly based on MALLS, AUC and Shell modelling (as outlined above). 5.1. Surface plasmon resonance Surface plasmon resonance (SPR) is a useful way of discovering interactions between ECM molecules. The technology involves attaching one of the interacting pair of molecules to a sensor chip (ligand). A sample containing one or more interacting molecules is passed over the sensor chip surface, and binding of molecules to the ligand generates a response which is proportional to the bound mass. Binding is monitored in real time which
allows values such as specificity of an interaction, the affinity and kinetics of an interaction, and even the concentration of the bound ligand (Fig. 9). Automation allows the screening of a large number of interactions in one experiment. Detailed kinetic experiments can be run to determine the kinetics of these interactions. It can be used to study protein-protein, protein-glycosaminoglycan, or any other molecule that can be immobilized onto the sensor chip surface, such as potential drug. Nucleic acids are also easy to immobilize onto the sensor chip surface, and interactions have been reported using larger complexes such as viruses and even whole cells. The most widely used system for measuring SPR, is the Biacore supplied by GE Healthcare, which have a variety of different systems based on SPR, each of which provides different levels of automation and number of interactions measured, ranging from simple single interactions to multiple arrays. One of the most widely used machines for ECM research is the Biacore 3000, as well as its predecessor Biacore 2000 and 1000 instruments. SPR on the Biacore 3000 instrument involves attachment or immobilisation of your ligand of choice onto one of several different sensor chip surfaces via either a covalent linkage, high affinity capture or hydroscopic adsorption.
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Fig. 9. The principles of immobilisation and binding by surface plasmon resonance using Biacore. (i) The sensor chip consists of a gold surface which is coated with a carboxylated dextran matrix. (ii) The carboxylated matrix is then activated (1) then the ligand is adsorbed to the surface then crosslinked (2), before the remaining activated sites are blocked (3). The real time sensorgram is shown in magenta indicating the 3 stages (above). (iii) When an analyte is passed over the ligand and interaction occurs, the response on the sensorgram is increased until saturation occurs or injection ends (4). After injection, the analyte begins to disssociate from the ligand and the sensorgram response drops (5).
5.1.1. Immobilization protocols The most common method of immobilisation is to covalently link your protein of interest to a modified dextran matrix which is attached to the gold surface of the sensor chip. The usual methods are to link via primary amine groups, thiol groups or active disulphides or to immobilise using aldehyde coupling by oxidising carbohydrate groups of you ligand. The next most common method of ligand immobilisation is the high affinity capture of biotin to a covalently linked streptavidin molecule. The key advantage of SPR over other interaction methods such as solid phase binding or calorimetry is the low sample amount needed. For the immobilised ligand, this is even more so with most experiments needing as little only a microgram of protein, making the use of commercially available recombinant proteins economically viable. For the analyte, more reagent is needed but preliminary studies can be carried out with as little as 10 lg of protein, making the process economical with precious or hard to purify samples. 5.1.2. Interaction of MAGP-1 with fibrillin-1 To map the interaction of MAGP-1 with fibrillin-1, MAGP-1 was immobilized onto a CM5 sensor chip using amine coupling, fragments of fibrillin-1 was then passed over the surface of the chip and the response of the analyte scan was measured [33] (Fig. 10A and B). The identified binding fragments were then used in a more detailed analysis to determine the binding kinetics. This is achieved by injection of several concentrations of the fibrillin-1 fragments, the resultant curves could then be fitted to a mathematical model of 1:1 binding kinetics or, if this is not possible, the equilibrium binding constant (KD) can be calculated by plotting the maximum binding response against
concentration. (Fig. 10C). Using this analysis the KD of the interaction was calculated to be 242 nM. 5.1.3. Interaction of fibrillin-1 with heparin SPR is not only limited to protein-protein interactions, and glycosaminoglycans such as heparin, heparan sulphate or chondroitin sulphate, once biotinylated can be immobilised using a streptavidin immobilised SA sensor chip. Glycosaminoglycans were biotinylated using various methods, but it was found that coupling the biotin molecule at the end of the heparin, which mimics the attachment to glycoproteins, produced a higher affinity binding [34]. Using this method, several heparin binding sites were identified on fibrillin-1, and detailed kinetic data was obtained. It was found that heparin bound to the same N-terminal region of fibrillin-1 that also bound MAGP-1 (see above). This binding allowed inhibition analysis using the Biacore with immobilised heparin, where increasing concentrations of MAGP-1 were found to inhibit fibrillin-1 binding. The inverse was also found to be true, that increasing concentrations of heparin inhibited fibrillin-1 binding to immobilised MAGP-1 (Fig. 10D). 6. Molecular composition of elastic fibre complexes 6.1. Mass spectrometry analysis of the composition of matrix complexes The advent of mass spectrometers has allowed the identification of protein complexes, at biological levels. It allows non-selective discovery of proteins, without the need of antibodies or other predetermined markers, making it an ideal tool for analysing samples, or tissues with unknown
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Fig. 10. (A) Diagram showing recombinantly expressed fibrillin-1 protein fragments used in binding studies using surface plasmon resonance, indicating the fibrillin-1 domains they contain. The scheme uses the same symbols as shown in Fig. 1. (B) Interaction of MAGP-1 with fibrillin-1 fragments. Fibrillin1 fragments were injected at 200 nM (black) and 40 nM (gray) over a MAGP-1-immobilised surface, and the maximum response difference (Resp. Diff.) for each experiment is shown in resonance units (RU). (C) Fibrillin fragment PF1 was injected over a MAGP-1-immobilised surface. Each sensorgram shows six different analyte concentrations of 0.5, 1, 5, 10 (duplicated), 15, and 20 lg/ml. One representative experiment is shown. The inset shows the saturation binding curve, calculated using the maximum response values for each concentration, plotted against concentration. Using nonlinear regression, KD and Bmax values were calculated using GraphPad Prism version 2.0. Time is shown in seconds (s). (D) To illustrate that Biacore may also be used to perform inhibition analysis, MAGP-1 and a heparan oligosaccharide dp24 were both found to inhibit each other from binding the N-terminal region of fibrillin-1 (fragment PF1). For inhibition of the fibrillin-1 PF1 interaction with heparin dp24 by MAGP-1 (j), PF1 (1 lg/ml) was incubated with MAGP-1 (0.4–1200 nM), before addition to immobilised dp24. For inhibition of PF1 with MAGP-1 by dp24, PF1 (1 lg/ml) (N), was incubated with dp24 (0.4–4000 nM) before addition to immobilised MAGP-1. The response difference was normalised to a percentage of the response for no inhibitor added.
