From the Department of Cell Biology and Physiology,
Washington University School of Medicine, St. Louis, Missouri 63110, the § Department of Clinical Chemistry, Hoshi University
School of Pharmacy, Tokyo 142-850, Japan, and the
¶ Department of Anatomy and Cell Biology, McGill University,
Montreal, Quebec H3A 2B2, Canada
Received for publication, December 13, 2002, and in revised form, March 3, 2003
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ABSTRACT |
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Elastic fiber assembly is a complicated process
involving multiple different proteins and enzyme activities. However,
the specific protein-protein interactions that facilitate elastin polymerization have not been defined. To identify domains in the tropoelastin molecule important for the assembly process, we utilized an in vitro assembly model to map sequences within
tropoelastin that facilitate its association with fibrillin-containing
microfibrils in the extracellular matrix. Our results show that an
essential assembly domain is located in the C-terminal region of the
molecule, encoded by exons 29-36. Fine mapping studies using an exon
deletion strategy and synthetic peptides identified the hydrophobic
sequence in exon 30 as a major functional element in this region and
suggested that the assembly process is driven by the propensity of this sequence to form The inherent complexity of the elastic fiber, combined
with the unique physical properties of its component proteins, has made
understanding elastin structure and assembly one of the most difficult
problems in matrix biology. Although technical advances in cell and
molecular biology have given us new information about elastin gene
expression and elastin synthesis, surprisingly little is known about
how the elastic fiber is assembled at the molecular level. The major
component of the mature fiber is a covalently cross-linked polymer of
the protein elastin. Elastin is secreted from the cell as a soluble
monomer called tropoelastin. In the extracellular space, lysine
residues within tropoelastin are specifically modified to form covalent
cross-links between tropoelastin chains. This cross-linked polymer has
a high degree of reversible distensibility, including the ability to
deform to large extensions with small forces.
The tropoelastin molecule is characterized by a series of tandem
repeats, each including a lysine-containing cross-linking region
followed by a hydrophobic motif (1). Cross-linking is initiated by the
extracellular enzyme(s) lysyl oxidase, which catalyzes the
oxidative deamination of lysyl Recently, two inherited diseases, autosomal dominant cutis laxa and
supravalvular aortic stenosis
(SVAS),1 have been linked to
mutations within the elastin gene that may alter the ability of the
elastin precursor to undergo normal assembly (5, 6). Each of these
diseases has a distinct clinical phenotype and may be caused by
fundamentally different molecular mechanisms. Autosomal dominant cutis
laxa is characterized by redundant, loose, sagging, and inelastic skin
with variable systemic organ involvement. The known elastin mutations
in autosomal dominant cutis laxa are single nucleotide deletions in
exons 30 and 32 that, depending on exon splicing, give rise to a
missense peptide sequence extending into the 3'-untranslated region (6,
7). Although the exact pathomechanism of this disease is not clear, it
is thought that alterations at the C terminus of the secreted mutant
protein interfere with the deposition of normal elastin in a
dominant-negative fashion (5, 6).
SVAS is characterized by thickening of the arterial wall and either
focal or diffuse narrowing of the aorta and, frequently, of other major
arteries. Mutational analysis has identified a wide spectrum of
mutations associated with SVAS, including large deletions,
translocations, inversions, and, most frequently, point mutations
(8-12). Accumulating evidence suggests that the pathomechanism of SVAS
is haploinsufficiency (13), which can result from the deletion of one
complete copy of the elastin gene (as occurs in Williams syndrome) or
through functional hemizygosity arising from loss-of-function mutations
in one elastin allele.
The objective of this study was to better characterize elastic fiber
assembly and to identify the domains on the tropoelastin molecule that
mediate this process. We were also interested in determining whether
proteins from transcripts with SVAS-like mutations are capable of
incorporating into elastic fibers and, if so, whether they interfere
with normal fiber function. Using an in vitro model system
of fiber assembly together with expression of tropoelastin constructs
in transgenic mice, we provide evidence for a critical assembly domain
located in the region between exons 29 and 36 that mediates the
interaction of tropoelastin with microfibrils in the extracellular
matrix. Studies with synthetic peptides and tropoelastin deletion
constructs suggest that the hydrophobic sequence encoded by exon 30 is
a major functional element in this region. These findings also provide
an explanation for how SVAS mutations could lead to haploinsufficiency
when the truncated product of the mutant allele is secreted without
this critical assembly domain.
Materials--
All reagents were purchased from Sigma unless
otherwise noted.
Antibodies--
The antibody used to detect bovine elastin in
in vitro experiments with pigmented epithelial (PE) cells is
a polyclonal antibody generated against recombinantly produced human
tropoelastin. The only exception occurred in experiments in which
matrices were probed concurrently for tropoelastin and fibrillin-1. In
those experiments, BA4, a monoclonal antibody reactive against bovine tropoelastin (14), and Fib-1 CT, a polyclonal antibody generated against the recombinant FBN1 C-terminal domain (15), were used. Tissue
immunofluorescence studies made use of BA4 and anti-mouse recombinant
tropoelastin antibody N6-17, a polyclonal antibody generated against
recombinantly produced murine tropoelastin exons 6-17. BA4 will detect
bovine and human elastin, but does not interact with mouse
tropoelastin. In addition, CTe, a polyclonal antibody directed against a folded peptide encoded by exon 36 of bovine elastin
(16), was used in Western blotting to probe for proteolytic fragments
containing the tropoelastin C terminus.
