From the Divisione di Oncologia Sperimentale 2, Centro di Riferimento Oncologico di Aviano, 33081 Aviano, Italy,
§ Department of Biochemistry, Genetics and Microbiology,
University of Regensburg, Regensburg, Germany D-8400, ¶ Istituto
di Istologia, Università di Padova, 35100 Padova, Italy, and
Dipartimento di Scienze e Tecnologie Biomediche,
Università di Udine, 33100 Udine, Italy
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ABSTRACT |
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EMILIN (elastin
microfibril interface located
protein) is an extracellular matrix glycoprotein abundantly
expressed in elastin-rich tissues such as blood vessels, skin, heart,
and lung. It occurs associated with elastic fibers at the interface
between amorphous elastin and microfibrils. Avian EMILIN was extracted
from 19-day-old embryonic chick aortas and associated blood vessels and
purified by ion-exchange chromatography and gel filtration. Tryptic
peptides were generated from EMILIN and sequenced, and degenerate
inosine-containing oligonucleotide primers were designed from some
peptides. A set of primers allowed the amplification of a 360-base pair
reverse transcription polymerase chain reaction product from chick
aorta mRNA. A probe based on a human homologue selected by
comparison of the chick sequence with EST data base was used to select
overlapping clones from both human aorta and kidney cDNA libraries.
Here we present the cDNA sequence of the entire coding region of
human EMILIN encompassing an open reading frame of 1016 amino acid
residues. There was a high degree of homology (76% identity and 88%
similarity) between the chick C terminus and the human sequence as well
as between the N terminus of the mature chick protein where 10 of 12 residues, as determined by N-terminal sequencing, were identical or
similar to the deduced N terminus of human EMILIN. The domain organization of human EMILIN includes a C1q-like globular domain at the
C terminus, a collagenous stalk, and a longer segment in which at least
four heptad repeats and a leucine zipper can be identified with a high
potential for forming coiled-coil Elastic fibers are major constituents of the extracellular matrix
(ECM),1 confer to connective
tissues the properties of resilience and elastic recoil, and are
remarkable for the diverse range of tissues in which they are found.
Elastic fibers can be identified in the ECM of many tissues as solid
branching and unbranching fine and thick rod-like fibers (in elastic
ligaments) or as concentric sheets of lamellae (in blood vessels) or in
three-dimensional meshworks of fine fibrils (in elastic cartilage)
or as a combination of these (in skin and lung) (1). Electron
microscopy has provided additional insights into the structure of
elastic fibers, which are composed of two morphologically
distinguishable components: an amorphous core lacking any apparent
regular or repeating structure (2) and a microfibrillar component (3)
consisting of fibrils of 12-13 nm in diameter that are located
primarily around the periphery of the amorphous core but to some extent
are also interspersed within it.
Despite this simple morphology, elastic fibers are now known to be
highly complex structures (4) consisting of several specific proteins,
including elastin, fibrillin 1 and 2 (5, 6), microfibril-associated
proteins 1 to 4 (7-10), latent-transforming growth factor We had originally isolated from chick aorta a novel glycoprotein
component associated with the ECM of blood vessels, gp115 (18), later
christened EMILIN (19). The major characteristics of this protein were
the following. EMILIN was preferentially extracted from tissues using
buffers containing guanidine HCl and reducing agents; it formed a
fibrillar network in the ECM of aorta; the amino acid composition was
characterized by a high content of glutamic acid and arginine (18).
Subsequent studies have established that (i) EMILIN is broadly
expressed in connective tissues and is particularly abundant in blood
vessels, skin, heart, lung, kidney, and cornea, whereas it is
undetectable in the serum (20-22); (ii) the protein is synthesized by
aortic smooth muscle cells and by tendon fibroblasts, and it is
deposited extracellularly as a fine network (18, 23); (iii) soon after
secretion, EMILIN undergoes intermolecular cross-linking by disulfide
bonds, giving rise to high molecular weight aggregates (23); (iv)
EMILIN is a component of elastic fibers and is localized mainly at the
interface between amorphous elastin and microfibrils (19); (v) finally and more important for the functional significance of EMILIN, the
process of elastin deposition in vitro is perturbed by the addition of anti-EMILIN antibodies in the culture medium (19). Therefore, given the close co-distribution of elastin and EMILIN, the
fine localization at the interface between elastin and microfibrils, and the interference with the deposition of elastin in
vitro, it is likely that EMILIN plays a fundamental role in the
process of elastogenesis also in vivo. To initiate
addressing the question of the functional role of EMILIN directly, we
have sought to clone its gene.
We report here the cDNA sequence and the analysis of the deduced
amino acid sequence of human EMILIN. This glycoprotein, a new member of
the C1q/TNF superfamily of proteins, is characterized by a gC1q-like
C-terminal domain, a short collagenous domain, two leucine zippers, and
an extended coiled-coil region that is uniquely shared with another
member of this superfamily, multimerin (24). At the N terminus of these
two members of the superfamily there is a short region of homology
including a partial epidermal growth factor-like motif. In addition,
the isolated recombinantly produced EMILIN gC1q-like C-terminal domain
is able to support cell adhesion.
