From the Divisione di Oncologia Sperimentale 2,
Centro di Riferimento Oncologico, 33081 Aviano and
§ Dipartimento di Scienze e Tecnologie Biomediche,
Università di Udine, 33100 Udine, Italy
Received for publication, December 22, 2000
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ABSTRACT |
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EMILIN (elastin
microfibril interfase located
Protein) is an elastic fiber-associated glycoprotein
consisting of a self-interacting globular C1q domain at the C
terminus, a short collagenous stalk, an extended region of potential
coiled-coil structure, and an N-terminal cysteine-rich domain (EMI
domain). Using the globular C1q domain as a bait in the yeast
two-hybrid system, we have isolated a cDNA encoding a novel
protein. Determination of the entire primary structure demonstrated
that this EMILIN-binding polypeptide is highly homologous to EMILIN.
The domain organization is superimposable, one important difference
being a proline-rich (41%) segment of 56 residues between the
potential coiled-coil region and the collagenous domain absent in
EMILIN. The entire gene (localized on chromosome 18p11.3) was isolated
from a BAC clone, and it is structurally almost identical to that of
EMILIN (8 exons, 7 introns with identical phases at the exon/intron
boundaries) but much larger (about 40 versus 8 kilobases)
than that of EMILIN. Given these findings we propose to name the novel
protein EMILIN-2 and the prototype member of this family EMILIN-1
(formerly EMILIN). The mRNA expression of EMILIN-2 is more
restricted compared with that of EMILIN-1; highest levels are
present in fetal heart and adult lung, whereas, differently from
EMILIN-1, adult aorta, small intestine, and appendix show very low
expression, and adult uterus and fetal kidney are negative. Finally,
the EMILIN-2 protein is secreted extracellularly by in
vitro-grown cells, and in accordance with the partial
coexpression in fetal and adult tissues, the two proteins shown
extensive but not absolute immunocolocalization in
vitro.
The elasticity of many tissues such as lung, dermis, and large
blood vessels depends on the presence of a high content of elastic
fibers in the ECM.1 These
structures are composed of two distinct morphological elements, a more
abundant amorphus core of which elastin, responsible for the elastic
properties, is the major constituent, and microfibrillar structures of
about 10-12-nm diameter, which are located around the periphery of the
amorphus component and consist primarily of fibrillin-1 and/or -2 (1,
2). Whereas the amorphous elastic core is apparently poorly organized,
fibrillin-containing microfibrils are highly organized structures.
Several components that contribute to the elastic fiber organization
have been identified and cloned, including microfibril-associated
proteins 1 to 4 (3-6), latent transforming growth factor EMILIN differs from all other elastin-associated proteins and has a
unique multimodular organization (20); it includes a C1q-like globular
domain at the C terminus, endowed with cell-adhesion-promoting functions, a short uninterrupted collagenous stalk, a long segment of
about 650 residues with a high potential for forming coiled-coil Yeast Two-hybrid Library Screening--
The
Saccharomyces cerevisiae strain EGY48 (p8op-lacZ;
MAT Library Screening--
The entire coding sequence of the
cDNA isolated in the yeast two-hybrid system was determined by
cDNA library screening. The insert from one selected yeast
two-hybrid system clone (about 1000 bp) was labeled by the random
primer method with a 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 Isolation and Characterization of a Human Genomic DNA
Clone--
A human genomic BAC library was screened for specific
clones at Genome System using a cDNA insert corresponding to the
5'-end of the EMILIN-2 (see below) cDNA. Two positive clones
were identified, and one was further characterized. It was
authenticated by successful PCR reamplification of insert fragments
with primer pairs derived from the EMILIN-2 cDNA sequences, and it
was partially characterized by restriction enzyme mapping and Southern
blot analysis. Appropriate restriction fragments were gel-purified and
subcloned in the pGEM 7z+ vector and then sequenced.
Dot Blot Analysis--
RNA expression analysis was
performed using a human multiple tissues blot from
CLONTECH. A 32P-labeled probe was
synthesized using as template the EMILIN-binding protein-1 (EBP-1)
clone and the multiprime labeling kit (Amersham Pharmacia Biotech).
Hybridization was performed at 65 °C in Rapid-hyb buffer. After film
exposure the blot was stripped and hybridized with a
32P-labeled EMILIN-1 probe. All the other procedures
were performed using standard techniques.