Transfection Constructs--
The transfection constructs used
for this study were derived from a full-length bovine cDNA that has
been previously characterized (17). It carries a deletion of exons 13 and 14. When attempts were made to amplify the missing exons from
cultured fetal bovine chondrocytes using RT-PCR, cDNA containing
the exons could not be identified, suggesting that the
To generate the full-length transfection construct, bovine tropoelastin
cDNA was cloned into the MluI-XbaI
site of the pCIneo vector (Promega). Construction of the 1-28
truncation was achieved by PCR amplification of the appropriate exons
from the full-length cDNA. The forward primer contains an
MluI site and the ATG from exon 1, whereas the reverse
primer contains a stop site and the XbaI restriction site
for cloning. Primer sequences are as follows: exon 1F,
5'-GCACGCGTATGCGGAGTCTGACGGCTCGG-3'; and exon 28R,
5'-GCTCTAGAGTCATGCCACACCACCTGGAATGCC-3'. Generation of the remaining
constructs required a pCIneo vector lacking the manufacturer-provided
NotI site (pCIneo*). This was generated by cutting the
vector with NotI and filling the resultant linearized vector
with Klenow fragment, followed by self-ligation. Full-length
tropoelastin was then cloned into the pCIneo* vector at the
XbaI site (FL*). Only one NotI site is present in
FL*, located at the beginning of exon 29. To generate the truncation constructs 1-29 and
Several steps were required to generate construct Tissue Culture--
PE cells (20) were maintained in Dulbecco's
modified Eagle's medium (Cellgro) supplemented with nonessential amino
acids (Cellgro), sodium pyruvate (Cellgro), penicillin/streptomycin (Washington University Tissue Culture Supply Center), and 10% cosmic
calf serum (Hyclone Laboratories). Cells were maintained in a 37 °C
humidifying CO2 incubator.
Transfection--
Stable transfection of PE cells was performed
using Lipofectin (Invitrogen) according to the manufacturer's
instructions. Briefly, 2 × 105 cells were plated in a
six-well plate. When cells reached 60-80% confluence, 1 µg of the
chosen construct and 1 µl of reagent complex were added to the cells
in Opti-MEM serum-free medium (Invitrogen). Sixteen hours later, the
transfection reagents were removed, and fresh Dulbecco's modified
Eagle's medium containing 10% cosmic calf serum was added. After
48 h, cells were then placed under selection with 500 µg/ml active Geneticin (G418 sulfate, Invitrogen).
Immunofluorescence--
To detect bovine tropoelastin in the
matrix of stable pools of G418-selected PE cells, the cells were plated
on glass coverslips in six-well plates and allowed to reach confluence.
Seven days after visual confluence, the medium was removed, and the
cell layer was washed with phosphate-buffered saline (PBS) three times to remove all non-cell layer-associated proteins. Cells were then fixed
with ice-cold methanol for 60 s and washed with PBS to remove any
remaining alcohol. The cells were treated with blocking solution consisting of PBS containing 1% gelatin and 0.1% Tween 20. The anti-human recombinant tropoelastin antibody was used at a dilution of
1:250 in blocking solution. Fluorescent secondary antibody (goat
anti-rabbit 488, Molecular Probes, Inc.) was used at a
concentration of 1:2000 in blocking solution. After secondary antibody
incubation, the cells were washed before removal from the
six-well plate and inversion of the coverslip onto a glass slide.
Fluorescence was protected using anti-fade mounting medium (Gelmount,
Biomedia Corp.). A Zeiss Axioscope microscope was used for fluorescence microscopy, and the images were captured on an Axiocam digital camera
using Axiovision software. All images are shown at magnification ×40
except where noted.
Metabolic Labeling and Immunoprecipitation--
Conditioned
media from confluent monolayers of fetal bovine chondrocytes,
untransfected PE cells, and stably transfected PE cells that
were metabolically labeled with
L-[4,5-3H]leucine (1 mCi/ml; ICN
Pharmaceuticals, Irvine, CA) were immunoprecipitated for tropoelastin
as described previously (19). Immune complexes were pelleted, washed,
and resuspended in 35 µl of Laemmli sample buffer containing 100 mM dithiothreitol. Samples were electrophoresed on
SDS-polyacrylamide gels, fixed, treated with EN3HANCE
(PerkinElmer Life Sciences) for 1 h, dried, and exposed to X-Omat
AR film (Eastman Kodak Co.).
Transgenic Animals--
Animals were generated expressing either
full-length bTE or bTE-(1-28). To generate the full-length bTE and
bTE-(1-28) vectors, the pCIneo constructs described above were
linearized, yielding two constructs that contained all of the pCIneo
vector components in addition to the appropriate tropoelastin sequence.
To generate construct RT-PCR--
RNA was isolated from wild-type and transgenic
animals using RNAzol (Tissue Tek) according to the
manufacturer's instructions. Heart, lung, kidney, and liver total RNAs
were screened in all founders tested. RT-PCR was performed on 1 µg of
each resultant RNA under the following conditions. Residual DNA was
removed by treating the RNA with RQ1 RNase-free DNase in first-strand
RT reaction buffer (Invitrogen) containing 2 µl of 0.1 M
dithiothreitol for 30 min at 37 °C. The enzyme was heat-inactivated
at 72 °C for 10 min. Reverse transcription was performed with
Superscript II reverse transcriptase (Invitrogen) according to the
manufacturer's instructions in the presence of dNP6 random primers
(Roche Applied Science) and dNTPs (Promega). PCR was then performed on
the resultant cDNA with bovine tropoelastin-specific forward
(5'-GGAATTGGAGCCATTCCCACATTTGGG-3') and reverse
(5'-GAGCCACGCCGACTCCAGG-3') primers. Thirty-five cycles of
amplification were performed with a 67 °C annealing temperature and
a 30-s extension time. The products of this reaction were then analyzed
by gel electrophoresis.
Immunofluorescence Analysis--
Tissues from transgenic and
wild-type animals were dissected and washed with sterile PBS before
being embedded in blocks containing OCT freezing medium (Tissue
Tek). The tissues were then frozen over dry ice, and 10-µm sections
were cut using a microtome.