molecular compositions. Mass spectrometers consist of a source which generates gaseous ions from the sample, and an analyser. Gaseous ions can be manipulated by electrical and magnetic fields, which allows mass and charge states to be determined by the analyser. Further data can also be obtained if the machine contains a fragmentation device which can break peptide ions into small fragments usually around the peptide bond. There are two common types of source for analysing biological samples, a MALDI (Matrix Assisted Laser Desorption/Ionisation) source generated ions by firing a laser at your sample which has been mixed with matrix that converts the laser energy into excitation energy. The other common source is by electrospray ionization (ESI), where charged droplets are sprayed from a capillary tube, then the solvent in the droplet evaporates off leaving the charged sample ions. The mass to charge ratio is then determined using the analyser, which includes one or a combination of different types. These types include
time of flight (TOF), Quadropole, or Ion Trap, each manipulating the ions in a different way. Accuracy of the mass determination is greater if the peptide ion is lower in mass, so proteins are usually digested before with the protease trypsin. Multiple peptide ions per protein allowing a greater chance of identification, and these peptide ions may also be further fragmented to give some sequence information about each peptide, thereby increasing further the identification accuracy. To aid the analysis of peptides, separation using reverse phase chromatography, prior to injection, decreases the amount of peptide ions analysed at any one time. Separation of peptides can also be further achieved by including a second dimension to the chromatography, which is usually ion exchange. Greater mass accuracy, speed of analysis and methods of detection over time have allowed the analysis of more and more complex samples, making it a powerful technique in biological research.
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Fig. 11. Fibrillin-1 domain structure, showing analysis of the pattern of tryptic peptides obtained from the human zonular microfibril preparations. (i) The number of times each tryptic peptide was identified; (ii) the number of peptides identified in each domain. The seven TB motifs are numbered in an N- to C-terminal direction. Domains with peptide hits only after guanidine HCl (denaturing) extraction are shown with hatching.
6.2. Composition of fibrillin-rich microfibrils To investigate the composition of fibrillin-rich microfibrils, fibrils were isolated from ciliary zonules, aorta and skin, using a non-denaturing method of collagenase digestion, or a denaturing method using guanidine solubilisation [35]. The microfibrils were then isolated by size exclusion chromatography, followed by concentration by freeze drying then digested using trypsin in reducing conditions. The study identified that the microfibrils predominantly consisted of fibrillin-1. MAGP-1 was also detected, but no other predicted microfibril-associated components such as LTBPs or fibronectin were detected. The pattern of tryptic peptides identified from fibrillin-1 was not as complete as that obtained from identically treated recombinant fibrillin-1 fragments. Whilst tryptic peptides from certain domains of the molecule were found in multiple microfibril purifications, other regions had less or no detectable peptides (Fig. 11). Further investigation of the trypsin-digested microfibrils by AFM revealed an undigested part corresponding to the bead structure, which is thought to be non-reducibly crosslinked by transglutaminase. 6.3. Mass spectrometry to investigate novel interactions To investigate novel fibrillin interactions, mass spectrometry was used to identify proteins that were co-purified during fishing experiments; for these studies, we used proteins secreted into culture medium or deposited within the extracellular matrix of cell cultures known to form fibrillin microfibrils. Recombinant proteins, including overlapping fibrillin-1 fragments, MAGP-1, fibulin 1, fibulin 5 and lysyl oxidase were used as bait proteins; each contained a six-histidine tag which allowed easy selection of protein complexes. The bait proteins were incubated with the growth media and secreted matrix from human dermal fibroblasts (HDF) and human retinal pigment epithelial cells (ARPE 19). Purified protein complexes were analysed using tandem mass spectrometry, and the results collated into a large database which allowed the analysis of primary and secondary interactions of the complexes. Over 200 interacting species were identified using two separate search
engines. Several protein groups that interacted with elastic fibre molecules were identified, including collagens, growth factors, other elastic fibre components, and the core proteins of proteoglycans. 7. Conclusions The biology of fibrillin microfibrils and elastic fibres has proved difficult to unravel due to their multimolecular nature, their hierarchical assembly process, and their complex macromolecular and tissue organisation. However, biomolecular analyses have recently provided critical new insights into how they assemble and function. Biophysical approaches such as multi-angle laser light scattering and analytical ultracentrifugation have allowed determination of the shape and mass of fibrillin-1 assembly intermediates, thereby forming a basis for modelling the microfibril assembly process for the first time. In parallel, Biacore analysis has revealed the high affinity fibrillin-1 interactions that support linear and lateral assembly into microfibrils, and the key interactions between elastic fibre molecules that support elastic fibrillogenesis. Knowledge of the molecular composition of tissue purified microfibrils and elastic fibres is being revealed using mass spectrometry. References [1] C.M. Kielty, Expert Rev. Mol. Med. 8 (2006) 1–23. [2] L. Pereira, M. D’Alessio, F. Ramirez, J.R. Lynch, B. Sykes, T. Pangilinan, J. Bonadio, Hum. Mol. Genet. 2 (1993) 961–968. [3] R.S. Smallridge, P. Whiteman, J.M. Werner, I.D. Campbell, P.A. Handford, A.K. Downing, J. Biol. Chem. 278 (2003) 12199–12206. [4] C. Baldock, V. Siegler, D.V. Bax, S.A. Cain, K.T. Mellody, A. Marson, J.L. Haston, R. Berry, M.C. Wang, J.G. Grossmann, M. Roessle, C.M. Kielty, T.J. Wess, Proc. Natl. Acad. Sci. USA 103 (2006) 11922–11927. [5] C.M. Kielty, S. Stephan, M.J. Sherratt, M. Williamson, C.A. Shuttleworth, Philos. Trans. R Soc. Lond. B. Biol. Sci. 362 (2007) 1293–1312. [6] H.A. Lucero, H.M. Kagan, Cell. Mol. Life Sci. 63 (2006) 2304–2316. [7] C.M. Kielty, E. Hanssen, C.A. Shuttleworth, Anal. Biochem. 25 (1998) 108–112. [8] P. Schuck, M.A. Perugini, N.R. Gonzales, G.J. Howlett, D. Schubert, Biophys. J. 82 (2002) 1096–1111. [9] V. Bloomfield, W.O. Dalton, K.E. Vanholde, Biopolymers 5 (1967) 135–148.