Purification of Avian EMILIN and Peptide Sequences--
EMILIN
was purified as described previously (18). Briefly, aortas and
associated blood vessels were excised from 19-day-old chick embryos and
dropped into ice-cold 20 mM sodium phosphate, pH 7.4, 150 mM NaCl containing the following protease inhibitors: 25 mM EDTA, 2 mM phenylmethylsulfonyl fluoride, 5 mM N-ethylmaleimide, and 1 mM
p-aminobenzamidine hydrochloride. All subsequent
procedures were carried out at 4 °C. The tissues were homogenized,
and the pellet obtained after centrifugation at 12,000 × g for 45 min was re-extracted twice in the same buffer and
twice in 0.1 M Tris-HCl, pH 7.5, containing 6 M
guanidine HCl and protease inhibitors (guanidine buffer). The last
pellet was extracted twice in guanidine buffer containing 25 mM dithioerythritol. The last two supernatants were extensively dialyzed against distilled water and lyophilized. The
lyophilized extract was reduced and alkylated, and it was then
fractionated by DEAE-cellulose chromatography followed by agarose gel
filtration. The EMILIN-containing fractions, as assessed by an
enzyme-linked immunosorbent assay and by immunoblotting with a specific
monoclonal antibody, 147H11 (21), were pooled, dialyzed against
distilled water, and lyophilized. An aliquot of the pooled fractions
was analyzed by SDS-polyacrylamide gel electrophoresis to check for the
presence of contaminating proteins.
EMILIN was then resolved on a 4-10% SDS gradient gel. The
Coomassie-stained band corresponding to EMILIN was excised, cut into
small pieces, and washed with 0.2 M
NH4HCO3 followed by 0.2 M
NH4HCO3, acetonitrile (1:1). This procedure was
repeated twice. The gel pieces were lyophilized for 2 h and then
rehydrated by adding three portions of 0.2 M
NH4HCO3 containing 0.002% Tween 20 at 5-min
intervals. The first aliquot added contained 1 mg of trypsin/100-µl
gel pieces, and only as much liquid was used as was necessary to
restore the original gel volume. Protease digestion was performed
overnight at 37 °C, and the peptides were extracted twice with 5%
trifluoracetic acid and once with 2.5% trifluoracetic acid in 50%
aqueous acetonitrile. Alternatively, the gel pieces were repeatedly
washed with 50 mM Tris buffer, pH 9.0, and 50 mM Tris buffer, acetonitrile (1:1), and cleavage was
carried out with a lysine-specific protease from
Achromobacter (Wako) at pH 9.0 and 30 °C. The peptides
were then separated by high performance liquid chromatography on a
reversed phase Nucleosil-120 C18 column using a linear gradient of
0-70% aqueous acetonitrile in 20 mM ammonium acetate
buffer. Sequence analysis was done on a Procise protein sequencing
system (Applied Biosystems) according to the manufacturer's
instructions. The amino acid sequences obtained were used to search the
Swiss-Prot protein sequence data base (Geneva University Hospital and
University of Geneva, Geneva, Switzerland).
Primers Design--
Four of the nine chick EMILIN peptides
sequenced appeared suitable for the purpose of degenerate primers
design. A set of degenerate inosine-containing oligodeoxynucleotides
based on peptides 4, 6, 8 and 9 were synthesized on an ABI-381A
synthesizer (Applied Biosystem) in both the sense and the antisense
orientations (Fig. 1). The primers were purified by native
polyacrylamide gel electrophoresis (20% gel) using a standard
sequencing apparatus.
PCR-based Cloning Strategy and Sequencing of Chick and Human
EMILIN--
Total RNA from aortas of 19-day-old chick embryos was
isolated using RNA fast (Molecular System, San Diego, CA). The poly(A)+ RNA fraction was purified from total RNA with the use of Oligotex kit
(Qiagen GmbH, Germany). The first-strand cDNA was synthesized starting from 1 µg of poly(A)+ RNA primed with hexanucleotides and
reverse-transcribed with 20 units of AMV-RT (Promega Corp., Madison,
WI). PCR was performed on a Robocycler Gradient Apparatus (Stratagene,
La Jolla, CA) with the degenerate inosine-containing oligonucleotides
used as primers in all the possible different combinations and at
different annealing temperatures ranging from 48 to 72 °C with
2 °C steps. The amplification products were cloned into the pGEM-T
vector (Promega Corp.) and sequenced by the dideoxy- nucleotide
chain termination method using the modified T7 polymerase (Sequenase,
Amersham Pharmacia Biotech).