For immunofluorescence analysis, sections were thawed to room
temperature, fixed in cold ethanol, and etched to enhance antigenicity with 1 mg/ml hyaluronidase in 0.1 M sodium acetate buffer
(pH 5.5) containing 0.85% NaCl for 30 min at 37 °C. After
hyaluronidase treatment, the tissues were treated as described above
for tissue culture cells. The antibodies used were BA4 and N6-17. Both
were used at a 1:500 dilution. Goat anti-mouse 594 and goat anti-rabbit 488 secondary antibodies (Molecular Probes, Inc.) were used for primary
antibody detection.
Quick-freeze Deep-etch Electron Microscopy and Congo Red Staining
of Exon 30 Peptides--
A peptide encoding exon 30 of bovine
tropoelastin (GLGGVGGLGVGGLGAVPGAVGLG) was synthesized by solid-phase
peptide synthesis using a 431 A peptide synthesizer (ABI) running
Fast-Moc chemistry and dissolved at 10 mg/ml in 7 M
guanidine HCl. A second, scrambled version (LVGGGGGGLPVLGGAGALGGVGV)
was also synthesized as a control for this assay. The peptide stock
solution (10 µl) was diluted with 200 µl of PBS and 200 µl of
water and subjected to slow rotation overnight at room
temperature. Precipitated peptide was pelleted by centrifugation in a
microcentrifuge, smeared on a glass slide, fixed in 95% ethanol, and
stained with 0.05% Congo red in 50% glycerol. Evaluation of Congo red
staining by polarization microscopy was performed using a Zeiss
Axioscope equipped with optimally aligned cross-polarizers. For
electron microscopy, freshly prepared mica flakes were added to
solutions containing peptide filaments, followed by freeze-drying and
platinum replication according to established procedures (18, 19).
Purification of Recombinant Tropoelastin--
Full-length bovine
tropoelastin was cloned into the pQE vector (QIAGEN Inc.), expressed in
bacteria, and purified with nickel-nitrilotriacetic acid resin (QIAGEN
Inc.) according to the manufacturer's instructions. Bound protein was
then eluted with 6 M urea buffer (pH 4.0) and dialyzed in
0.1% acetic acid. Protein concentration was quantified by amino acid
analysis, and aliquots of 200 µg were lyophilized.
Protease Treatment and Western Blotting--
Aliquots (200 µg)
of recombinant tropoelastin were resuspended in 350 µl of PBS
containing the protease inhibitors EDTA (1 mM) and
pepstatin (1 mM) to block proteolysis by non-trypsin-type proteases. A sample was taken prior to the addition of plasmin at time
0. Then, 1 µl of 0.2 µg/ml plasmin was added, and 50-µl samples
were taken at 30, 60, and 120 min. Plasmin activity was stopped by
adding 1× SDS-PAGE sample buffer containing 0.1 M
dithiothreitol and boiling for 5 min. The sample was electrophoresed on
SDS-polyacrylamide gels and transferred to nitrocellulose (Schleicher & Schüll). Nitrocellulose blots were blocked in 5% (w/v) nonfat
milk in 50 mM Tris (pH 7.5), 171 mM NaCl, and
0.05% (v/v) Tween 20 (TTBS). The CTe primary antibody (18)
was diluted 1:250 in TTBS containing 2.5% (w/v) nonfat milk and
incubated for 1 h at room temperature. The blot was then washed
and incubated with peroxidase-conjugated donkey anti-rabbit IgG
secondary antibody (Amersham Biosciences) diluted 1:2000.
Chemiluminescence detection was performed using ECL Western blot
detection reagents (Amersham Biosciences) and exposed to XAR-5 x-ray
film (Eastman Kodak Co.).
The Presence of the Tropoelastin C Terminus Is Required for Its
Deposition into the Extracellular Matrix of PE Cells--
To determine
which regions of tropoelastin are necessary for its association with
the extracellular matrix, we generated expression constructs consisting
of full-length elastin as well as mutant and deletion forms of the
molecule. These constructs were then transfected into PE cells, and the
ability of the expressed transgene to associate with microfibrils in
the PE cell matrix was determined by immunofluorescence microscopy. PE
cells are a cell line derived from the pigmented epithelial cells in
the ciliary body of the bovine eye (20). They are known to lay down an
elaborate fibrillar matrix composed of fibrillin-1, fibrillin-2, and
MAGP1 (microfibril-associated glycoprotein-1), but do not produce
endogenous elastin (17). Because these cells produce all of the
components necessary to form the scaffolding for elastic matrices, but
not tropoelastin itself, they provide a useful system for studying the
early stages of elastic fiber assembly.
After stable transfection of PE cells with the full-length bovine
tropoelastin construct, microfibrils in the PE cell matrix became
decorated with elastin expressed from the transgene (Fig. 1). In this experiment, elastin was
detected using the anti-human recombinant tropoelastin antibody, which
has been shown to cross-react with the bovine protein. As expected,
untransfected cells did not contain elastin in their extracellular
matrix (Fig. 2A). Because earlier studies had suggested that the C terminus of tropoelastin contains a critical assembly site (16), our truncation and mutation constructions focused on this region. Exon 36 encodes a conserved sequence that contains the molecule's only 2 Cys residues and a
terminal Arg-Lys-Arg-Lys sequence. The cysteine residues have been
shown to form a disulfide-bonded loop structure that creates a highly
charged "pocket" at the end of the molecule believed to facilitate
interactions between tropoelastin and highly acidic microfibrils. It
was an antibody to this exon 36 sequence that disrupted elastin fiber
assembly in studies by Brown-Augsburger et al. (16). To
assess the importance of this region of the protein to elastin
assembly, we generated tropoelastin constructs in which first one and
then both of the cysteines residues were mutated to alanines, as well
as one construct in which the Arg-Lys-Arg-Lys sequence was deleted. A
fourth construct in which exon 36 was deleted in its entirety (
We next assayed a series of C-terminal truncations that systematically
deleted individual exons beginning with exon 36. Like construct Expression of Full-length and Truncated Tropoelastin as Transgenes
in Mice--
To determine how the absence of the C-terminal region of
tropoelastin alters its incorporation into existing elastic fibers, we
generated transgenic animals expressing either full-length bTE or
bTE-(1-28). Using species-specific antibodies, it was possible to
determine whether either of the transgenes was able to incorporate into
elastic fibers made by the mouse and whether either acted through a
dominant-negative mechanism to disrupt normal mouse elastic fiber
assembly. The constructs consist of linearized forms of the
transfection constructs used in the in vitro experiments. They contain the immediate-early components of the cytomegalovirus promoter to guide expression of the transgene and a SV40-derived 3'-untranslated region and poly(A) signal.
