From the Divisions of Molecular and Cellular
Biochemistry and § Structural Biology, Department of
Biochemistry, University of Oxford, South Parks Road, Oxford OX1 3QU,
United Kingdom
Received for publication, July 21, 2000, and in revised form, November 16, 2000
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ABSTRACT |
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Human fibrillin-1, an extracellular matrix
glycoprotein, has a modular organization that includes 43 calcium-binding epidermal growth factor-like (cbEGF) domains arranged
as multiple tandem repeats. A missense mutation that changes a highly
conserved glycine to serine (G1127S) has been identified in cbEGF13,
which results in a variant of Marfan syndrome, a connective tissue
disease. Previous experiments on isolated cbEGF13 and a cbEGF13-14 pair indicated that the G1127S mutation caused defective folding of cbEGF13
but not cbEGF14. We have used limited proteolysis methods and
two-dimensional NMR spectroscopy to identify the structural consequences of this mutation in a covalently linked cbEGF12-13 pair
and a cbEGF12-14 triple domain construct. Protease digestion studies of
the cbEGF12-13 G1127S mutant pair indicated that both cbEGF12 and 13 retained similar calcium binding properties and thus tertiary structure
to the normal domain pair, because all identified cleavage sites showed
calcium-dependent protection from proteolysis. However,
small changes in the conformation of cbEGF13 G1127S, revealed by
the presence of a new protease-sensitive site and comparative
two-dimensional NOESY data, suggested that the fold of the mutant
domain was not identical to the wild-type, but was native-like.
Additional cleavage sites identified in cbEGF12-14 G1127S indicated
further subtle changes within the mutant domain but not the flanking
domains. We have concluded the following in this study. (i) Covalent
linkage of cbEGF12 preserves the native-like fold of cbEGF13 G1127S and
(ii) conformational effects introduced by G1127S are localized to
cbEGF13. This study demonstrates that missense mutations in
fibrillin-1 cbEGF domains can cause short range structural effects in
addition to long range effects previously observed with a E1073K
mutation in cbEGF12.
The epidermal growth factor-like
(EGF)1 domain is a widely
distributed module found in transmembrane and extracellular proteins (1) where it may occur as multiple tandem repeats. The module is
characterized by six highly conserved cysteine residues, which normally
disulfide bond in a 1-3, 2-4, 5-6 arrangement and stabilize the
global fold of the domain. A subset of these domains are distinguished by the presence of a calcium binding consensus sequence
(D/N)X(D/N)(E/Q)Xm(D/N*)Xn(Y/F), where m and n are variable, and an asterisk indicates a possible Fibrillin-1, a 350-kDa extracellular matrix glycoprotein, is a major
structural component of 10-12-nm connective tissue microfibrils (16).
It has a modular organization and is mainly composed of multiple tandem
arrays of EGF domains, the majority of which contain calcium binding
sites (Fig. 1). In addition there are
seven transforming growth factor
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
-hydroxylation site (2-4). In tandem repeats of fibrillin
calcium-binding EGF (cbEGF) domains, the bound Ca2+
performs a key structural role in restricting interdomain flexibility, which may facilitate protein-protein interactions (5, 6) and also
protect the modules against proteolytic cleavage in vitro (7, 8). The biological importance of the cbEGF domain is highlighted by
the number of diseases that are caused by missense mutations within
this domain type. These include Marfan syndrome (MFS) and related
disorders, congenital contractural arachnodactyly, hemophilia B,
familial hypercholesterolemia, "CADASIL" (cerebral autosomal
dominant arteriopathy with subcortical infarcts and leukoencephalopathy) (mutations in fibrillin-1 (9), fibrillin-2 (10),
factor IX (11), low density lipoprotein receptor (12), and Notch 3 (13), respectively), and protein S (14) and protein C deficiencies
(15).