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[10] G. Binnig, C.F. Quate, C. Gerber, Phys. Rev. Lett. 56 (1986) 930–933. [11] C. Baldock, M.J. Sherratt, C.A. Shuttleworth, C.M. Kielty, J. Mol. Biol. 330 (2003) 297–307. [12] M.J. Sherratt, C. Baldock, J.L. Haston, D.F. Holmes, C.J. Jones, C.A. Shuttleworth, T.J. Wess, C.M. Kielty, J. Mol. Biol. 332 (2003) 183–193. [13] S. Stephan, M.J. Sherratt, N. Hodson, C.A. Shuttleworth, C.M. Kielty, J. Biol. Chem. 279 (2004) 21469–21477. [14] S. Alexander, L. Hellemans, O. Marti, J. Schneir, V. Elings, P.K. Hansma, M. Longmire, J. Gurley, J. Appl. Phys. 65 (1989) 164–167. [15] G. Meyer, N.M. Amer, Appl. Phys. Lett. 53 (1988) 1045–1047. [16] M.J. Sherratt, D.F. Holmes, C.A. Shuttleworth, C.M. Kielty, Biophys. J. 86 (2004) 3211–3222. [17] M.J. Sherratt, D.V. Bax, S.S. Chaudhry, N. Hodson, J.R. Lu, P. Saravanapavan, C.M. Kielty, Biomaterials 26 (2005) 7192–7206. [18] M.J. Sherratt, C. Baldock, A. Morgan, C.M. Kielty, Matrix Biol. 26 (2007) 156–166. [19] H. Wang, A. Fertala, B.D. Ratner, E.H. Sage, S.Y. Jiang, Anal. Chem. 77 (2005) 6765–6771. [20] E. Balnois, K.J. Wilkinson, Colloids Surf. A Physicochem. Eng. Asp. 207 (2002) 229–242. [21] Z. Sun, L.A. Martinez-Lemus, A. Trache, J.P. Trzeciakowski, G.E. Davis, U. Pohl, G.A. Meininger, Am. J. Physiol. 289 (2002) H2526– H2535. [22] J.S. Graham, A.N. Vomund, C.L. Phillips, M. Grandbois, Exp. Cell Res. 299 (2004) 335–342.
[23] D.J. Stokes, Phil. Trans. 361 (2003) 2771–2787. [24] M. Toth, C.J. Lobo, W.R. Knowles, M.R. Phillips, M.T. Postek, A.E. Vladar, Nano Lett. 7 (2007) 525–530. [25] L. Muscariello, F. Rosso, G. Marino, A. Giordano, M. Barbarisi, G. Cafiero, A. Barbarisi, J. Cell. Physiol. 205 (2005) 328–334. [26] M. Baguneid, D. Murray, H.J. Salacinski, B. Fuller, G. Hamilton, M. Walker, A.M. Seifalian, Biotech. Appl. Biochem. 39 (2004) 151–157. [27] A. Miller, Macromol. Symp. 222 (2005) 109–114. [28] A. Motta, C. Migliaresi, F. Faccioni, P. Torricelli, M. Fini, R. Giardino, J. Biomat. Sci. 15 (2004) 851–864. [29] J.M. Manero, F.J. Gil, E. Padros, J.A. Planell, Microsc. Res. Tech. 61 (2003) 469–480. [30] S.C. Rizzi, D.J. Heath, A.G. Coombes, N. Bock, M. Textor, S. Downes, J. Biomed. Res. 55 (2001) 475–486. [31] G.D. Danilatos, Adv. Elect. Electron Phys. 78 (1990) 1–102. [32] N. Hodson, D.V. Bax, A.C. Lomas, J. Ghosh, M.G. Walker, C.M. Kielty, Int. J. Exp. Pathol. 84 (2003) A23–A81. [33] M.J. Rock, S.A. Cain, L.J. Freeman, A. Morgan, K. Mellody, A. Marson, C.A. Shuttleworth, A.S. Weiss, C.M. Kielty, J. Biol. Chem. 279 (2004) 23748–23758. [34] S.A. Cain, C. Baldock, J. Gallagher, A. Morgan, D.V. Bax, A.S. Weiss, C.A. Shuttleworth, C.M. Kielty, J. Biol. Chem. 280 (2005) 30526–30537. [35] S.A. Cain, A. Morgan, M.J. Sherratt, S.G. Ball, C.A. Shuttleworth, C.M. Kielty, Proteomics 6 (2006) 111–122.