After founder lines were stabilized, animals were tested for expression
of the transgene. RT-PCR performed on RNA isolated from full-length and
exon 1-28 transgenic animals showed that message from the transgene
was being transcribed and was stable in these animals (lung tissue
shown in Fig. 4). Using an antibody that
detects human and bovine elastin, but not mouse elastin, and one that
detects only mouse elastin, we assayed frozen sections from wild-type
(non-transgenic) and transgenic lines. As expected, wild-type
non-transgenic lungs showed no staining with the bovine-specific antibody (Fig. 4A), but significant elastic fiber staining
with the mouse antibody was observed (Fig. 4D). Analysis of
the four founders expressing the full-length bovine tropoelastin
transgene found uniform deposition of bovine elastic fibers in the
heart and developing aorta (data not shown), consistent with past
studies showing strong expression of the cytomegalovirus promoter in
these tissues (21-23). In other tissues, deposition of bovine elastic fibers differed among founders, most likely resulting from
transcriptional differences associated with positional effects
surrounding the location of transgene insertion. Commonly positive
tissues included lung (Fig. 4B), kidney, bladder, and small
blood vessels of the liver (data not shown). In all tissues in which
the full-length transgene was expressed in a given animal, the bovine
protein was found to incorporate into existing mouse elastic fibers
with no obvious alteration of fiber structure (Fig. 4E). In
contrast to what was found with the full-length bovine transgene, the
product of the exon 1-28 transgene did not associate with elastic
fibers in any of the mRNA-positive tissues when assayed in multiple
founders (Fig. 4C). The tissue expression pattern for the
exon 1-28 transgene, based on mRNA analysis, was essentially the
same as that observed for the full-length construct.
The Amino Acid Sequence Encoded by Exon 30 Aggregates to Form
Amyloid-like Fibers with Deletion of Exon 30 Decreases, but Does Not Ablate, Elastic Fiber
Formation--
The propensity of the exon 30 peptide to form
amyloid-like aggregates and the failure of tropoelastin truncation
constructs lacking exon 30 to associate with the PE cell matrix
suggested that the exon 30 sequence might facilitate deposition of
tropoelastin into the extracellular matrix and hence influence elastin
fibrillogenesis. This possibility was tested by expressing, in the PE
cell system, a tropoelastin construct with only exon 30 deleted
(
Expression of construct Identification of a Hypersensitive Protease Site That May Influence
Assembly--
In analyzing tropoelastin secreted into the culture
medium of transfected PE cells, we noticed several proteolytic
breakdown products associated with the full-length molecule and
mutation forms that were not evident in samples of the
bTE-(1-28) protein (Fig. 3). Further repetitions of the experiment
revealed varying degrees of degradation for all of the constructs from
experiment to experiment. The only construct for which fragmentation
was reproducibly decreased was bTE-(1-28). The molecular mass of the primary breakdown product of the full-length molecule was similar in
size to the intact bTE-(1-28) protein (i.e. ~55
kDa), suggesting that a cleavage site may be present near the exon 28 border that, when cleaved, leads to removal of the C-terminal assembly sequence.
To investigate susceptible sites for proteolytic cleavage, we treated
recombinant tropoelastin with plasmin for times ranging from 30 min to
2 h. Earlier studies determined that the degradation of
tropoelastin isolated from developing chick aorta could be largely
prevented by the inclusion of inhibitors of trypsin-like proteases (28,
29). Given that plasminogen, the pro form of the enzyme plasmin, is a
trypsin-like protease present in high concentrations in serum and that
blood vessels are a key location for elastin assembly, the choice of
this protease seemed to be a reasonable one. When recombinant
tropoelastin was treated with plasmin at 37 °C, a ladder of
fragments similar to the naturally occurring breakdown products was
detected (data not shown). At early time points, many fragments of
various sizes were present, but by 2 h of treatment, only one
resistant C-terminal fragment remained (Fig.
8), as evidenced by Western blotting of
the proteolytic fragments probed with an antibody specific for the
extreme C terminus of tropoelastin (CTe) (16).
N-terminal sequencing of the various breakdown products revealed two
major tropoelastin cleavage sites: one in exon 26 (R Using an in vitro assembly assay together with
expression studies in transgenic mice, we have shown that the
C-terminal region of elastin, encoded by exons 29-36, contains an
important assembly domain that directs the association of tropoelastin
with microfibrils. Our focus on the C-terminal region of tropoelastin
was guided by previous studies from our laboratory suggesting that the
C terminus of the molecule is important for its assembly into
fibers. In these experiments, accumulation of elastin in the
extracellular matrix of fetal bovine chondrocyte cells was inhibited
when an antibody to the 17 amino acids encoded by the final exon of the elastin gene (exon 36) was added to the cultured cells. An antibody to
a sequence in the N-terminal end of the molecule (exon 4/5) had no
effect (16). Our initial mutation and deletion constructs expressed in
PE cells were focused on exon 36, but we found that these amino acids
are not required for elastin secretion or for its deposition into the
extracellular matrix. In fact, mutation of one or both of the cysteine
residues to alanine, deletion of the terminal RKRK sequence, or
deletion of the entire exon 36 had no observable effect on the ability
of the mutant protein to associate with microfibrils. Rather, a large
portion of the C-terminal region consisting of exons 29-36 had to be
deleted before elastin accumulation in the extracellular matrix could be entirely inhibited. Because the truncated molecules were effectively secreted and were stable in the culture medium, these findings suggest
that sequence 29-36 contains epitopes, exclusive of exon 36, necessary
for mediating the association of tropoelastin with microfibrils in the
extracellular matrix.