-binding protein-like (TB) domains,
two hybrid domains, a proline-rich region, and N and C termini with
homology to other matrix proteins (17). Mutations within the
fibrillin-1 gene (FBN-1) result in a spectrum of connective
tissue disorders (fibrillinopathies), which range in severity from MFS,
Shprintzen-Goldberg syndrome, and ectopia lentis to familial ascending
aortic aneurysm (18). Of particular interest are the structural
consequences of a missense mutation, which changes a highly conserved
glycine to a serine in cbEGF13 of human fibrillin-1. The G1127S
mutation produces a variant of the MFS phenotype and has been
identified as a risk factor for ascending aortic aneurysm and
dissection (19). Interestingly, the same mutation has been identified
in human factor IX (G60S) where it is associated with mild hemophilia B
(20). The location of G1127S within the "neonatal" region of
fibrillin-1 (Fig. 1), where MFS causing mutations are associated with
extreme phenotypic diversity, suggests that structural investigations of this region may yield important insights into the mechanism of
disease.
View larger version (11K):
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Fig. 1.
Schematic illustration of the domain
organization of fibrillin-1. The positions of the neonatal region
and the cbEGF domain constructs studied here are indicated. The G1127S
mutation occurs in cbEGF13, highlighted by an
asterisk.
A previous study has shown that the G1127S mutation resulted in
defective folding in vitro when introduced into the single cbEGF domain 13. When present in the covalently linked cbEGF13-14 domain pair, the C-terminal cbEGF14 domain adopted the native fold and
retained calcium binding properties despite the fact that the adjacent
domain 13 was misfolded. This suggested that the effects of the
mutation were localized (21). Here the structural consequences of the
G1127S mutation in the cbEGF12-13 domain pair and also in the triple
domain fragment, cbEGF12-14, have been investigated, using a
combination of protease digestion studies and two-dimensional NMR
methods. The data indicate that covalent linkage of cbEGF12 moderates
the effect of G1127S in cbEGF13 resulting in localized changes to the
structure and calcium binding properties of domain 13, but not cbEGF12
or 14. The implications of such short range effects for the pathogenic
mechanism of MFS are discussed.
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EXPERIMENTAL PROCEDURES |
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Cloning of Wild-type and Mutant cbEGF Domain Constructs from Human Fibrillin-1-- DNA fragments (nucleotides 3338-3595 and 3338-3721 of human fibrillin-1 cDNA) encoding the wild-type sequences of the cbEGF12-13 domain pair and the cbEGF12-14 triple construct (residues 1069-1154 and 1069-1196 respectively, numbering according to Ref. 22) were amplified by standard polymerase chain reaction techniques using Pfu polymerase (Stratagene). The forward primer in cbEGF12 and the reverse primers for cbEGF13 and cbEGF14, together with the cloning procedures used, were as described previously (21, 23).
The G1127S mutation was introduced into the pQE30 recombinant plasmids containing the cDNA sequences of the cbEGF12-13 double or cbEGF12-14 triple constructs by PCR-based site-directed mutagenesis. The plasmids were amplified using a forward primer: 5'-GAGGTAGTGTTTGCCATAAC-3' and a reverse primer: 5'-GGCATAGGAGAGGATC-3'. The amplified DNA was purified from a 0.7% agarose gel with a Qiaex II gel extraction kit (Qiagen) and ligated and transformed into Escherichia coli NM554[pREP4]. Clones were sequenced to confirm the mutation had been introduced with no other changes into the inserted fragments.
Protein purification, refolding, and His-tag cleavage were carried out as described previously (21), except that the cleaved cbEGF12-14 triple construct was further purified on a fast protein liquid chromatography (FPLC) MonoQ column in 50 mM Tris, pH 7.5, using a 0-0.5 M NaCl gradient prior to a final reverse phase HPLC step. The identity of the purified products was confirmed by mass spectrometry (Table I).
Proteolysis of Wild-type and Mutant cbEGF Domain Constructs-- Peptides were dissolved to give a final protein concentration of 3.2 mg/ml in a total volume of 100 µl containing 100 mM NaCl, 50 mM Tris pH 7.5, and either 10 mM EGTA or 10 mM CaCl2. Incubation with trypsin (EC 3.4.21.4; bovine pancreatic; TPCK-treated; Sigma; 1:1000, w/w) or endoproteinase GluC from Staphylococcus aureus (EC 3.4.21.19; Sigma; 1:100, w/w) was carried out at 37 °C. At the times indicated, samples (10 µl) were withdrawn, and the reaction was stopped by the addition of 2× SDS sample buffer containing 100 mM dithiothreitol and heating at 95 °C for 4 min. The reaction mixtures were analyzed by 16% SDS-PAGE and visualized by Coomassie Blue staining.