Methods 45 (2008) 42–52 www.elsevier.com/locate/ymeth
Biomolecular analysis of elastic fibre molecules Stuart A. Cain, Bertrand Raynal, Nigel Hodson, Adrian Shuttleworth, Cay M. Kielty * Wellcome Trust Centre for Cell-Matrix Research, Faculty of Life Sciences, University of Manchester, Michael Smith Building, Oxford Road, Manchester M13 9PT, UK Accepted 30 January 2008
Abstract Elastic fibres are macromolecular extracellular matrix assemblies that endow dynamic connective tissues such as arteries, lungs and skin with the property of elastic recoil. Here, we describe how we have purified elastic fibre molecules and then analysed them using a range of biochemical and biomolecular approaches. Such approaches have provided powerful insights into the complex hierarchical processes of extracellular matrix assembly. We outline molecular interaction and kinetics assays using Biacore, biophysical approaches such as multi-angle laser light scattering and analytical ultracentrifugation which provide information on molecular and macromolecular shape and mass in solution, the visualisation of molecules and assemblies using microscopy approaches such as atomic force microscopy and environmental scanning electron microscopy, and compositional analysis of macromolecular complexes using mass spectrometry. Data from these in vitro analytical approaches can be combined to develop powerful new models of elastic fibre assembly. Ó 2008 Elsevier Inc. All rights reserved. Keywords: Elastic fibres; Multi-angle laser light scattering; Analytical ultracentrifugation; Biacore; Mass spectrometry; Atomic force microscopy
1. Introduction Elastic fibres are large insoluble extracellular matrix assemblies comprising a central cross-linked core of elastin surrounded by a mantle of fibrillin microfibrils [1]. They endow dynamic tissues such as arteries, lungs, skin and ligaments with elastic recoil. Their assembly, which is developmentally regulated, has proved difficult to resolve due to their multimolecular composition and complex tissuespecific organization. Fibrillin microfibrils, which have a complex beaded morphology, assemble pericellularly and interact with a number of microfibril-associated molecules such as MAGP-1. In tissues, they form loose microfibrillar bundles that provide long-range tissue elasticity and, during elastic fibre formation, act as a template for elastin deposition. This higher-order assembly process involves several other molecules including lysyl oxidase cross-linking enzymes, and fibulins -4 and -5. Before describing biomolecular analyses of microfibril and elastic fibre assembly, *
Corresponding author. Fax: +44 161 275 5082. E-mail address: [email protected] (C.M. Kielty).
1046-2023/$ - see front matter Ó 2008 Elsevier Inc. All rights reserved. doi:10.1016/j.ymeth.2008.01.005
we first provide a brief summary of the molecules of the elastic fibre system. 1.1. Elastic fibre molecules The major structural components of elastic fibres are elastin and fibrillin-1 (for recent review, see [1]). Heritable diseases and mouse models have shown that several other molecules are also important in the assembly of functional elastic fibres. They include fibulins -4 and -5 which may regulate the deposition of tropoelastin on the microfibril template, and lysyl oxidase enzymes that cross-link and stabilize assembled elastin. Other molecules associated with microfibrils and elastic fibres include MAGP-1 and the glycosaminoglycan heparan sulphate. Each of these molecules has a unique molecular chemistry and interaction signature. In this article, we focus on the major elastic fibre molecules, namely fibrillin-1 and elastin. 1.1.1. Fibrillin-1 Fibrillin-1 is a large multidomain glycoprotein which contains 47 epidermal growth factor-like domains (EGF)
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domains, 43 of which are calcium-binding (cbEGF). These domains are interspersed with 8-cysteine containing (TB) modules, and there is an N-terminal proline-rich region that may act as a ‘hinge’ region [2] (Fig. 1.) In the presence of calcium, contiguous cbEGF domains adopt a relatively rod-like, although non-linear, conformation [3,4]). Fibrillin-1 has 14 N-glycosylation sites and, upon secretion, is processed at N- and C-termini by furin proteases. We have used mass spectrometry to determine the molecular composition of microfibrils, and biophysical and molecular interaction analyses to determine how fibrillin-1 molecules assemble to form tissue microfibrils. 1.1.2. Tropoelastin Tropoelastin is the soluble, secreted form of elastin. It comprises alternating hydrophobic and lysine-rich crosslinking domains [1] (Fig. 2). It acts as a ‘block co-polymer’ in vitro, undergoing a unique form of self-assembly, termed coacervation, as temperature increases. The rate of selfassembly and its initial reversibility, and the macromolecular form of the resultant multimers are regulated by salt concentration and rate of temperature change. In vitro, elastic multimers can form porous ‘gels’ that can be stabilized by synthetic crosslinks [5]. In vivo, assembled elastin is stabilized by lysyl-derived crosslinks catalyzed by members of the lysyl oxidase family, especially the lysyl oxidase (LOX) isoform [6]. We have used environmental electron scanning microscopy (ESEM) and mass spectrometry to analyze elastin coacervates, and how fibrillin-1 influences the coacervation process, and biophysical and molecular interaction analyses to determine how fibrillin microfibrils
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act as a template for tropoelastin deposition during elastic fibre formation. 2. Fibrillin isolation and purification 2.1. Cesium chloride density gradient centrifugation One of the problems encountered during the purification of cell matrix molecules is their large size. Therefore, we used a combination of density gradient and size exclusion chromatography strategies to purify fibrillin from cell media. Typically, fibrillin-1 molecules were purified on a density gradient, as previously achieved for microfibrils [7]. Caesium chloride was added to medium to give a starting density of 1.35 g/ml, and centrifuged in a Beckman Ti45 rotor at 125,000g for 70 h at 15 °C in 6 100 ml tubes. Tubes were emptied from the top and 20 fractions (5 ml) were collected. This strategy used the characteristic density of fibrillin-1 molecules (1.35 g/ml) which is larger than most of the globular proteins (1.20 g/ml). Fibrillin-1 containing fractions, detected by immuno-blotting, were pooled and concentrated by dialysis against Aquacide II (CalbiochemÒ) which decreased the volume from 125 ml to 5 ml. This 5 ml fraction was dialyzed against 0.1 M NaCl, 1 mM CaCl2, 50 mM Tris pH 8.0, and separated on a Superdex 200 size exclusion column eluted in 0.1 M NaCl, 1 mM CaCl2, 50 mM Tris pH 8.0 at a flow rate of 0.5 ml/min. Due to the characteristic large size of fibrillin-1 (350 kDa), these molecules elute in the dead volume of the column, whereas any smaller molecules (<200 kDa) would be included within the column volume.
Fig. 1. Schematic of the domain structure of fibrillin-1, including a key showing the different domains, N-linked glycosylation sites and C-terminal furin cleavage site.
Fig. 2. Schematic of the domain structure of human tropoelastin, indicating the corresponding exon numbers. Human tropoelastin consists of alternating hydrophobic (oval) and crosslinking domains (rectangle); there are two types of cross-linking domain, lysine–proline rich (KP) and lysine–alanine rich (KA).