Although the PE cell assay provides an excellent means to assess
whether a given form of tropoelastin can associate with microfibrils in
the extracellular matrix, its reliance on immunofluorescence co-localization provides only limited information about the quality of
the resultant elastic fiber. We were not able to determine by this
method, for example, whether any of the deletion constructs found to
co-localize with microfibrils associated less efficiently than the
full-length protein or bound in a way that precluded later assembly
steps. However, that there may be quantitative differences is suggested
by preliminary experiments in which desmosine levels were found to be
lower in constructs lacking exon
36.2 This result is
consistent with data reported by Hsiao et al. (30) showing
decreased cross-linked elastin in the matrices of cells to which
tropoelastin lacking exon 36 had been added.
The importance of the C-terminal sequence to tropoelastin assembly was
confirmed using transgenic mice, which also allowed us to determine
whether tropoelastin molecules bearing C-terminal mutations would lead
to defects in elastic tissues through dominant-negative effects.
Characterization of the different mouse lines showed that animals
expressing wild-type bovine tropoelastin transgenes successfully
incorporated the protein into the mouse elastic fiber. In contrast, no
bTE-(1-28) protein could be detected in any of the tissues in
which mRNA for the transgene was readily identified. Incorporation
of a transgenic construct lacking exon 30 into elastic fibers was
observed, but at levels significantly lower than those of the
full-length protein. Each of these findings is consistent with results
found in PE cells, where construct Fine mapping of the matrix-binding activity of tropoelastin using
synthetic peptides or expression constructs with single exon deletions
localized sequences in and around exon 30 as being the major
interactive site. The sequence encoded by exon 30 contains a tandem
repeat (GGLG(V/A)) that resembles sequences found in other proteins
that aggregate via The presence of an assembly site centered on exon 30 has important
implications for understanding both normal elastin assembly and the
molecular mechanisms of diseases arising from mutations within the
elastin gene. For example, the characteristic mutations reported for
autosomal dominant cutis laxa are single nucleotide deletions in exons
30 and 32 that result in out-of-frame sequence extending into the
3'-untranslated region with notable loss of the cysteine residues in
exon 36. It has been speculated that this abnormal sequence might
disrupt assembly of normal elastin in a dominant-negative fashion. In
contrast, the majority of mutations in isolated SVAS are point
mutations that produce premature termination sites. Urbán
et al. (13, 38, 39) have shown that many of these mutations
produce an unstable mRNA transcript that is rapidly degraded
through nonsense-mediated decay, resulting in elastin haploinsufficiency at the RNA level. In one case, however, mRNA and
protein from a mutant elastin gene were identified in cells from an
SVAS individual, although at reduced levels compared with the wild-type
allele (13). If mRNA from a gene with a truncation mutation escapes
nonsense-mediated decay, the absence of a C-terminal assembly domain
may preclude its incorporation into the growing fiber. The result would
be haploinsufficiency at the protein level.
Removal of the C-terminal domain by proteolytic events outside the cell
also appears to be a mechanism for regulating elastin assembly in
instances of normal tissue growth and remodeling. For example, closure
of the ductus arteriosis shortly after birth involves dissolution of
the vessel's elastic laminae and ingrowth of intimal cushions. At the
same time, tropoelastin secreted by ductus arteriosis smooth muscle
cells is inhibited from forming new elastic fibers by proteolytic
removal of the C-terminal domain (40). Although the exact cleavage site
within the tropoelastin molecule was not characterized in the ductus
arteriosis study, the size of the truncated protein (52 kDa) is similar
to that of the fragment expected when tropoelastin is cleaved with
plasmin at exon 26. This site in exon 26 has previously been described as being susceptible to cleavage by kallikrein and thrombin (41) and
clearly defines a location on the surface of the tropoelastin molecule
that is accessible to trypsin-like proteases. The identification of a
hypersensitive protease site and the finding in the ductus arteriosis
that the C-terminal region of tropoelastin can be specifically removed
by proteolysis to inhibit assembly provide evidence for a possible
mechanism for extracellular control of elastic fiber formation.
It has long been assumed that coacervation of tropoelastin is a crucial
step in assembly of the elastic fiber (42-44). Our data suggest,
however, that coacervation may not be involved in the initial steps of
elastin assembly and that coacervation by itself cannot drive elastin
assembly in tissues. Coacervation, an entropically driven, inverse
temperature transition caused by the interaction of the hydrophobic
domains in the molecule, occurs as tropoelastin monomers associate to
form large aggregates. Several laboratories have shown that the large
hydrophobic sequences in the middle of the molecule play a dominant
role in the intermolecular interactions that occur during coacervation
(43). If coacervation were the only requirement for assembly, then all
of our deletion constructs would form fibers (or at least aggregates)
because they all contain the critical middle hydrophobic sequences.
This was clearly not the case, as constructs bTE(1-28) and -sheet structure. Tropoelastin molecules lacking the C-terminal assembly domain expressed as transgenes in mice did not
assemble nor did they interfere with assembly of full-length normal
mouse elastin. In addition to providing important information about
elastin assembly in general, the results of this study suggest how
removal or alteration of the C terminus through stop or frameshift mutations might contribute to the elastin-related diseases
supravalvular aortic stenosis and cutis laxa.