For N-terminal sequence analysis of the cleavage sites, samples were digested for 30 min in the presence of EGTA or calcium, the reaction was stopped by boiling for 4 min, and the digest products were purified under non-reducing conditions by reverse phase HPLC. Samples were analyzed by SDS-PAGE, and N-terminal sequencing was carried out using automated Edman chemistry. The samples were absorbed onto PVDF (polyvinlidene difluoride (0.2-µm porosity) using a ProSorb cartridge (PE Biosystems, Warrington, UK.) following the manufacturer's protocol. The membrane-bound samples were then excised from the ProSorb cartridge, and N-terminal sequencing was performed on an Applied Biosystems 494A `Procise' sequencer (PE Biosystems). Cysteine residues reported in the N-terminal sequences were inferred from the published sequence (22).
Structural and Calcium Binding NMR Studies of the cbEGF12-13
Domain Pair--
All NMR spectra were recorded on a home-built/GE
Omega spectrometer equipped with self-shielded pulsed field gradients
at 600 MHz. The wild-type and G1127S mutant cbEGF12-13 domain pair protein samples were dissolved in 550 µl of 90% H2O,
10% 2H2O, 5 mM Tris-HCl, pH 6.5, to yield final protein concentrations of 2.00 and 1.93 mM,
respectively. Added NaCl was not utilized in these investigations
because high ionic strength may compromise spectral quality.
Two-dimensional NOESY spectra (24, 25) were acquired for both samples
at 0 mM CaCl2 and 12.5 mM
CaCl2 to allow qualitative assessment of calcium binding.
At 12.5 mM CaCl2, the two calcium binding sites
of the wild-type domain pair are saturated based on previous calcium
binding studies of the cbEGF12-13 domain pair (26), and these
calcium-loaded spectra were also used to assess the structural
consequences of the G1127S mutation. All spectra were recorded with a
mixing time of 150 ms at T = 33 °C. Water suppression was
achieved using field gradients (27). Data were processed using Felix
2.3 (Biosym, Inc.). 1024 complex points were acquired in F2
and F1 for each experiment with a spectral width of 8000 Hz
in each dimension. Spectra were referenced with respect to the
H2HO resonance and were zero-filled to 8 K in the
F2 dimension to yield a digital resolution of 0.98 Hz/pt.
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RESULTS |
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Expression and Purification of Wild-type and Mutant cbEGF
Domain Constructs--
The predicted structure of the cbEGF12-14
triple construct analyzed in this study, modeled on the coordinates of
the cbEGF32-33 domain pair from human fibrillin-1, is shown in Fig.
2 (5, 23). This model has been validated
by NMR structural studies of the cbEGF12-13
pair.2 The G1127S mutation in
domain 13 was introduced into the cbEGF12-13 double and cbEGF12-14
triple constructs at the position indicated. The wild-type and mutant
domain pairs and triple constructs were expressed and purified as
described previously (21). After reduction and refolding in
vitro followed by reverse phase HPLC, the cbEGF12-13 domain pairs
and cbEGF12-14 triple constructs had slightly different elution times.
However, there was no difference in the elution profile of each mutant
from the corresponding wild-type construct. On in vitro
refolding in the presence of Ca2+ each gave one major
species, the molecular mass of which was confirmed by mass spectrometry
(Table I). Together, the characteristic change in the elution profile on refolding of the pair and triple constructs, the observed Ca2+-dependent
protection against proteolysis, and the NMR analysis of the wild-type
cbEGF12-13 domain pair (see below) were all indicative of correctly
folded cbEGF domains (28).
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Protease Digestion of the cbEGF12-13 Domain Pairs--
Digestion
and subsequent SDS-PAGE analysis of both the wild-type and G1127S
mutant cbEGF12-13 constructs by trypsin in the presence of EGTA (10 mM) or Ca2+ (10 mM) showed that
there was significant protection by calcium against proteolysis (Fig.