To clone the human EMILIN cDNA, the FASTA computer program (25)
located at the EBI server (26) was used to search the GenBankTM data
base for expressed sequence tags (ESTs) containing sequences homologous
to those of chick EMILIN. Several human and mouse entries showed a
significative homology to the 3' end of the chick EMILIN. Two primers,
ESTH-N (5'-ATTATGATCCAGAGACAGGC-3') and ESTH-R
(5'-CCGAGTGCGCCAGCTGCCCC-3'), were designed based on the entry HSAA1823
and used in PCR to obtain a human EMILIN-specific probe. The reaction
was performed on a template constituted by total kidney RNA primed with
hexanucleotides and reverse-transcribed with 20 units of avian
myeloblastosis virus reverse transcriptase. The amplification product
was cloned into the pGEM-T vector (EST-H clone) and sequenced to
confirm its identity. The EST-H insert (290 bp) was labeled by the
random primer method with the multiprime kit (Amersham Pharmacia
Biotech) and utilized to screen, by the plaque hybridization method,
about 300,000 clones of a human kidney cDNA library in the Rapid Amplification of cDNA Ends (RACE)--
To determine
the sequence extending toward the 3' end, the RACE method, using the
5'-3' RACE kit (Roche Molecular Biochemicals) was applied. Reverse
transcriptase of chick aorta poly(A)+ RNA was performed with the use of
exanucleotides, and the product was subjected to PCR using the EMILIN
specific sense primer 5'-GGAGCCGCTCACCATCTTCAGCGGGGCCC-3' in
combination with the anchor-poly(dT) from the kit.
Production of Recombinant Prokaryotic gC1q-like Domain of Human
EMILIN--
The EMILIN gC1q-like domain was amplified by PCR from
Clone K1, ligated in-frame in the 6-His-tagged pQE-30 expression vector (Qiagen GmbH), and grown in M15 cells. To check for errors generated by
PCR, all the cloned fragments were sequenced in both directions. M15
cells were centrifuged at 4000 × g for 20 min, and the
cell pellet was resuspended in sonication buffer (50 mM
sodium phosphate, pH 8.0, 0.3 M NaCl) at 2-5 volumes/g of
wet weight. The sample was frozen in a dry ice/ethanol bath, thawed in
cold water, and sonicated on ice (1-min bursts/1-min cooling/2-300
watts), and cell breakage was monitored by measuring the release of
nucleic acids at A260 nm. The cell lysate was
centrifuged at 10,000 × g for 20 min, the supernatant
was collected, and purification of the EMILIN C1q-like domain was
performed by affinity chromatography on nickel nitrilotriacetic acid
resin (Qiagen GmbH) under native conditions (sonication buffer). The
recombinant protein was eluted from the affinity column in sonication
buffer, pH 6.0, containing 10% glycerol and 0.2 M
imidazole. After dialysis against cold phosphate-buffered saline, the
C1q-like domain was used for CD spectra analysis and cell adhesion assays.
Circular Dichroism Spectroscopy--
The purified polyhistidine
EMILIN gC1q-like peptide was used for CD spectroscopy analysis. A Jasco
J-600 CD/ORD spectrophotometer interfaced to an Olidata computer for
data collection was used for all the measurements. Calibration of the
instruments was performed with D(+)-10-camphorsulfonic acid
at 290 nm. Standard conditions were 25 mM
Na3PO4, 150 mM NaCl, pH 6.0, 10 °C, using a 0.2-cm path-length cuvette. The temperature was
controlled by a water bath. UV circular dichroism spectra are presented
as millidegrees of ellipticity. The reported results are the smoothed
average over 10 measurements. To calculate the secondary structural
content, data were transformed in terms of mean residue molecular
ellipticity ( Cell Adhesion--
The capability of isolated recombinant human
EMILIN gC1q-like domain to support cell attachment was evaluated using
several cell lines and the centrifugal assay for fluorescence-based
cell adhesion (CAFCA) (28, 29). Briefly, specifically devised six-well strips of flexible polyvinyl chloride (CAFCA miniplates; TECAN Polyfiltronics, Inc., Boston, MA) covered with double-sided tape (bottom miniplates) were coated overnight with recombinant EMILIN gC1q-like fragment and subsequently incubated with 1% heat-denatured bovine serum albumin for 2-4 h at room temperature to block uncovered areas of the plastic. Two human smooth muscle (SK-UT-1 and SK-LMS-1 leiomyosarcomas) and a human fibroblastic (HT-1080 fibrosarcoma) cell
lines were fluorescently labeled by incubation with the vital fluorochrome calcein AM (Molecular Probes Europe BV, Leiden, The Netherlands), rinsed extensively in Ca2+- and
Mg2+-free phosphate-buffered saline, and then aliquoted
into the bottom CAFCA miniplates at a density of 1-3 × 104 cells/well. Cell adhesion to substrates was assayed in
phosphate-buffered saline containing 0.1% bovine serum albumin, 1 mM MgCl2 and CaCl2, and 2% India
ink as a fluorescence quencher. CAFCA miniplates were placed on
specifically devised hard plastic holders (TECAN Polyfiltronics, Inc.)
and centrifuged at 142 × g for 5 min at 37 °C to
synchronize contact of the cells with the substrate. The miniplates
were incubated for 30 min at 37 °C and then mounted together with a
similar CAFCA miniplate lacking double-sided tape (top miniplate) such
as to create communicating chambers to be reverse-centrifuged. The
relative number of cells bound to the substrate (i.e.
remaining bound to the bottom miniplates) and unbound cells (in wells
of the top miniplates) was estimated by top/bottom fluorescence
detection in a computer-interfaced SPECTRAFluor microplate fluorometer
(TECAN Polyfiltronics, Inc.). Fluorescence values were analyzed by
custom CAFCA software (TECAN Polyfiltronics, Inc.) to determine the
percentage adherent cells of the total cell population analyzed
according to a previously published formula (28, 29).