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
-amino groups. Under normal
conditions, the cross-linking process is extremely efficient, with all
but ~5 of the ~40 lysine residues in tropoelastin participating in
covalent linkages that form the functional polymer. How the cross-linking sites within the monomer are aligned prior to
cross-linking is unclear. It has long been assumed that microfibrils
provide a scaffold or template for elastin assembly by binding and
aligning tropoelastin monomers so that lysine-containing regions are in register for cross-linking. This idea evolved from electron microscopic images showing that the appearance of microfibrils is the first ultrastructural indication of the elastic fiber (2-4) and that microfibrils are associated with elastin throughout the elastogenic period.
EXPERIMENTAL PROCEDURES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
13-14 form
is the dominant transcript in these elastogenic cells. Numbering
associated with deletion constructs refers to sequences encoded by the
exons that make up the bovine tropoelastin cDNA.
36, PCR products were amplified using the exon
27F primer (5'-CCCTGGCCGCAGCTAAAGCAGCCAAGTTCG-3') and the exon
29R primer (5'-GCTCTAGAGTCAAAATTGGGCTTTGGCGGCA-3') or the exon 35R
primer (5'-GCTCTAGAGTCAAAATTGGGCTTTGGCGGCA-3'). The reverse primers contain both the stop site for the molecule and an
XbaI site for cloning purposes. After amplification, FL* and
the PCR products were cut with NotI and XbaI and
ligated to one another. Similarly, the
RKRK construct (representing
the deletion of the last 4 amino acids from the molecule) was generated
by amplification using the above exon 27F primer and the
RKRK-R
primer that deletes these C-terminal amino acids
(5'-GCTCTAGAGAAAATTGGGCTTTGGCGGCA-3'). This product was also cut with
NotI and XbaI and inserted into the similarly
cleaved FL* vector. The internal deletion
33 was constructed by
RT-PCR amplification of total RNA isolated from fetal bovine ligament
cells. Fetal bovine ligament cells are known to express an
alternatively spliced form of tropoelastin in which exon 33 has been
spliced out (1). The PCR primers used for the amplifications include
the exon 27F primer as well as the exon 36R primer that leads to
amplification of the entire 36th exon as well as its stop codon
(5'-GCTCTAGAGTCACTTTCTCTTCCGGCCACA-3'). The product of the
amplification was then cleaved with NotI and XbaI
and ligated into the FL* vector cut with the same enzymes. To generate
the Cys-to-Ala mutations, oligonucleotide-directed mutagenesis was
performed using a QuikChange site-directed mutagenesis kit (Stratagene)
with oligonucleotides C755A-F
(5'-TGGGGAAATCCGCTGGCCGGAAGAG-3') and
C755A-R (5'-CTCTTCCGGCCAGCGGATTTCCCCA-3') and
oligonucleotides C751A-F
(5'-CCAGGTGGGGCCGCCCTGG-3') and C751A-R
(5'-ATTCCCCACCGCGGCCCCACCTGG-3') according
to the manufacturer's recommendations.
30. First, exons
28-36 were amplified by PCR. The primers used were exon 28F
(5'-GGAATTCAGATCTTGGTGGAGCCG-3') and the exon 36R primer listed above. The exon 28F primer contains an EcoRI site
such that the product can be digested with EcoRI and
XbaI and ligated into a similarly digested pUC-19 shuttle
vector (pUC-28-36). To generate the
30 insert, exon 30 was deleted
by PCR amplification of a 113-bp fragment using the exon 28F primer and
the exon 29/31R primer
(5'-AACTGCAGCTGGAGACACACCAAATTGGGCGGCTTTGGCGGC-3') encoding a PstI site in exon 31. The
EcoRI-PstI-restricted fragment was ligated into
the similarly digested pUC-28-36 plasmid, resulting in the pUC
30
plasmid. pUC
30 was then digested with NotI and XbaI, and the bTE
30 insert was ligated into the
NotI-XbaI-restricted FL* plasmid, resulting in
pCIneo-bTE
30. All alterations were verified by nucleotide sequencing.
30, the pCIneo construct was linearized, and
extraneous plasmid sequences (neo cassette, etc.) were
removed by restriction digestion with ClaI. In all cases,
the appropriate fragment was purified using a QIAGEN gel extraction
kit. Each linearized product contained, at a minimum, the tropoelastin
sequence flanked by the cytomegalovirus immediate-early promoter and
the SV40-derived 3'-untranslated region and poly(A) signal. Each
fragment was resuspended at 2 µg/ml in injection buffer composed of 5 mM Tris-HCl (pH 7.4), 0.25 mM EDTA (pH 8.0),
and 5 mM NaCl and was injected into B6C3/F1 mouse
fertilized eggs, which were implanted into the uteruses of
pseudopregnant foster mothers. After birth, potential founders were
screened for the presence of the transgene using PCR and bovine
tropoelastin-specific forward (5'-GGGGTACCAGGAGCTGTTCC-3') and reverse
(5'-CCTTGGGCTTGACTCCTGCTC-3') primers. Once detected, animals positive
for the transgene were mated to wild-type animals to stabilize the line.
RESULTS
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
36)
was also generated. Interestingly, protein from all of the mutant
constructs localized to the fibrillar matrix of PE cells (Fig.
2C), indicating that the sequence encoded by exon 36 is not
required for the initial association of tropoelastin with
microfibrils.
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Fig. 1.
Co-localization of tropoelastin and
fibrillin-1 in PE cell matrices. PE cells were transfected
with bovine tropoelastin cDNA, and the ability of the resultant
protein to associate with microfibrils was assessed by
immunofluorescence microscopy. Double labeling with antibodies reactive
against elastin (BA4) (A) and fibrillin-1 (anti-C-terminal
domain antibody) (B) showed that the two sets of fibers
co-localized in the extracellular matrix (C).
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Fig. 2.