Production of Recombinant Prokaryotic gC1q of Human EMILIN-2
(gC1q-2) and Preparation of Monoclonal Antibodies--
The sequence
corresponding to the C-terminal domain of EMILIN-2 (gC1q-2) was
amplified from the yeast two-hybrid system template (see above) with
the following primers: sense,
5'-GGGGATCCGGGCGGGGTCTGCCGCG-3'; and antisense,
5'-GGGGTACCTTAGAGGTGGGAAAGGAAAGGAT, where the underlined nucleotides correspond to appended BamHI (sense) and
KpnI (antisense) restriction enzyme recognition sites plus
two additional protective nucleotides. The amplified gC1q-2 fragment
was then ligated in frame in the 6His-tagged pQE-30 expression vector
(Qiagen) and transformed in M15 cells. Positive clones were isolated,
and the cloned fragment was sequenced in both directions to check for errors generated by PCR. 500 ml of liquid culture grown at 0.6 A600 nm was induced with 2 mM
isopropyl-1-thio- Immunofluorescence--
Indirect immunofluorescence of cells
grown on tissue culture glass chamber slides (Nunc Inc., Naperville,
IL) was carried out on cells fixed in 4% (v/v) paraformaldehyde in
phosphate-buffered saline for 30 min before incubation with the a
polyclonal rabbit anti-EMILIN-1 antiserum or a murine anti-EMILIN-2
(gC1q-2 domain) monoclonal antibody. These two antibody reagents are
specific for their respective antigens and did not show any
cross-reactivity (data not shown). The slides were then incubated with
fluorescein-conjugated goat anti-rabbit IgG (for EMILIN-1) or with
rhodamine-conjugated goat anti-mouse IgG (for EMILIN-2) and examined
under the confocal laser (MRC-1024, Bio-Rad laboratories, Hercules, CA)
scanning microscope (Diaphot 200, Nikon Inc., Melville, NY).
Identification of a Binding Partner for EMILIN-1 in the Yeast
Two-hybrid Assay--
The two hybrid system was used to screen for
potential interactors with the gC1q domain of EMILIN. A segment
spanning the gC1q domain of EMILIN (residues 845 to 995 of the
published sequence; see Ref. 20 and GenBankTM/EBI Data Bank
accession number AF 088916) was fused to the LexA DNA-binding domain in
pLex, and EGY48 S. cerevisiae cells were transformed
with this plasmid. A library of human kidney cDNAs in the
pB42-activating domain was then introduced to the transformants, and
the colonies growing in the absence of the Leu/His/Trp/Ura markers were
selected. Among the 5 × 106 cells transformed in
total, 87 colonies were Leu-positive. After the
The characterized human cDNA spans about 3877 bp and has an open
reading frame of 1053 amino acids,
starting with a Met codon whose surrounding sequences fit into the
eukaryotic translation start sites (28).The 3'-untranslated region of
715 nucleotides includes one putative polyadenylation signal. The
predictions with the highest probabilities for the initial residue of
the mature protein are between positions The Novel Protein Belongs to the EMILINs Family--
We had
previously reported on the isolation and characterization of EMILIN
(20), a multimodular protein composed by a C-terminal gC1q domain, a
short collagenic domain, a long region with high propensity to form
coiled-coil structures and, at the N terminus, a characteristic
cysteine-rich domain (EMI domain; see Ref. 21). The primary structure
of the novel protein reported here, including the lack of a
transmembrane segment, the presence of a putative secretory signal
peptide, and the overall sequence composition and domain organization
suggests that this gene product is an EMILIN-related ECM protein that
may also form oligomers via its C-terminal gC1q domain and the
potential coiled-coil domain (see Fig. 1, top). Based on the
primary sequence, on the domain composition, and on the gene structure
organization (see below), EMILIN and EBP were renamed EMILIN-1 and
EMILIN-2, respectively. The novel protein includes an N-terminal EMI
domain and a C-terminal gC1q domain, which both have up to 70%
sequence similarity with the corresponding human EMILIN-1 domains.
Pairwise alignment of the EMILINs further emphasizes the close kinship
between these two members. In the C1q domain, for example, both protein
present an insertion of 10 amino acids in comparison to all the other members of the C1q-containing proteins family, one of which,
ACRP30/Adipo Q, is shown for comparison in Fig.