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These combined approaches permit a pure fraction of fibrillin-1 molecules to be obtained. Having purified the protein of interest, its relative molecular mass (Mr) and N-glycosylation status can be assessed by denaturing SDS–PAGE in reducing and non-reducing conditions. The identity of the purified molecules can be confirmed using immunoblotting, using monoclonal antibodies directed to the protein of interest, for example fibrillin-1. 3. Monomer and multimer biophysical analysis For biomolecular analysis of purified molecules and multimers, it is important to establish whether the proteins of interest are monomeric, multimeric or non-specifically aggregated in native state. The size distribution of purified native proteins can readily be determined using non-denaturing agarose gel electrophoresis, or by size fractionation in native conditions. We routinely use Sephadex 200 columns for proteins in the size range 25–100 kDa. For structural and molecular interaction studies, high quality uniform monomer preparations are usually required. Extracellular matrix molecules are usually large, with a capacity to oligomerize, making it difficult to measure
molecular mass accurately. Due to their size, they are often also difficult to characterize in solution using techniques such as small angle X-ray scattering (SAXS). Furthermore, oligomers are difficult to separate and purify, making it essential to be able to analyze complex mixtures. Studies of molecular assembly intermediates and large multimers require determination of the shape and mass of a range of assemblies within a single sample. Size fractionation, laser light scattering and analytical ultracentrifugation are highly effective protocols, singly and in combination, for analyzing these parameters. For a detailed strategy for the biophysical analysis of molecules or multimers of interest, see Fig. 3. As an example, we have gained new insights into the process of fibrillin-1 microfibril assembly by defining the size distribution, molecular mass and shape of assembly intermediates of fibrillin-1 that were purified from cell culture medium, exploiting the powerful combination of analytical ultracentrifugation and multi-angle laser light scattering. The data were analysed using Sedfit software [8] which provides a means of accurately calculating the mass of individual molecular species within a complex velocity sedimentation profile. Below, we use this example
Fig. 3. Flow diagram showing potential strategies for biophysical analysis of oligomeric and non-oligomeric molecules using analytical ultracentrifugation, multi-angle laser light scattering (MALLS) and quasi-elastic light-scattering (QELS). These combined approaches allow the determination of molecular mass (Mr), size and shape of molecules and multimers in solution.
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to highlight the potential of these combined approaches to dissect the assembly pathways of other extracellular matrix multimers.
3.1. Multi-angle laser light scattering Multi-angle laser light scattering (MALLS) allows the measurement of the average molecular mass (Mr), as well as the radius of gyration (Rg) which gives size information. This can be coupled with Quasi-elastic Light Scattering (QELS) to measure hydrodynamic radius (Rh) giving further information about the size of the molecule of interest. Prior to measurement, samples are separated by size exclusion chromatography, and measurements are performed on line, allowing the determination of Mr, Rg and Rh over the peak where polydisperse sample will show the range of Mr, Rg and Rh over the peak (Fig. 4). This enabled the separation of monomers from aggregates and assemblies. Interestingly, analysis of the relationship between Rg and the Mr of the polydisperse sample provides
Fig. 4. Characterisation of purified fibrillin-1 monomers and multimers by MALLS/QELS. (A) Size exclusion chromatography of purified fibrillin-1 in 0.1 M NaCl, 1 mM CaCl2, 50 mM Tris/HCl pH 8.0, using a G5000PW size fractionation column. The column effluent was monitored on-line with MALLS, a refractometer to measure refractive index, and with QELS. Line the refractive index signal, squares show measured Mr (blue). (B) Rg (blue) and Rh (red) dependence on elution volume. (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)
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details of the size and shape of the molecule, and how these parameters are affected by an increase in total mass. Rg ¼ KM ar
ð1Þ
where a is a parameter sensitive to the shape of the macromolecules, and K is an empirical constant. For a single molecule, a rod-like conformation gives an approximated value of 1 for a, whereas a compact symmetrical structure gives an approximated value of 0.33. Analysis of the fibrillin-1 assembly intermediates presented in Fig. 4 shows the shape change towards rod-like conformation with oligomerisation. 3.2. Analytical ultracentrifugation A second approach to measure the molecular mass as well as the size (Rh) and shape of the molecule of interest is to use analytical ultracentrifugation. Sedimentation equilibrium experiments allow the measurement of molecular mass (Mr), and provide a means to analyze association. Sedimentation velocity experiment allow the measurements of sedimentation value that permit the determination of size (Rh) and shape through the calculation of the frictional ratio (f/fO). These values can then be used to model the overall shape of the molecule or multimer, using different
Fig. 5. Sedimentation characteristics and model of fibrillin-1 monomers and multimers. (A) Sedimentation values of purified fibrillin-1 in 0.1 M NaCl, 1 mM CaCl2, 50 mM Tris/HCl pH 8.0 Arrows highlight the detected species (Table 1). (B) Shell modeling (Beads model) of some of the detected species, which predicts how a fibrillin microfibril may assemble.
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Table 1 Characterisation, identification and modelling of detected fibrillin-1 species detected by MALLS/QELS and analytical ultracentrifugation Identified number of monomer 1* 1 2 4 6 10 18 22
Theorotical Mr (kDa)
Measured S
S20,w
f/f0H
Calculated S20,w from modelling
340 340 680 1360 2040 3400 6120 7480
6.3 12.3 20.4 26.4 32.5 43.5 56.7 67.7
6.5 12.7 21.1 27.3 33.6 45.0 58.7 70.0
2.94 1.49 1.43 1.75 1.87 1.96 2.23 2.13
6.2 11.8 20.8 25.3 35.1 42.8 60.6 69.6
The number of fibrillin-1 monomers was calculated using MALLS/QELS, apart from * which were calculated by Sedfit analysis. The frictional ratios (f/f0H) were calculated, taking into account hydration of 0.328 g H2O/g for fibrillin-1, as calculated directly from the amino acid sequence.