Immunostaining of elastin in the
extracellular matrix of PE cells transfected with bovine tropoelastin
constructs. Various mutation and deletion tropoelastin cDNA
constructs were transfected into PE cells, and their ability to be
deposited into the extracellular matrix was assessed by
immunofluorescence microscopy. A schematic of the various constructs
used for transfection of PE cells is shown. The presence (+) or absence
( ) of the transgenic protein in the extracellular matrix is
indicated, as is minimal incorporation of tropoelastin (+/
).
A-F show PE cells stained with the anti-human recombinant
tropoelastin antibody. A-D and F are
shown at magnification ×40, whereas E is ×10.
Untransfected PE cells contained no elastin in their extracellular
matrix (A). PE cells were transfected with full-length
(FL) bovine tropoelastin (B), full-length bovine
tropoelastin containing the C751A (CtoA) mutation affecting
intrachain disulfide bonds encoded by exon 36 (C), exons
1-30 of bovine tropoelastin (D), exons 1-29 of bovine
tropoelastin (E), and exons 1-28 of bovine tropoelastin
(F). All constructs with the exception of 1-28 and 1-29
were incorporated into the extracellular matrix of PE cells.
bTE-(1-28) was absent from the matrix, whereas bTE-(1-29) was
minimally incorporated.
36,
protein from constructs lacking exons 31-36 (Fig. 2D) was
deposited onto microfibrils. When exon 30 was then deleted (i.e. the expressed protein contained only exons 1-29), the
association of protein with microfibrils was substantially decreased
(Fig. 2E) relative to the full-length control. Deletion of
exon 29 (resulting in construct 1-28) completely abolished elastin
assembly into the matrix (Fig. 2F). Protein from all of the
constructs was detected at high levels in PE cell-conditioned medium
(Fig. 3), confirming their synthesis and
secretion.
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Fig. 3.
Immunoprecipitation of tropoelastin from
transfected PE cell media. Immunoprecipitation of metabolically
labeled conditioned media from stably transfected PE cells (see Fig. 1)
confirmed that all of the transgenes were expressed and secreted.
Un, medium from untransfected PE cells; FBC,
conditioned medium from fetal bovine chondrocytes, a cell line that
secretes bovine tropoelastin and served as a positive control;
FL, PE cells transfected with full-length bovine
tropoelastin; CtoA, C751A mutant; 1-29, bovine
tropoelastin containing exons 1-29; 1-30, bovine
tropoelastin containing exons 1-30; 33, bovine
tropoelastin with exon 33 deleted;
36, bovine
tropoelastin with exon 36 deleted; RK, bovine tropoelastin
with the last 4 amino acids deleted (
RKRK). The major band at ~55
kDa is a stable degradation product resulting from cleavage by
trypsin-like enzymes at the end of exon 26.
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Fig. 4.
RT-PCR data and immunostaining of transgenic
animals expressing bovine tropoelastin. RT-PCR performed on RNA
derived from the lungs of transgenic animals showed the presence of
bovine transcripts from both the full-length (FL) and exon
1-28 transgenes. To assure the absence of genomic DNA contamination,
RT-PCR was performed in the presence (+) and absence ( ) of reverse
transcriptase. Frozen sections of lung tissue derived from wild-type
(A and D), full-length bTE transgenic
(B and E), and bTE-(1-28) transgenic
(C and F) animals stained either with BA4 to
visualize bovine tropoelastin (A-C) or with anti-mouse
recombinant tropoelastin antibody (N6-17) to visualize murine
elastin (D-F). All tissues were positive for murine
tropoelastin, but only the full-length transgenic animals incorporated
bovine tropoelastin.
-Sheet Structure--
To better understand
the mechanism of C-terminal based tropoelastin deposition, we used
synthetic peptides to investigate whether particular sequences in this
region contribute to tropoelastin aggregation. One sequence in
particular was suggested by earlier studies of Robson et al.
(24, 25), who pointed out similarities between structural motifs in
exon 30 of tropoelastin and other proteins that aggregate via
-sheet/
-turn structures. To determine whether the exon 30 sequence might form similar structures, synthetic exon 30 peptide was
dissolved in water and analyzed at 6-h intervals for the formation of
precipitates. By 12 h, fine rod-like fibers were visible in the
solution; and by 24 h, a substantial precipitate had formed.
Staining of the precipitate with Congo red and analysis under polarized
light showed a characteristic apple green birefringence associated with
intercalation of the dye into regions of
-structure (26). Electron
microscopy of the exon 30 aggregate (Fig.
5) revealed that the peptide formed
filamentous aggregates with a diameter of ~7-10 nm, similar to those
seen for lamprin and other amyloid-forming sequences (27). Duplicate
experiments were performed in buffers containing physiologic salt
(PBS). Although fibers were visible by eye in these experiments, the
salt crystals that formed during the experiment inhibited the
visualization of precipitated peptide by the various microscopy
methods. Additionally, a change in incubation temperature from room
temperature to 37 °C did not substantially change the quantity or
quality of fibers formed (data not shown). No fibers were ever observed
with the exon 30 scrambled peptide.
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Fig. 5.
Exon 30 peptide forms amyloid-like
fibers. A synthetic peptide encoded by exon 30 was dissolved in
water and allowed to form aggregates overnight. Precipitates were
pelleted and prepared for quick-freeze deep-etch microscopy. The
micrograph shows that exon 30 peptides polymerized into fibers.
Scale bar = 50 nm.
30). Relative to the full-length construct (Fig.
6A), deposition of the
30 protein into matrix fibers was greatly reduced (Fig. 6B).
Rare fields were present, however, in which deposition of
30 did
take place. Expression of a construct containing an internal deletion of a different hydrophobic exon in the C-terminal domain, exon 33 (
33), yielded an elastin product that was deposited into fibers in
the extracellular space. Although the fibers may be qualitatively different from those produced by the full-length construct (Fig. 6C), the fact that they were deposited shows that the result
found for
30 was not simply a consequence of exon deletion in
general.