3B. The EMI domain at the N terminus is much more conserved between the two proteins in comparison to the other EMI domain-containing gene products (Fig. 3A).
The short collagenic stretch of 17 triplets present in EMILIN-1 is conserved in EMILIN-2, although in the latter there are four
imperfections. Although lacking any detectable sequence homology in the
extended central region, EMILIN-1 and EMILIN-2 have structural motifs, consisting of heptad repeats in which positions 1 and 4 are
preferentially occupied by aliphatic moieties and positions 5 and 7 are
filled with polar residues, in common. The presence of these repeats, which are characteristic of coiled-coil Chromosomal Localization and Analysis of the EMILIN-2
Gene--
The exon/intron boundaries of the protein-coding region of
the EMILIN-2 gene were identified by comparison between a human BAC
isolated with an EMILIN-2 probe carrying the entire EMLIN gene and the
cDNA sequence. As for the EMILIN-1 gene, each intron is in phase 1 with the exception of the first two introns, which are in phase 2, and
all the sequences at the exon/intron boundaries are in full agreement
with the consensus rules established for the splice sites of vertebrate
genes (32). The gene consists of 8 exons and 7 introns (Fig.
5) as in the EMILIN-1 gene (31). However,
whereas the exon structure is remarkably similar between the two genes,
several introns of EMI-2 are much larger than those of the EMI-1 gene
resulting in an overall gene size of the EMILIN-2 gene around 40 kilobases as compared with the highly compact EMILIN-1 gene (8 kilobases) (31). The C1q-like domain is split in exons 7 and 8, the
latter containing also the 3'-untranslated sequence; the collagenic
region is encoded by exon 6 and part of 5, the latter also encoding the
EMILIN-2-specific proline-rich region. Finally, characteristic is the
presence in both genes of a very uncommon large exon of about 1900 bp,
in which the coiled-coil regions potentially involved in interchain
interaction are clustered. The present amino acid sequences of EMILIN-1
and EMILIN-2 and the strong similarities of exon organization indicates
that they are the products of closely related but distinct genes likely to be derived from a common ancestor. While this study was under way, a
GenBankTM search using sequences originated from the BAC
clone retrieved a cluster of partially characterized human genomic
clones localized on chromosome 18p11.3 between the markers D18S476 and
D18S481. One of the clones, corresponding to GenBankTM entry AC015958, allowed the complete characterization of the 3'-end of the gene, from
exon 5 to the 3'-untranslated region. A partial overlap exists between
the very end of the EMILIN-2 gene (AF270513) and GenBankTM
entry AL117592, corresponding to a cDNA of about 2100 bp. Moreover, a predicted gene (KIAA0249) with a transcript of about 6000 bp lays very close to the 3'-end of the EMILIN-2 gene on the opposite
strand.
EMILIN-1 and EMILIN-2 Are Differentially Expressed in a Variety of
Tissues--
Distribution of EMILIN-1 and EMILIN-2 mRNAs in
various adult and fetal human tissues was studied by RNA blot
hybridization on a multiple tissue blot containing 50 different tissues
and developmental stages. Many tissues express EMILIN-1 mRNA in
different amounts, with the highest levels in the adult small
intestine, aorta, lung, uterus, and appendix and in the fetal spleen,
kidney, lung, and heart; intermediate expression was detected in adult liver, ovary, colon, stomach, lymph node, and spleen; adult heart, bladder, prostate, adrenal gland, mammary gland, placenta, and kidney
showed low expression whereas a series of other adult tissues, including skeletal muscle and different regions of adult brain, did not
express EMILIN-1 mRNA at all. (Fig.
6). The mRNA expression for EMILIN-2
resulted much more restricted, with a relatively high expression in
fetal heart and adult lung, intermediate levels in peripheral
leukocytes, placenta, and spinal cord and low expression in fetal
brain, spleen, thymus, and lung and in adult heart, aorta, testis, bone
marrow, small intestine, thymus, lymph node, and appendix. Although RNA
spotted amounts are accurately normalized allowing a semiquantitative
comparative analysis among the tissue mRNAs hybridized with the
same probe, a direct quantitative comparison between EMILIN-1 and
EMILN-2 mRNA expression is not feasible because of possible
differences in hybridization efficiency between the two probes.