3A). Although minor
differences were apparent between the wild-type and mutant constructs
in the presence of EGTA, the degree of protection by Ca2+
appeared equivalent. Similar SDS-PAGE analysis of endoproteinase GluC digests also demonstrated protection by calcium (data not shown). Digestion products obtained in the presence of Ca2+
and EGTA were purified under non-reducing conditions by HPLC, and the
N-terminal sequences of each were determined. The amount of each N
terminus expressed relative to the authentic N-terminal sequence is
shown in Table II. In both the mutant and
wild-type cbEGF12-13, three trypsin and one endoproteinase GluC
susceptible cleavage sites were located in cbEGF12. The cbEGF12-13
G1127S mutant contained an additional endoproteinase GluC site
(1134GSYRC) in cbEGF13. Calcium-dependent
protection from proteolysis of all sites in the mutant pair, including
this additional site in the mutant domain, was found, and the degree of
protection was the same as in the wild-type pair (Table II).
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Protease Digestion of the cbEGF12-14 Triple Domain Constructs-- Trypsin digestion and subsequent SDS-PAGE analysis of both the wild-type and mutant cbEGF12-14 triple constructs in the presence of EGTA (10 mM) and Ca2+ (10 mM) again showed the protection against digestion afforded by calcium (Fig. 3B). The G1127S mutation did not appear to significantly affect the calcium binding properties of the construct, because the calcium-dependent protection from proteolysis was indistinguishable from that of the wild-type on SDS-PAGE. Calcium protection was also evident on digestion by endoproteinase GluC (data not shown).
N-terminal sequence analysis of HPLC-purified digestion products
obtained in the presence of EGTA or Ca2+ identified the
cleavage sites shown in Table II. On endoproteinase GluC digestion, an
extra site 1134GSYRC, not present in the wild-type
construct, was identified in the mutant. This site, located at the turn
of the central two-stranded anti-parallel -sheet, was the same site
revealed in the cbEGF12-13 G1127S pair. In the case of trypsin, two
additional sites, 1126GSVCH and 1138CECPP, were
seen upon digestion of the mutant construct. The additional site at
1126GSVCH is adjacent to the mutated residue whereas the
other protease sensitive site, 1138CECPP, occurs distal to
the mutation but still within cbEGF13 (Fig.
4). The comparison of the amount of each
of these N termini identified on digestion in EGTA or calcium, and
expressed relative to the authentic N-terminal sequence is shown in
Table II. Calcium-dependent protection from proteolysis of
all cleavage sites, including those in the mutant domain, was observed.
All cleavage sites were represented in comparable amounts indicative of
efficient cleavage of a single population of molecules.
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Structural and Calcium Binding NMR Studies of the cbEGF12-13 Domain
Pair--
Comparative analysis of the calcium-saturated
two-dimensional NOESY spectra for the wild-type and G1127S mutant
cbEGF12-13 constructs indicated that, unlike the case for the
cbEGF13-14 wild-type and mutant spectra (21), a significant number of
peaks corresponding to domain 13 were unaffected or only mildly
affected by the presence of the G1127S mutation. The fingerprint
regions of these spectra are shown in Fig.
5. Most resonances appeared unaltered
between the two spectra, and a subset of peaks were only slightly
shifted. For example, of the two well resolved sets of triplet peaks in
the lower left-hand corner of the spectra, the chemical shifts of the
upper set of peaks were identical, whereas the positions of resonances
in the lower triplet were slightly shifted. Because these peaks involve
connectivities to downfield-shifted CH, which are typically involved
in
-structure, the data suggest that domain 12 in the mutant
cbEGF12-13 pair is unaffected by the presence of the G1127S mutation,
and that the
-sheet region of domain 13 is only mildly affected.
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To examine calcium binding properties of each construct, the aromatic
regions of the two-dimensional NOESY spectra acquired at 0 and 12.5 mM CaCl2 were overlaid for both the wild-type
and G1127S mutant cbEGF12-13 domain pair (Fig.
6). Chemical shift changes of the H*
resonances of the consensus, aromatic, calcium binding residues for the
N- and C-terminal domains of each pair, i.e.