Purification and Peptide Sequences of Chick EMILIN--
A chick
aorta EMILIN preparation obtained following differential extraction
procedures, DEAE ion-exchange, and size exclusion chromatography was
analyzed by SDS-polyacrylamide gel electrophoresis under reducing
conditions and found to be composed of a single major band with an
estimated Mr of about 115,000. This finding was
in accord with previous results (18), and the few low
Mr contaminants represented less than 10% of
the stained material as judged by quantitative scanning of the gel
(data not shown). The protein was cleaved by trypsin and by a
lysine-specific protease, the peptides were separated by reverse phase
chromatography on a Nucleosil-120 C18 column, and several components
were selected for sequencing (Fig.
1).
Cloning Strategy--
Based on the peptide sequences, several
degenerate oligonucleotides in the sense and antisense orientations
were synthesized and used to amplify fragments from poly(A)+ RNA of
chick aortas. Several amplification products of different lengths were
obtained using the various combinations of oligonucleotides. The
sequence of the cloned products revealed that all but one contained
stop codons in all the possible frames. On the contrary, the
384-bp-long amplification product obtained with the use of the primers
corresponding to peptides 9 (sense orientation) and 6 (antisense
orientation) encompassed an open reading frame (clone D1) that
contained peptide 1, thus suggesting that the amplified sequence
corresponded to that of genuine EMILIN protein (Fig. 1).
After the initial successful search of the GenBankTM for EST-containing
homologous sequences to those of clone D1 of chick EMILIN, five partly
overlapping cDNA clones were sequentially isolated from Nucleotide and Predicted Amino Acid Sequences--
The partial
chick cDNA corresponds to an open reading frame of 128 amino acids
(data not shown). The coding sequence and the deduced amino acid
sequence of the human EMILIN cDNA (GenBankTM accession number AF
088916) is shown in Fig. 3. Several
partially overlapping EST entries (HSAA23367, HSAA1823, HS1269888)
showed an almost perfect match with the 3' end of the human EMILIN
transcript, including the last 465 bp of the coding sequence and the
entire 3'-untranslated region. However, numerous substitutions and gaps have also been detected: C2772-GAP; C2823G; C2826G; G2827T; C2857A; C2877T; C2883-GAP; G2895-GAP. Independent sequencing of BAC clones confirmed the present sequence.2
The open reading frame of the human EMILIN begins with a Met codon
whose surrounding sequences fit into the eukaryotic translation start
sites (30) and is preceded by an in-frame stop codon (data not shown).
The human cDNA spans about 3400 bp and has an open reading frame of
1016 amino acids. The predictions with the highest probabilities for
the initial residue of the mature protein are between position 2 (Ser,
Y value of 0.508) and 3 (Tyr, Y value of 0.580) of the present
sequence. Although the best prediction for the cleavage site of the
signal peptide is at position 3, alignment between the N-terminal
peptide sequence of the chick mature EMILIN and the deduced N terminus
of the human EMILIN (see below) indicates the position 1 (Ala) as the
most likely candidate for the initiation of the mature protein.
Therefore, residues Comparison between Chick and Human Sequences--
The alignment of
the 128-residue-long stretch of chick EMILIN and the C-terminal region
of human EMILIN showed a high degree of homology (Fig.
4, panel C), the overall
degree of amino acid sequence identity and identities plus similarities
in this domain was calculated to be about 76 and 88%, respectively;
furthermore, a partial sequence of the N terminus of chick EMILIN
compares very well (9 residues are identical, and 2 are similar) with
the deduced N terminus of the human EMILIN (Fig. 4, panel
A). The high level of identities and similarities between chick
and human EMILIN at both C and N termini indicates that the two
sequences identify the same protein in these species.
Domain Structure--
The predicted domain structure of human
EMILIN is composed, starting from the C-terminus, of a globular domain
(gC1q-like), an uninterrupted stretch of 17 Gly-Xaa-Yaa triplets,
indicating that EMILIN possesses a straight collagen stalk, and a
641-long amino acid sequence in which there are several heptad repeats separated by unrelated sequences with the potential for forming coiled-coil Coiled-coil Prediction--
The 3-4-3-4 spacing of hydrophobic
residues predicts that the region of human EMILIN spanning residues 152 and 792 (Figs. 2 and 3) will form an Alignment of Sequences at the C-terminal Domain--
The C
terminus of EMILIN exhibits a striking homology to a gC1q-like domain
of a number of proteins including the A, B, and C chains of human and
mouse complement C1q protein (39, 40), the Adhesion-promoting Activity of the Recombinant C1q-Domain of Human
EMILIN--
The gC1q domain of C1q complement component has been shown
to promote cell attachment of mononuclear cells (51), platelets (52),
and endothelial cells (53). Therefore, the potential cell adhesive
capacity of EMILIN gC1q-like domain in comparison with that of
fibronectin, a prototype adhesive molecule, was assessed using the
smooth muscle cell lines SK-UT-1 and SK-LMS-1 and the fibroblast cell
line HT1080. The EMILIN gC1q-like domain was cloned in the pQE30
expression vector, and the protein was purified under native conditions
and coated at different concentrations. The native state of the EMILIN
gC1q-like domain was examined by CD spectra analysis. The peptide (Fig.