Nevertheless, the conclusion can be reached that (i) EMILIN-1 is more
widely distributed in both fetal and adult tissues; (ii) EMILIN-1 is
expressed at higher levels in fetal heart and fetal lung compared with
adult tissues (or any other tissue); (iii) EMILIN-2 is much more
expressed in fetal than in adult heart; (iv) conversely, adult lung
shows the highest expression for EMILIN-2 as compared with fetal lung
and all the other tissues; and (v) finally, in uterus only EMILIN-1 is
expressed.
Codistribution of EMILIN-1 and EMILIN-2 in
Vitro--
Confocal microscopy analysis was performed on several tumor
cell lines, and in a number of them a positive expression of EMILIN-2 was detected. As shown in Fig. 7 for the
leiomyosarcoma cell line SK-LMS-1, EMILIN-2-specific immunofluorescence
was extracellular with a diffuse meshwork pattern. In colabeling
experiments it partially colocalized with EMILIN-1, although in some
areas a predominant deposition of EMILIN-2 or EMILIN-1 could also be
detected.
Candidate interactors for human EMILIN-1 were investigated by the
yeast two-hybrid system. One ligand, EMILIN-2, that is secreted extracellularly and is deposited in vitro in the ECM with a
meshwork pattern, was identified using as a bait the gC1q-1 domain of
EMILIN-1; its cDNA and gene and a preliminary mRNA
tissue distribution pattern are reported. The structural
characteristics and the predicted domain organization of EMILIN-2
replicate closely those recently established for EMILIN-1 (20). As a
result, the structural/functional criteria defining the EMILIN members
are beginning to emerge; accordingly, they are expected to be
constituted of four structurally distinct regions preceded by a signal
peptide. In fact, both EMILIN-1 (20) and EMILIN-2 display the newly
identified EMI domain at their N terminus. This domain is consistently
found at the N terminus downstream of the signal peptide in all EMI
domain-containing proteins, except for multimerin, which also has a
propeptide upstream of the EMI domain that is cleaved before secretion
of the mature protein (33). Using both qualitative and quantitative
yeast two-hybrid systems, the EMI-1 domain was recently found to
interact with the gC1q-1 domain and even more strongly with the gC1q-2 domain (21). This finding suggests that, in addition to the gC1q-1/gC1q-2 interaction that was instrumental in isolating the first
EMILIN-2 clone from the library, the heterotypic EMI-1/gC1q-2 interaction detected in vivo in the two-hybrid system might
be related to the macroassembly and tissue organization of EMILINs. The
EMI-2 domain is followed by an extended discontinuous sequence with the
potential of forming amphipathic coiled-coil EMILIN-2 is slightly larger than EMILIN-1 and harbors, right upstream
of the collagenous domain, a unique proline-rich motif of 53 residues.
The proline-rich region is also of potential interest for EMILIN-2
interactions and assembly. For instance and by analogy to proteins such
as dystrophin, which has an overall extended conformation interrupted
by proline-rich sequences representing sites of protein-protein
interaction (36) or may allow bending of the protein, the proline-rich
region of EMILIN-2 could provide some flexibility that is not present
in EMILIN-1. Among the elastic/microfibril-associated glycoproteins
fibrillin-1 has one proline-rich region of equivalent length. However,
different from the proline-rich region of fibrillin-1 that is largely
hydrophobic (30%) and thus unlikely to form a surface loop (37, 38),
in EMILIN-2 this region is highly hydrophylic and potentially exposed
to the solvent and thus available for interactions with other ligands.
While displaying four interruptions of the Gly-X-Y triplets
not detected in EMILIN-1 (20), the collagenous domain of EMILIN-2 could
still form a trimeric collagen-like region, as shown for instance in
type IV collagen (39). This sequence could participate in trimerization
providing additional binding strength to the trimers. At variance from
the rigid stalk that the collagenous domain would form in EMILIN-1,
these imperfections could confer to the collagenous domain of EMILIN-2
more flexibility or bending capability. Considering that upstream of
this domain there is the hydrophylic proline-rich domain, a flexible
rod could confer a higher probability of protein-protein interaction
with potential ligands.