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Fig. 6.
Deletion of exon 30 decreases, but does not
completely inhibit, elastin deposition. PE cells transfected with
the full-length (A) or 30 (B) expression
construct were stained with an antibody to detect bovine tropoelastin.
Deposition of the
30 protein into the extracellular matrix was
significantly reduced relative to the full-length protein. A construct
containing an internal deletion of exon 33 (
33), another hydrophobic
exon in the C-terminal domain, yielded an elastic matrix similar in
appearance to that generated by the full-length construct
(C).
30 as a transgene in mice gave results
similar to those observed with PE cells. The
30 protein was detected
in several tissues and organs of the transgenic mice, but the amount of
incorporated protein was much less than what was found for the
full-length transgene (Fig. 7).
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Fig. 7.
Immunostaining of bovine tropoelastin in
full-length and 30 transgenic animals.
The lungs (A) and hearts (C) from transgenic
animals expressing full-length bovine tropoelastin showed significant
deposits of bovine tropoelastin, whereas the lungs (B) and
hearts (D) from
30 animals revealed only minimal
staining.
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Fig. 8.
Bovine tropoelastin contains a
plasmin-resistant C-terminal fragment. Shown are Western blots of
recombinant bovine tropoelastin cleaved with plasmin for 0, 30, 60, and
120 min at 37 °C. The blots were probed with antibody
CTe, which is reactive against exon 36 and therefore
detects fragments with an intact C terminus. Multiple fragments were
initially generated by cleavage with plasmin; but by 2 h, only a
single C-terminal fragment remained.
AAAGLPAGVGP) corresponding to a relatively stable 55-kDa fragment and one in exon 6 (YK
AAAKAGAAG), where the arrow is the site of cleavage by plasmin.
Once cleavage is initiated at these sites, complete degradation of the
remaining molecule is rapid in the in vitro assay.
DISCUSSION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
30 was found to associate with
microfibrils at low levels relative to the full-length transgene
product, and construct 1-28 was found only in the culture medium.
Because phenotypic and histopathological assessment of all the
transgenic lines found no adverse effects of transgene expression, our
results demonstrate that incorporation of the full-length bovine
protein into the mouse fiber did not disrupt the assembly or function
of the endogenous mouse elastin. Similarly, expression of
assembly-incompetent forms of the protein (1-28 and
30) did not
interfere in a dominant-negative fashion with deposition of normal
elastin. Similar results were obtained by Sechler et al.
(31), who showed that transgenic mice expressing rat constructs
(full-length and the naturally occurring splice variant
13-15) as
transgenes yield healthy animals with no observable harmful health effects.
-sheet/
-turn structures. Examples include
lamprin (a matrix protein of the lamprey annular cartilage) (24),
spidroin (a spider dragline silk protein) (32), and various
matrix proteins of the chorion or eggshell membrane of insects
(33-35). These proteins assemble through the interdigitation of side
chains belonging to residues present in short stretches of
cross-
-conformation (
-turn/
-sheet) (25, 36). Based on these
structural similarities, Robson et al. (24) have suggested that sequences of this type contribute to self-aggregation of elastin
monomers and alignment of polypeptide chains for cross-linking. Results
presented in this report confirm that exon 30 does indeed contribute to
tropoelastin assembly, but most likely in the context of interaction
with microfibrils. Previous studies in our laboratory have demonstrated
interactions between tropoelastin and small expression constructs
containing the Pro- and Gly-rich regions of fibrillin-1 and
fibrillin-2, respectively (37). Although we did identify the amino acid
sequence responsible for tropoelastin binding in the fibrillin
fragments, it is interesting to note that the glycine-rich region of
fibrillin-2 contains several repeats of the GGXGX
sequence that could interact with exon 30 of tropoelastin via
-sheet/
-turn structures.
30 did not undergo assembly when expressed in either PE cells or transgenic mice. Instead, our results argue for a model of nucleated assembly in
which the tropoelastin monomer interacts with microfibrils in a process
mediated by the exon 30 assembly domain. This process is initiated by
-structure interactions between exon 30 of tropoelastin and
-structure-containing regions on microfibrillar proteins such as the
glycine-rich portion of fibrillin-2. Consequently, we believe that
microfibrils are required to initiate or greatly enhance the rate of
the assembly process through an interaction with the C-terminal region
of tropoelastin. We cannot rule out a role for coacervation in
directing tropoelastin self-interaction in later stages of fiber
assembly; however, we do not know at what point microfibrils are no
longer required.
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ACKNOWLEDGEMENTS |
---|
We thank Clarina Tisdale for technical assistance in the generation of antibodies and cutting of frozen sections, Chris Ciliberto for maintenance and genotyping of transgenic animals, and Tom Broekelmann for additional technical support. We also acknowledge Ron McCarthy and the Program in Lung Biology at the Washington University School of Medicine for the production of the transgenic animals used in this study.
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FOOTNOTES |
---|
* This work was supported by Grants HL53325, HL62295, and HL61006 from the National Institutes of Health and by a grant from the National Marfan Foundation.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.
To whom correspondence should be addressed: Dept. of Cell
Biology and Physiology, Washington University School of Medicine, Campus Box 8228, 660 South Euclid Ave., St. Louis, MO 63110. Tel.: 314-362-2254; Fax: 314-362-2252; E-mail:
bmecham@cellbiology.wustl.edu.
Published, JBC Papers in Press, March 6, 2003, DOI 10.1074/jbc.M212715200
2 B. A. Kozel, H. Wachi, E. C. Davis, and R. P. Mecham, unpublished data.
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ABBREVIATIONS |
---|
The abbreviations used are: SVAS, supravalvular aortic stenosis; PE, pigmented epithelial; RT, reverse transcription; bTE, bovine tropoelastin; PBS, phosphate-buffered saline.
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REFERENCES |
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