7) exhibited a classic The results provided in this report concern a new human cDNA
whose major structural elements were a gC1q-like C-terminal domain, a
short uninterrupted collagenous domain, and an extended domain containing sequences with the potential of forming amphipathic coiled-coil Evidence that chick EMILIN stained strongly with PAS (18) and that in
biosynthetic studies a treatment with tunicamycin reduced the apparent
molecular mass of about 20-25 kDa (23) indicated that chick EMILIN was
highly glycosylated. The present identification of seven potential
N-glycosylation sites in the human EMILIN sequence is in
accord with the previous experimental data using the chick system (18,
23). Similarly, the presence of 20 cysteine residues with a high
potential for intermolecular S-S bonding is also in accord with the
finding that newly synthesized and secreted chick EMILIN migrated as a
monomer in SDS gels under reduced conditions but was present as a large
aggregate that did not enter the gel in the absence of reducing agents
(23).
The domain organization of EMILIN is unique; it bears features shared
with several other members of the C1q/TNF superfamily (Fig.
9), i.e. C1q (A, B, C),
collagens VIII, X, saccular collagen, ACRP-30/AdipoQ, and HP-27, such
as the gC1q-like domain and a collagenous domain, but also EMILIN
displays an extended discontinuous and potentially coiled-coil region
that is absent in all the other members of the C1q/TNF superfamily,
except multimerin, a large soluble glycoprotein found in platelets
helices. At the N terminus there
is a cysteine-rich sequence stretch similar to a region of multimerin,
a platelet and endothelial cell component, containing a partial
epidermal growth factor-like motif. The native state of the
recombinantly expressed EMILIN C1q-like domain to be used in cell
adhesion was determined by CD spectra analysis, which indicated a high
value of
-sheet conformation. The EMILIN C1q-like domain promoted a
high cell adhesion of the leiomyosarcoma cell line SK-UT-1, whereas the
fibrosarcoma cell line HT1080 was negative.
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
-binding
protein 1 to 4 (11-14), fibulin (15), and MAGP-2 (8, 16). Elastin and
fibrillins are the main components of the amorphous core and
microfibrils, respectively. Fibrillin-containing microfibrils are
highly organized structures, and microfibril-associated proteins and
probably latent-transforming growth factor
-binding proteins are
predominantly associated with them. Fibrillin-containing microfibrils
are also found as elastin-free bundles in tissues where elastic fibers
cannot be morphologically distinguished such as in ocular zonule,
oxitalan fibers of the cornea, kidney, and spleen (17).
EXPERIMENTAL PROCEDURES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
gt10
vector (CLONTECH Laboratories Inc., Palo Alto, CA).
The longest insert of the five positive plaques identified, K1, was
used to rescreen the library. The second screening yielded only one
positive clone, K2, constituted by a fusion between a specific EMILIN
cDNA and an unrelated cDNA. Three additional rounds of
screening of a human aorta cDNA library resulted in the isolation
of clones A1, A2, and A3. Also in this library several clones appeared
to be cloning artifacts carrying short EMILIN-specific sequences fused
to unrelated cDNAs. The human sequences were performed using the
Big Dye terminator cycle sequencing kit and a model 310 DNA sequencing
system (Perkin-Elmer Applied Biosystem). To correct for possible TAQ
polymerase errors, all sequences were determined from both strands and
were repeated on clones obtained from independent PCR products. All
human cDNA sequences were confirmed by sequencing the EMILIN gene
by an independent analysis of a BAC clone, used to characterize the
EMILIN gene.2
) (deg × cm2 × dmol
1),
based on a mean residue weight of 104.3. Then, the spectra were
analyzed using the Menendez-Arias program (27).
RESULTS
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
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Fig. 1.
Sequences of chick EMILIN peptides.
Peptides were generated as described under "Experimental
Procedures." Amino acid sequences are indicated in 1-letter code,
with X representing unidentified residues; the sequences of
degenerate and inosine-containing oligonucleotides derived from
peptides 4, 6, 8, and 9 are in small letters and
underlined; y, c/t; h, a/c/t;
w, t/a; n, a/g/c/t; r, a/g;
s, g/c; i, inosine.
gt10
human kidney and aorta libraries using a human specific PCR-derived
probe based on the EST entries and probes derived from subclones at the
5' end of each successively isolated clone (Fig.
2). Remarkably, walking toward the 5' end
of the human cDNA was hampered by the surprisingly high number of
fused clones isolated from both libraries.
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Fig. 2.
A schematic diagram with the cloning strategy
and the domain structure of chick and human EMILIN. The various
types of clones (solid lines) used in determining the
sequence are shown above (chick) and below (human) the respective
diagrams. Each clone is identified by a capital letter
followed by a number; letters refer to the tissue
of origin (A, aorta; K, kidney) or the technique
used (D, clones obtained by amplification with degenerate
primers). 3'-unt indicates the 3'-untranslated region. The
different domains are designated according to Bork and Koonin (54).
EGF, epidermal growth factor; CC, coiled-coil;
LZ, leucine zipper; COL, collagenous domain; C1q, gC1q-like domain.