models such as prolate, spherical, circular cylinder or Shell modelling [9]. Development of new analysis software such as SEDFIT permits the analysis of complex mixtures and determination of their sedimentation characteristics, Mr and f/fO. In the example presented in Fig. 5A, the oligomerisation of fibrillin-1 has been studied. Fibrillin-1 was purified by CsCl centrifugation and size exclusion chromatography on a Sephacryl S200 column. The data show the presence of multiple oligomers that can be characterized by the Rh, f/fO and sedimentation values (Fig. 5A; Table 1). As the number of fibrillin-1 monomers increases within a multimer, there is a corresponding increase in stiffness and length, as shown by the increase in f/fOH (Table 1). Every oligomer shape has then been modelled using Shell modeling [9] techniques, and an example of such modelling data is shown in Fig. 5B. 4. Monomer and multimer imaging
brils and collagen VI microfibrils [11,12] to individual protein subunits (e.g. collagen (VIII) alpha1 subunits [13]. A fine tip, usually with a radius of curvature of around 10 nm, which is located at the end of a flexible cantilever is scanned across the surface of the sample (Fig. 6). In intermittent or ‘‘tapping” mode, the cantilever is oscillated at its resonant frequency during the scanning, which results in minimal damage to the specimen due to little or no lateral force. Repulsive forces between the tip and the sample cause the cantilever to be deflected as it scans across surface features. Laser light reflected off the back of the cantilever is collected by photodiodes and deflections of the cantilever caused by these features changes the amount of light received by the photodetectors; it is this output that is used to produce the image [14,15]. Imaging of air-dried specimens requires little sample preparation. An aliquot of sample is deposited onto a substrate surface, often freshly cleaved mica, and then allowed to air-dry. However, both substrate surface charge and buffer conditions affect the physical properties of the sample, including shape and dimensions. For example, we showed that the characteristic double beads of type VI collagen (white arrows) were apparent when the microfibrils were deposited on to mica coated with poly-L-lysine (PLL) but absent from those on untreated mica (Fig. 7) [16]. Fibrillin microfibrils (the large beaded structures in Fig. 7) also exhibit different morphology on mica or mica-PLL. A later study showed that biological functions such as cell adhesion were markedly affected by choice of substrate [17]. Calcium-bound fibrillin microfibrils have very different dimensions when adsorbed on mica or in physiological solution [18]. Our studies employ a Multimode AFM with a Nanoscope IIIa controller and an ‘‘E” scanner (Veeco Instruments), giving a scan area of 12 12 lm, and a height range up to 10 lm. Routinely, the Nanoscope software is used to determine cantilever oscillation frequency and drive
Whilst biophysical approaches provide quantitative data on molecular shape and mass, complementary microscopy approaches provide the means to visualize directly extracellular matrix molecules and assemblies. 4.1. AFM Atomic force microscopy is a powerful technique that allows the direct imaging of purified recombinant monomers and multimers in hydrated or air-dried states, without metal or carbon coating. The protein is adsorbed onto freshly cleaved mica, or to other flat surfaces such as graphite. Visualisation allows direct measurements of characteristics such as shape, molecular length, assembly periodicities and molecular organization. Developed by Binnig and colleagues [10] as an alternative method to scanning tunnelling microscopy, AFM has been used in our group to visualize a range of isolated extracellular matrix components, from fibrillin-1 microfi-
Fig. 6. Operation of an atomic force microscope. The diagram shows the arrangement of the cantilever, tip, photodetector and sample.
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Fig. 7. Atomic force microscopy of a tissue isolated sample containing fibrillin microfibrils and type VI collagen microfibrils. Samples were adsorbed onto freshly cleaved mica, or onto mica that had been pretreated with poly-L-lyine (PLL). The thin filaments (white arrows) are type VI collagen microfibrils, and the wider diameter microfibrils with prominent beads are fibrillin microfibrils. Bar = 100 nm.
amplitudes, but we adjust the setpoint to just below the point where tip-sample interaction is lost. Scanning is usually performed at rates of 1.49 Hz with scan sizes of either 5 or 2 lm2. First order flattening of images is performed using the Nanoscope software prior to exporting data to other packages for analysis. In addition to direct imaging, molecular or multimer height data can be readily obtained by AFM. For example, Sherratt and coworkers have identified age-related deterioration of fibrillin microfibrils isolated from skin biopsies by measuring changes in interbead lengths following mechanical stretching. Height data can also be used to investigate specific points of interaction, for example, to determine the location of SPARC binding sites on collagen I and procollagen I [19]. When using data derived from height measurements, it should be noted that, due to the geometry of the tip, it is often necessary to correct for the inherent widening effect that is seen [17,20]. Tip correction is a straight-forward process achieved using a calibration specimen of known dimensions, such as colloidal gold. In addition to direct imaging of unshadowed, unstained molecules, AFM can also be used as a force spectrometer. By coating the tip with specific bioactive species (such as monoclonal antibodies), it is possible to determine the binding forces between a cell surface receptor and extracellular matrix ligand. Sun et al. used this approach to study mechanical interactions between fibronectin and a5b1-integrin on vascular smooth muscle cells [21]. Graham et al. used a similar approach to acquire force elongation/relaxation profiles for human type I collagen microfibrils [22]. 4.2. ESEM The environmental scanning electron microscope (ESEM) is a relatively recent development in electron microscopy that enables the imaging of hydrated biological material without pre-treatment [23], and with a high spatial
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resolution, in some cases in the order of 1–2 nm [24]. One of the main areas where the technique has made an impact is in the field of biomaterials and tissue engineering. In particular, the ability to study cellular material in situ on a scaffold material without subjection to elaborate fixation/ dehydration procedures whilst maintaining fully hydrated state, makes the ESEM a valuable tool (Fig. 8A) [25]. Our recent ESEM studies have focused on tissue engineered arterial grafts [26] and analysis of recombinant elastin coacervates in the presence or absence of fibrillin-1 or other elastic fibre molecules [5]. Other groups have used ESEM to study hydrogels [27,28], cell growth on bioactive matrix surfaces [29] and osteoblast attachment to polymer/ hydoxyapatite composites [30]. ESEM differs from conventional scanning electron microscopy in its use of pressure limiting apertures along the length of the microscope column, such that a high vacuum is maintained at the electron gun whilst the specimen chamber is under poor vacuum. The chamber is filled with a gas, usually water vapor, and it is this feature that maintains the hydrated state of the sample whilst also providing, via the cascade effect [31], the imaging medium. Briefly, when the primary electron beam is scanned across the surface of the sample, secondary electrons are released from the surface. These secondary electrons then collide with water vapor molecules which results in further secondary electrons being released from the water molecules. These electrons then collide with further water molecules, and a cascade amplification results. The resulting secondary electron signal is collected by a specialized type of detector known as gaseous secondary electron detector, which may even form the final aperture of the microscope column itself. Positive ions produced as a result of the collisions are drawn towards the specimen surface and neutralize the charging effects caused by the non-conductive nature of the sample. We use ESEM to image a variety of extracellular matrix and fibrin-based support materials used in tissue engineering and regeneration [13,32]. These range from electro-spun polycaprolactone to biomaterials such as tropoelastin aggregates. In the latter case, a solution of coacervated and crosslinked tropoelastin was deposited onto an ESEM specimen mount and then imaged using a FEI Quanta 200 with an accelerating voltage of 10– 30 keV (Fig. 8B). The sample was held at a temperature of 5 oC using a Peltier cooling stage and, at a pressure ca 3.5 Torr, highly ordered linear arrays were observed. It is important to note that the conditions used for ESEM are more likely to result in specimen damage from the electron beam than conventional scanning electron microscopy, and so care must be observed in image acquisition and interpretation. 5. Monomer and multimer interaction analysis The processes of fibrillin-1 microfibril assembly and elastic fibre formation have been extensively probed, in
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Fig. 8. Environmental scanning electron microscopy. (A) The arrangement of microscope column, electron beams and hydrated sample; (B) ESEM analysis of coacervated recombinant human tropoelastin (globules and linear arrays).