Phe1093 and Tyr1136, respectively, were used to
assess calcium binding. The overlay of the wild-type NOESY spectra at
zero and saturating calcium showed the expected, native binding
properties previously observed when Kd values were
determined for the two sites in this domain pair (26). Comparison of
the wild-type spectral overlay with that of the G1127S mutant revealed
that both domain 12 and 13 retained calcium binding properties in the
mutant cbEGF12-13 pair. Domain 12 appeared to retain native calcium
binding properties because the Phe1093 peak movement was
identical to that seen in the wild-type spectrum. The peak movement
seen for Tyr1136 in cbEGF13 was slightly altered, however,
with a larger chemical shift change associated with binding by the
mutant domain. Collectively, these comparative data indicate that when
preceded by cbEGF12, the G1127S mutant cbEGF13 domain preserves a
native-like but not identical fold.
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DISCUSSION |
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In a previous study, it was shown that G1127S caused misfolding of isolated domain 13 with loss of calcium binding properties, whereas, in a cbEGF13-14 domain pair, cbEGF14 was unaffected. In this study, the effects of the G1127S mutation on the cbEGF12-13 domain pair have been assessed, and the results indicate that domain 12 is unaltered, and the consequences of this mutation are again localized to domain 13. The demonstration that domain 13 in the 12-13 pair retains the ability to bind calcium is unlike the situation previously seen for both the single mutant cbEGF13 domain and the mutant cbEGF13-14 domain pair. As calcium binding by cbEGF domains is used as a probe for correct refolding, the fact that cbEGF13 continues to bind calcium in the mutant cbEGF12-13 pair suggests that this domain preserves a native-like fold. This hypothesis is also supported by a comparative analysis of the calcium-saturated wild-type and mutant two-dimensional NOESY spectra for the cbEGF12-13 pair, which show only minor differences (see Fig. 5).
These NMR studies indicate that the G1127S mutation has a less severe
effect on folding when preceded by cbEGF12. Measurement of the calcium
binding affinities of the two sites in the cbEGF12-13 wild-type pair by
NMR and fluorescence spectroscopy has previously demonstrated that
covalent linkage of an N-terminal cbEGF domain had a stabilizing effect
on the adjacent C-terminal domain (26, 6). The observation in this
study that cbEGF12 moderates the effect of the G1127S folding mutation
also suggests an effect of N-terminal linkage, because structural
features associated with cbEGF domains (-sheet, calcium binding)
were absent in cbEGF13 or cbEGF13-14 mutant constructs.
In parallel with NMR analyses, protease digestion studies have also been used to probe the structural and calcium binding properties of the wild-type and G1127S-containing fibrillin fragments. It has previously been shown that tandem repeats of wild-type cbEGF domains are susceptible to proteolytic cleavage at specific sites on removal of calcium by EGTA, whereas in the presence of calcium, protection from proteolysis is observed (7, 29). An increased susceptibility to proteolysis in vitro also results from the introduction of specific calcium binding mutations in both recombinantly expressed polypeptides (30) and domain pairs (29). A comparison of protease digestion profiles together with the identification of how far reaching the loss of calcium-dependent protection from proteolysis is can therefore be used to assess the short versus long range consequences of various mutations. As this approach can be applied to relatively large fragments and only requires a small amount of material, it can be successfully applied to the investigation of large numbers of mutations in a more native setting.
On endoproteinase GluC digestion of the cbEGF12-13 pair, an additional cleavage site was revealed in cbEGF13 of the mutant fragment, which showed calcium-dependent protection from proteolysis. These results are consistent with the NMR analysis of this domain pair, suggesting that the mutant domain has a degree of disruption but is not severely misfolded. A comparison of the digestion patterns of the cbEGF12-14 triple construct with those of the corresponding wild-type provided additional insights. In addition to the endoproteinase GluC site in the mutant domain, also identified in the cbEGF12-13 pair, two tryptic sites were identified in the mutant triple construct indicative of further subtle changes to the mutant domain, which did not detectably affect its calcium binding properties.