Cysteines and potential glycosylation sites are indicated by
closed and open circles, respectively. The
positions of peptides derived from the chick molecule and the
corresponding degenerate primers are indicated by numbers
and arrows, respectively, in reverse type.
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Fig. 3.
Nucleotide and predicted amino acid sequence
of human EMILIN. First line, nucleotide sequence;
second line, deduced amino acid sequence. Plain
and bold numbers on the right indicate
nucleotides and amino acids, respectively. Amino acids are numbered
starting at the predicted beginning of the putative mature sequence.
The presumed N terminus of the mature protein is marked by a
closed arrow, and the UAG stop codon is indicated by a
star. The polyadenylation signal is bold and
underlined. Potential N-attachment sites for
oligosaccharides are boxed, and cysteine residues are
circled. Several structural features are
highlighted; the partial epidermal growth factor-like motif
is double-underlined; the coiled-coil sequences are
underlined by a broken line with the residues in
the a-position marked by a dot; the leucines in
the d-position of potential leucine zippers are indicated in
reverse types; the glycines (G) of the
collagenous domain are shown within triangles; the C1q-like
C-terminal domain is boxed.
21/
1 correspond most likely to a signal
peptide, because the sequence agrees very well with the classical
consensus sequence (31) and ends with a consensus signal cleavage site
(32). Thus, the calculated molecular mass for the mature protein is 104.5 kDa. The human EMILIN contains 7 potential
N-glycosylation sites and 20 cysteines with a number of them
clustered as doublets, separated by none, one, or two residues that
could be involved in intramolecular disulfide bonding. By applying the
3'-RACE, 383 additional bp corresponding to the 3'-untranslated
sequence could be obtained. The observation that the most 3' end EST
entry terminates 37 bp before the end of the present RACE product,
together with a potential polyadenylation sequence 16 bp before the end of our sequence, strongly suggests that our RACE product includes the
entire 3'-untranslated sequence.
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Fig. 4.
Sequence comparison. A,
N-terminal sequence. The chick sequence was obtained by sequencing a
purified preparation of aorta EMILIN (G. M. Bressan, unpublished
observation). B, gC1q-like domain. Identical/similar
residues are indicated in reverse type. C,
N-terminal region. The sequence of multimerin is from Ref. 24.
Cysteines are marked by a star.
-helices (see below). A 91-bp-long residue sequence that
includes two sequences corresponding to structures referred to as the
"leucine zippers," which are typical of several gene regulatory
proteins (33, 34), is located between the coiled-coil region and the
collagenous stretch. This finding is rather unusual especially for an
ECM protein, as there are only a few precedents in the literature for
the presence of leucine zippers in the extra nuclear compartments: the
Drosophila ECM protein pollux (35) and the cytoplasmic
protein dystrophin, whose leucine zipper has been reported to interact
with troponin (36). Although the leucine zipper pattern is far from
being specific, it is not clear at the moment what might be the
significance of its presence in the context of EMILIN. Finally, a
search of the NCBI data bank indicated the existence of a region of
homology between the N-terminal end of EMILIN and the platelet and
endothelial specific protein, multimerin (24), spanning amino acids 33 and 108 (Fig. 4, panel B). This region contains several
identical residues, including three conserved cysteines and a partial
epidermal growth factor-like consensus sequence.
-helical coiled-coil. When
analyzed by different algorithms (37, 38), the probability for
coiled-coil formation differed among the various heptad sequences.
Using the PairCoil program (38), the highest probability score (around 1.0) was located at the level of the first heptad repeat (Fig. 5), and similar results were obtained
when applying the Multicoil program (data not shown); furthermore, at
least two other heptad repeats (positions 497-539 and 615-684)
displayed a high probability of coiled-coil structures, and a fourth
one had intermediate values.
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Fig. 5.
The probability of coiled-coil
-helical structures. Several highly probable
coiled-coil regions are identified using the PairCoil program (37)
between residues 152 and 684.
1 and
2 chains of
type VIII (41, 42), and the
1 chain of type X (43) collagens,
precerebellin (44), multimerin (24), ACRP-30/AdipoQ (45, 46), the HP-27
protein from Siberian chipmunks (47), and a sunfish saccular collagen
(48). The length of this domain is included between 131 (gC1q-C) and
151 (EMILIN) residues, and the protein sequence comparison of the
C1q-like domain of EMILIN with the known similar domains indicates a
high level of conservation of several hydrophobic and uncharged
residues (Fig. 6). To obtain the best
sequence alignment of EMILIN with the other members of the superfamily,
it was necessary to allow for the insertion of a 10-residue sequence
that is unique for EMILIN and is missing in all the other members.
Nineteen residues are conserved in all 12 sequences analyzed, and 44 are conserved in at least half of the members. Initially, Fourier
transform infrared spectroscopy and structure prediction (49) of 15 gC1q-like sequences suggested a
-sheet secondary structure for this
domain. This prediction has been recently confirmed by the
analysis of the ACRP-30/AdipoQ crystal structure (50).
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Fig. 6.