our laboratory and others, at the molecular level using in vitro molecular interaction studies. The ultimate aims are to develop a model based on high and low affinity interactions that predicts how, and to what extent, these macromolecular complexes can undergo self-assembly. These predictions can then be tested in cell culture models, e.g. using knock-down and knock-in approaches, or in mouse models, to establish the roles for individual components in this hierarchical process. Molecular binding assays can also be used to validate models derived from biophysical analyses, e.g. our fibrillin-1 microfibril assembly based on MALLS, AUC and Shell modelling (as outlined above). 5.1. Surface plasmon resonance Surface plasmon resonance (SPR) is a useful way of discovering interactions between ECM molecules. The technology involves attaching one of the interacting pair of molecules to a sensor chip (ligand). A sample containing one or more interacting molecules is passed over the sensor chip surface, and binding of molecules to the ligand generates a response which is proportional to the bound mass. Binding is monitored in real time which
allows values such as specificity of an interaction, the affinity and kinetics of an interaction, and even the concentration of the bound ligand (Fig. 9). Automation allows the screening of a large number of interactions in one experiment. Detailed kinetic experiments can be run to determine the kinetics of these interactions. It can be used to study protein-protein, protein-glycosaminoglycan, or any other molecule that can be immobilized onto the sensor chip surface, such as potential drug. Nucleic acids are also easy to immobilize onto the sensor chip surface, and interactions have been reported using larger complexes such as viruses and even whole cells. The most widely used system for measuring SPR, is the Biacore supplied by GE Healthcare, which have a variety of different systems based on SPR, each of which provides different levels of automation and number of interactions measured, ranging from simple single interactions to multiple arrays. One of the most widely used machines for ECM research is the Biacore 3000, as well as its predecessor Biacore 2000 and 1000 instruments. SPR on the Biacore 3000 instrument involves attachment or immobilisation of your ligand of choice onto one of several different sensor chip surfaces via either a covalent linkage, high affinity capture or hydroscopic adsorption.
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Fig. 9. The principles of immobilisation and binding by surface plasmon resonance using Biacore. (i) The sensor chip consists of a gold surface which is coated with a carboxylated dextran matrix. (ii) The carboxylated matrix is then activated (1) then the ligand is adsorbed to the surface then crosslinked (2), before the remaining activated sites are blocked (3). The real time sensorgram is shown in magenta indicating the 3 stages (above). (iii) When an analyte is passed over the ligand and interaction occurs, the response on the sensorgram is increased until saturation occurs or injection ends (4). After injection, the analyte begins to disssociate from the ligand and the sensorgram response drops (5).
5.1.1. Immobilization protocols The most common method of immobilisation is to covalently link your protein of interest to a modified dextran matrix which is attached to the gold surface of the sensor chip. The usual methods are to link via primary amine groups, thiol groups or active disulphides or to immobilise using aldehyde coupling by oxidising carbohydrate groups of you ligand. The next most common method of ligand immobilisation is the high affinity capture of biotin to a covalently linked streptavidin molecule. The key advantage of SPR over other interaction methods such as solid phase binding or calorimetry is the low sample amount needed. For the immobilised ligand, this is even more so with most experiments needing as little only a microgram of protein, making the use of commercially available recombinant proteins economically viable. For the analyte, more reagent is needed but preliminary studies can be carried out with as little as 10 lg of protein, making the process economical with precious or hard to purify samples. 5.1.2. Interaction of MAGP-1 with fibrillin-1 To map the interaction of MAGP-1 with fibrillin-1, MAGP-1 was immobilized onto a CM5 sensor chip using amine coupling, fragments of fibrillin-1 was then passed over the surface of the chip and the response of the analyte scan was measured [33] (Fig. 10A and B). The identified binding fragments were then used in a more detailed analysis to determine the binding kinetics. This is achieved by injection of several concentrations of the fibrillin-1 fragments, the resultant curves could then be fitted to a mathematical model of 1:1 binding kinetics or, if this is not possible, the equilibrium binding constant (KD) can be calculated by plotting the maximum binding response against
concentration. (Fig. 10C). Using this analysis the KD of the interaction was calculated to be 242 nM. 5.1.3. Interaction of fibrillin-1 with heparin SPR is not only limited to protein-protein interactions, and glycosaminoglycans such as heparin, heparan sulphate or chondroitin sulphate, once biotinylated can be immobilised using a streptavidin immobilised SA sensor chip. Glycosaminoglycans were biotinylated using various methods, but it was found that coupling the biotin molecule at the end of the heparin, which mimics the attachment to glycoproteins, produced a higher affinity binding [34]. Using this method, several heparin binding sites were identified on fibrillin-1, and detailed kinetic data was obtained. It was found that heparin bound to the same N-terminal region of fibrillin-1 that also bound MAGP-1 (see above). This binding allowed inhibition analysis using the Biacore with immobilised heparin, where increasing concentrations of MAGP-1 were found to inhibit fibrillin-1 binding. The inverse was also found to be true, that increasing concentrations of heparin inhibited fibrillin-1 binding to immobilised MAGP-1 (Fig. 10D). 6. Molecular composition of elastic fibre complexes 6.1. Mass spectrometry analysis of the composition of matrix complexes The advent of mass spectrometers has allowed the identification of protein complexes, at biological levels. It allows non-selective discovery of proteins, without the need of antibodies or other predetermined markers, making it an ideal tool for analysing samples, or tissues with unknown