The presence of the cleavage site 1134GSYRC identified in
both the 12-13 and 12-14 mutant constructs (Fig. 4) and located distal to the mutation suggests a conformational change around the -sheet region of domain 13. Additional tryptic cleavage sites
1138CECPP and 1126GSVCH, observed only in 12-14 are supportive of this, although because of the proximity of these
sites to the mutation (in the case of C1138 via its
disulfide bond to C1124) we cannot formally exclude a
sequence-specific effect of the mutation that enhances substrate
recognition by the protease in the triple domain construct. However, an
alternative explanation that the addition of a C-terminal cbEGF domain
may influence the structure of the preceding mutant domain is also
possible. The position and calcium-dependent properties of
the cleavage sites in the flanking domains 12 and 14 were the same in
the mutant and wild-type constructs, indicative of a structural change
localized to cbEGF13. The protease data are therefore consistent with
the NMR data for the cbEGF12-13 (this study) and cbEGF13-14 (21) domain
pairs and validate this as a useful method for probing structural
effects of mutations. A summary of the structural and calcium binding
consequences of the G1127S mutation for cbEGF13, the cbEGF13-14 pair
(21), the cbEGF12-13 pair and the cbEGF12-14 triple construct (present
study) are schematically represented in Fig.
7. Further comparative NMR analyses of
the structure and dynamics of both the wild-type and mutant constructs
will allow the degree of disruption caused by the mutation to be
defined more precisely.
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The tryptic sites identified in cbEGF12 of both the pair and triple constructs were identical to those found in digests of a recombinantly expressed cbEGF10-22 construct containing a calcium binding mutation E1073K in cbEGF12 (30). E1073K is associated with neonatal MFS, the most severe form of the disease. In this case, the sites within mutant domain 12 showed enhanced susceptibility to proteolysis compared with the wild-type, in contrast to the present study in which the sites in mutant domain 13 containing the G1127S mutation were protected by calcium. In addition a cleavage site was also revealed N-terminal to cbEGF11, indicating a longer range structural effect of the calcium binding mutation. Collectively these data indicate that MFS-causing mutations in this region cause variable intramolecular effects on fibrillin-1 structure.
Because the effect of the G1127S mutation is confined to domain 13 and this domain retains a native-like fold, fibrillin-1 monomers containing this mutation are likely to be secreted by the cell and the effect of the mutation exerted either on or after incorporation into the microfibril. The small localized changes in structure and calcium binding in domain 13 could disrupt protein binding sites involved in the assembly process or the properties of the assembled microfibril. The protection afforded by calcium against proteolytic degradation of tandem repeats of cbEGF domains in fibrillin-1 (7, 29) suggests that missense mutations may result in increased proteolysis in vivo. Although cbEGF13 containing the G1127S mutation appears to be more susceptible to proteolysis than the wild-type in vitro, calcium-dependent protection was observed for all the additional protease sites revealed in the domain (Table II), therefore increased proteolytic susceptibility seems less likely to be involved in the pathogenic mechanism than for the E1073K mutation.
In summary, interdisciplinary studies utilizing both high and low
resolution methods have proved effective in identifying the structural
consequences of the G1127S mutation. Further studies will now focus on
identifying the functional effects of this and related mutations on
fibrillin assembly.
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ACKNOWLEDGEMENTS |
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We thank Tony Willis for amino acid analysis and N-terminal sequencing and Dr. Robin Aplin for mass spectrometry analysis.
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FOOTNOTES |
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* This work was supported in part by the Wellcome Trust.The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
¶ Members of the Oxford Centre for Molecular Sciences, which is funded by the MRC, BBSRC, and EPSRC.
Supported by the Medical Research Council and St. Johns
College, Oxford.
** A Wellcome Trust Senior Fellow.
To whom correspondence should be addressed. Tel.: 44 0 1865 285347; Fax: 44 0 1865 275259; E-mail: penny@bioch.ox.ac.uk.
Published, JBC Papers in Press, January 31, 2001, DOI 10.1074/jbc.M006547200
2 R. S. Smallridge, P. Whiteman, J. M. Werner, P. A. Handford, and A. K. Downing, manuscript in preparation.
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ABBREVIATIONS |
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The abbreviations used are: EGF, epidermal growth factor-like; cbEGF, calcium-binding epidermal growth factor-like; MFS, Marfan syndrome; NOESY, nuclear Overhauser enhancement spectroscopy; HPLC, high performance liquid chromatography; PAGE, polyacrylamide gel electrophoresis.
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