Sequence alignment of the gC1q-like
domains. Groups of similar residues are defined as follows: tiny,
S, G, A; aliphatic, I, L, V, M; aromatic, H, F, Y, W; positive, R, H,
K; negative, D, E; hydroxyl, S, T; amide, N, Q. Identical or similar
residues shared by 50% or more of the sequences are shaded
in gray, and those shared by more than 75% of the sequences
are in reverse type. An asterisk above the
sequences marks residue conservation in all the sequences. The
locations of the strands according to the crystal of the gC1q-like
domain of ACPR-30/AdipoQ (52) are indicated above the sequence by
arrows and capital letters. An insertion of 10 residues unique to EMILIN is boxed in gray. The
sequences of C1qA, C1qB, C1qC,
1 (VIII),
2 (VIII),
1 (X),
precerebellin (cerebellin), multimerin, ACRP-30/AdipoQ, sunfish
saccular collagen (sf sac coll), and HP-27 (chipmunk
(chip) HP-27) are from accession numbers P02745, K03430,
P02747, X57527, M60832, X65120, P23435 and P02682, U27109,
D45371, P38085, A41752, respectively. h, human;
A-H indicate the locations of the
strands.
-sheet CD
spectrum with a minimum at 217 nm. The evaluation of the content in
-sheet conformation for the peptide using the Menendez-Arias program
(27) gives a value higher than 70%, therefore confirming that also the
EMILIN gC1q-like domain fits very well with the structure determined
for ACRP-30/AdipoQ (50). Cell adhesion to fibronectin was high and
comparable for all cell lines with a cell binding of about 90%, the
EMILIN gC1q-like domain promoted a high level of adhesion of SK-UT-1
cells, whereas HT1080 were negative, and SK-LMS-1 bound with a low
percentage (Fig. 8A). However,
although adhesion to fibronectin induced a strong cell flattening, cell
adhesion to the EMILIN gC1q-like domain was not followed by a
significant level of cell spreading (Fig. 8B). The SK-UT-1
cells were for the most part round with small blebs or short
projections with only a very low percentage of cells displaying a flat
morphology. These results suggest either that cell adhesion to the
EMILIN gC1q-like domain uses different mechanisms/receptors than
adhesion to fibronectin and is not followed by cytoskeletal rearrangements or, less likely, that the standard cell adhesion conditions of time, temperature, and medium composition used to assay
adhesion to fibronectin are not appropriate to measure cell adhesion to
the C1q-like domain of EMILIN.
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Fig. 7.
Circular dichroism spectrum of the C1q-like
domain. The spectra were recorded at a protein concentration of
100 µM in 25 mM
Na3PO4, 150 mM NaCl, pH 6.0, at
10 °C and 0.2-cm path-length. The -sheet content was estimated
from the mean residue molecular ellipticity (
) (deg × cm2 × dmol
1), based on a mean residue weight
of 104.3 and analyzed using the Menendez-Arias program (27).
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Fig. 8.
Cell adhesion of SK-UT-1 and SK-LMS-1 smooth
muscle and HT1080 fibroblastic cell lines. Cell adhesion to intact
human fibronectin (a and b) and to the
recombinant human EMILIN gC1q-like domain (c and
d) purified under native conditions. The ligand proteins
were plated at 10 µg/ml, and the cell adhesion assay was carried out
in phosphate-buffered saline containing 1 mM
Ca2+ and 1 mM Mg2+. Adhesion to
wells coated with denatured bovine serum albumin alone was negligible.
Nomarski optics microscopy images of HT-1080 (left) and
SK-UT-1 (right) cells are shown.
DISCUSSION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
-helices. The determination of the primary structure through cDNA cloning and the assignment of this novel sequence to
human EMILIN was made possible by the very close identity at the C
terminus between the chick EMILIN sequence and that of the corresponding human cDNA. In fact, the finding that the deduced sequence of clone D1 of chick EMILIN contained peptide 1 of the tissue-purified EMILIN confirmed that the sequence amplified from chick
aorta mRNA corresponded to that of the genuine EMILIN described by
us (18-23) as an elastin-associated protein with undefined function. Thus, the sequence similarity at the gC1q-like domain, the near identity between the N-terminal residues of the mature chick protein, and the deduced amino acids of the presumed mature human protein support the conclusion that we are dealing with the same ECM
constituent in the two species.
-granules and endothelial Weibel-Palade bodies. Multimerin forms
disulfide-linked homomultimers of variable sizes (55) and interacts
with factor V, which is stored complexed with multimerin in the
-granules (56). There is experimental evidence that several members
of the C1q/TNF superfamily trimerize to form either heterotrimeric
collagen triple helices that are expressed as soluble plasma proteins
or type II membrane-bound molecules such as in C1q (A, B, C) (57, 58)
or to form homotrimers as in collagen X (59) and ACRP-30/AdipoQ (46).