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Fig. 10. (A) Diagram showing recombinantly expressed fibrillin-1 protein fragments used in binding studies using surface plasmon resonance, indicating the fibrillin-1 domains they contain. The scheme uses the same symbols as shown in Fig. 1. (B) Interaction of MAGP-1 with fibrillin-1 fragments. Fibrillin1 fragments were injected at 200 nM (black) and 40 nM (gray) over a MAGP-1-immobilised surface, and the maximum response difference (Resp. Diff.) for each experiment is shown in resonance units (RU). (C) Fibrillin fragment PF1 was injected over a MAGP-1-immobilised surface. Each sensorgram shows six different analyte concentrations of 0.5, 1, 5, 10 (duplicated), 15, and 20 lg/ml. One representative experiment is shown. The inset shows the saturation binding curve, calculated using the maximum response values for each concentration, plotted against concentration. Using nonlinear regression, KD and Bmax values were calculated using GraphPad Prism version 2.0. Time is shown in seconds (s). (D) To illustrate that Biacore may also be used to perform inhibition analysis, MAGP-1 and a heparan oligosaccharide dp24 were both found to inhibit each other from binding the N-terminal region of fibrillin-1 (fragment PF1). For inhibition of the fibrillin-1 PF1 interaction with heparin dp24 by MAGP-1 (j), PF1 (1 lg/ml) was incubated with MAGP-1 (0.4–1200 nM), before addition to immobilised dp24. For inhibition of PF1 with MAGP-1 by dp24, PF1 (1 lg/ml) (N), was incubated with dp24 (0.4–4000 nM) before addition to immobilised MAGP-1. The response difference was normalised to a percentage of the response for no inhibitor added.
molecular compositions. Mass spectrometers consist of a source which generates gaseous ions from the sample, and an analyser. Gaseous ions can be manipulated by electrical and magnetic fields, which allows mass and charge states to be determined by the analyser. Further data can also be obtained if the machine contains a fragmentation device which can break peptide ions into small fragments usually around the peptide bond. There are two common types of source for analysing biological samples, a MALDI (Matrix Assisted Laser Desorption/Ionisation) source generated ions by firing a laser at your sample which has been mixed with matrix that converts the laser energy into excitation energy. The other common source is by electrospray ionization (ESI), where charged droplets are sprayed from a capillary tube, then the solvent in the droplet evaporates off leaving the charged sample ions. The mass to charge ratio is then determined using the analyser, which includes one or a combination of different types. These types include
time of flight (TOF), Quadropole, or Ion Trap, each manipulating the ions in a different way. Accuracy of the mass determination is greater if the peptide ion is lower in mass, so proteins are usually digested before with the protease trypsin. Multiple peptide ions per protein allowing a greater chance of identification, and these peptide ions may also be further fragmented to give some sequence information about each peptide, thereby increasing further the identification accuracy. To aid the analysis of peptides, separation using reverse phase chromatography, prior to injection, decreases the amount of peptide ions analysed at any one time. Separation of peptides can also be further achieved by including a second dimension to the chromatography, which is usually ion exchange. Greater mass accuracy, speed of analysis and methods of detection over time have allowed the analysis of more and more complex samples, making it a powerful technique in biological research.
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Fig. 11. Fibrillin-1 domain structure, showing analysis of the pattern of tryptic peptides obtained from the human zonular microfibril preparations. (i) The number of times each tryptic peptide was identified; (ii) the number of peptides identified in each domain. The seven TB motifs are numbered in an N- to C-terminal direction. Domains with peptide hits only after guanidine HCl (denaturing) extraction are shown with hatching.
6.2. Composition of fibrillin-rich microfibrils To investigate the composition of fibrillin-rich microfibrils, fibrils were isolated from ciliary zonules, aorta and skin, using a non-denaturing method of collagenase digestion, or a denaturing method using guanidine solubilisation [35]. The microfibrils were then isolated by size exclusion chromatography, followed by concentration by freeze drying then digested using trypsin in reducing conditions. The study identified that the microfibrils predominantly consisted of fibrillin-1. MAGP-1 was also detected, but no other predicted microfibril-associated components such as LTBPs or fibronectin were detected. The pattern of tryptic peptides identified from fibrillin-1 was not as complete as that obtained from identically treated recombinant fibrillin-1 fragments. Whilst tryptic peptides from certain domains of the molecule were found in multiple microfibril purifications, other regions had less or no detectable peptides (Fig. 11). Further investigation of the trypsin-digested microfibrils by AFM revealed an undigested part corresponding to the bead structure, which is thought to be non-reducibly crosslinked by transglutaminase. 6.3. Mass spectrometry to investigate novel interactions To investigate novel fibrillin interactions, mass spectrometry was used to identify proteins that were co-purified during fishing experiments; for these studies, we used proteins secreted into culture medium or deposited within the extracellular matrix of cell cultures known to form fibrillin microfibrils. Recombinant proteins, including overlapping fibrillin-1 fragments, MAGP-1, fibulin 1, fibulin 5 and lysyl oxidase were used as bait proteins; each contained a six-histidine tag which allowed easy selection of protein complexes. The bait proteins were incubated with the growth media and secreted matrix from human dermal fibroblasts (HDF) and human retinal pigment epithelial cells (ARPE 19). Purified protein complexes were analysed using tandem mass spectrometry, and the results collated into a large database which allowed the analysis of primary and secondary interactions of the complexes. Over 200 interacting species were identified using two separate search
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