EMILIN is also likely to form similar trimers; it possesses a gC1q-like
domain, which is highly homologous to those of the other members of the superfamily and an uninterrupted collagenous domain, which can form a
collagen-like stalk region. Curiously, among the members of the family,
only EMILIN, ACRP-30/AdipoQ, and Hib27 possess an uninterrupted
collagenous domain. The EMILIN gC1q-like domain, when compared with the
other gC1q-like domains, has a much longer F
-strand because of a
10-residue insertion. However, the residues conserved throughout both
the C1q and TNF families of proteins and important in the packing of
the hydrophobic core of the individual monomer (50) are present in
EMILIN gC1q-like domain in the same relative positions, and this
appears sufficient to predict a similar trimeric and spatial
organization also for the domain of EMILIN. The potential structural
homology between the TNF family of growth factors and the gC1q-like
domains of the C1q family of proteins suggested that these diverse
members might derive from ancestral elements with close functional
activity (50). Likely targets for the proteins containing C1q/TNF
domains are cell surface receptors; these are well studied in TNF, but
initial data are also available for the C1q complement component
(51-53). In fact, several cell types are endowed with the capability
to attach to the C1q complement component via cell surface binding
sites; two types of structures have been described, a binding protein
that recognizes the collagenous domain (60) and another component that
binds to the gC1q domain (51). However, more recently the effective
nature of this second type of binding molecule has been disputed (61),
and further studies are necessary. The finding that EMILIN gC1q-like
domain displayed cell pro-adhesive capacity for some smooth muscle
cells but seemed to be much less reactive for fibroblastic cells is consistent with the above evidence and suggests that cell recognition of this domain might be exerted via specific cell surface receptors. The adhesion was high for SK-UT-1 cells but, within the time frame of
the cell adhesion assay, was not followed by a consistent spreading. Thus, it is possible that "receptors" distinct from classical integrins such as those recognized by fibronectin are involved here.
Neither the physiological significance of the observed adhesion is
clear yet nor whether this adhesion plays a primary or an auxiliary role. Close contacts between amorphous elastin and smooth muscle cells
in the aorta of 16-day embryos have been reported (62), and the
ultrastructural localization of EMILIN (19) does not exclude that the
interaction between the elastin amorphous core and the cells could also
take place via an EMILIN intermediate. As EMILIN was detected in early
stages of aorta development, in association with a network of thin
fibrils likely representing maturing microfibrils (19), EMILIN
deposition can be considered an early event in elastogenesis, and this
conclusion is reinforced by the observation that the process of elastic
fiber formation in vitro was greatly affected by the
addition of anti EMILIN antibodies (19). Whether the process of elastin
deposition and elastic fiber formation is regulated through cell
adhesion via the EMILIN gC1q-like domain remains to be seen.
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Fig. 9.
The C1q/TNF superfamily of proteins. The
different domains are designated as in Fig. 2. Bars within
the collagenous domains (COL) indicate short interruptions
or imperfections in the Gly-Xaa-Yaa sequence. The order in which the
different members are depicted highlights that EMILIN, in addition to
the C1q-like domain common to all the family members, shears a short
collagenous domain with only a few of the members and the
coiled-coil-containing region only with multimerin. EGF, epidermal
growth factor.
EMILIN, like multimerin (55), is heavily disulfide-linked and thus can
be found as large aggregates in the culture medium of aorta smooth
muscle cells (23). The possibility to form coiled-coil -helices
could further amplify its potential to associate into even larger
aggregates. In fact, one of the heptad repeat sequences has a
probability to form
-helices near 1.0, and in two other regions the
probability is above 0.6. Although formal proof that these heptad
repeats can form trimers is still lacking, the chances are high given
that the presumed trimerization process can initiate from the C1q-like
domain at the C terminus, proceeding then through the collagenous
domain next to it as in collagen X (59) and ACRP-30/AdipoQ (46).
Further studies are required to define how EMILIN subunits are
assembled into the large disulfide-linked multimers, i.e.
whether the EMILIN gC1q-like domain is a likely site for initial
interchain association and whether the heptad repeats associate only
intramolecularly into trimers or can also associate intermolecularly,
i.e. with other EMILIN trimers. To investigate these
possibilities, the preparation of full-length, truncated, and
point-mutated EMILIN recombinant molecules in eukaryotic cells is in
progress.3
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ACKNOWLEDGEMENTS |
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We thank Maria Teresa Mucignat and Gabriella Mungiguerra for technical assistance, Dr. Paola Spessotto for her help in performing the cell adhesion assays, and Dr. Gianluca Tell for performing the CD spectra analysis.
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FOOTNOTES |
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* This work was supported by grants from Associazione Italiana per la Ricerca sul Cancro, Telethon, MURST-Cofin 1997 (to A. C.), and Ricerca Sanitaria Finalizzata 793/03/97 (D.V.).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.
The nucleotide sequence(s) reported in this paper has been submitted to the GenBankTM/EMBL Data Bank with accession number(s) AF088916.
** To whom correspondence should be addressed: Divisione di Oncologia Sperimentale, Centro di Riferimento Oncologico, 33081 Aviano, Italy. Fax: 0039 0434 659 428; E-mail: acolombatti{at}ets.it.
2 R. Doliana, A. Canton, F. Bucciotti, M. Mongiat, P. Bonaldo, and A. Colombatti, manuscript in preparation.
3 M. Mongiat, R. Doliana, and A. Colombatti, unpublished observations.
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ABBREVIATIONS |
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The abbreviations used are: ECM, extracellular matrix; gC1q-like, globular C1q-like domain; TNF, tumor necrosis factor; PCR, polymerase chain reaction; EST, Expressed Sequence Tag; bp, base pair(s); RACE, rapid amplification of cDNA ends; CAFCA, centrifugal assay for fluorescence-based cell adhesion.
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REFERENCES |
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