The close identity between the EMILIN-1 and EMILIN-2 cDNAs is
further emphasized by their gene organization, which is almost identical. The exon size pattern and location of introns in the coding
sequence are very well conserved between the two genes, and the two
genes have probably evolved from a common ancestor. However, the intron
sequences have diverged, because the intron sizes in EMILIN-2 are much
larger, and the overall gene size is around 40 kilobases, five times
larger than the EMILIN-1 gene (20). The divergent evolution of the two
genes probably reflects random loss from and/or uptake of intervening
sequences into the noncoding regions of the genes after they
duplicated. Interestingly, the EMILIN-2 gene is located on
chromosome 18p11.3, centromerically positioned but close to the LAMA1
gene coding for the laminin The gC1q domains of both EMILINs have a high sequence
homology including a unique stretch of 10 residues absent in all other members of the C1q/tumor necrosis factor superfamily identified so far
(20). The gC1q-1 domain has been shown experimentally to promote
homotrimerization of EMILIN-1 (26); similarly, gC1q domains of other
members of the superfamily can form homo- or heterotrimers (22,
40-45). Thus, it is very likely that gC1q-2 will also form trimers.
The following question then arises. Given the finding that the gC1q-1
bait interacted with a gC1q-2 cDNA clone of the library, is there
the possibility that EMILIN-1 and EMILIN-2 can form heterotrimeric
assemblies, or are they compatible only with the formation of
homotrimers (Fig. 8)? Although the gC1q
domains are highly similar, the exclusive presence of the proline-rich
region in EMILIN-2, the detection of four imperfections in its
collagenous domain, and the fact that the in vitro
ECM-deposited EMILIN molecules display only a partial colocalization
favor the hypothesis that distinct homotrimers are formed.
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
-binding
proteins 1 to 4 (7-10), fibulins 1 and 2 (11, 12),
microfibril-associated glycoprotein-2 (13), and EMILIN (14). The latter
is synthesized in vitro, and it is deposited extracellularly
as a fine network (15, 16); it is broadly expressed in connective
tissues, and it is particularly abundant in blood vessels, skin, heart,
lung, kidney, and cornea (17-19). EMILIN is found at the interface
between amorphous elastin and microfibrils (14), and it might regulate
the formation of the elastic fiber given the finding that elastin
deposition in vitro is perturbed by the addition of
anti-EMILIN antibodies (14).
-helices, and a new cysteine-rich domain (EMI domain) at the N
terminus (21). The presence of a gC1q domain and the recent identification that gC1q is structurally homologous to the tumor necrosis factor family of growth factors (22) allowed the inclusion of
EMILIN in the C1q/tumor necrosis factor superfamily of proteins (21).
The gC1q-like domain is shared with several other ECM constituents
including type VIII and type X collagens in which it represents the
equivalent of the C propeptide of fibrillar collagens (23-25). Given
the tissue distribution of EMILIN, its proadhesive functions, and the
characteristics of its domains, it is likely that EMILIN plays a
fundamental role in the process of elastogenesis and might associate
with other ECM constituents. However, their identification is difficult
because of the low solubility of the tissue form (15) and by the very
large size of recombinant EMILIN (26) that makes it poorly suitable for protein-protein interaction studies. To bypass these problems we have
decided to isolate potential interactors of EMILIN by the yeast
two-hybrid system that allows the measurement of specific protein-protein interactions in vivo; a vector encompassing
the C-terminal gC1q-like domain that has previously been shown to interact with itself to form homotrimers (26) was constructed. In the
present study this vector was used as a bait to screen a human kidney
cDNA library and allowed the identification of a novel protein that
interacts with EMILIN via their gC1q domains. This gene product,
of which preliminary accounts were reported recently (21, 27) is
homologous to EMILIN, it is encoded by a distinct gene, and differs
in part from EMILIN in the tissue-specific expression pattern. Its
mRNA was detected in a variety of human organs including fetal
heart, lung placenta, and spinal cord. We propose to classify the
EMILINs as a new family of extracellular proteins and to name its
members as EMILIN-1 (formerly EMILIN) and EMILIN-2 (this study).
MATERIALS AND METHODS
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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
,his3,trp1,ura3,LexAop(x6)-LEU2) carrying
the reporter plasmid p8pop-LacZ and YM4271
(MATa,ura3-52,his3-200,lys2-801,ade2-101,ade5,trp1-901,leu2-3,112,tyr1-501,gal4-d512,gal80-D538,ade5-hisG) was used for all assays. Yeast cultures were grown at 30 °C in either YPD (1% yeast extract, 2% peptone, and 2% glucose) or SD minimal medium (0.5% yeast nitrogen base without amino acids, 2%
glucose, and 1% desired amino acid dropout solution). Growth and
manipulation of yeast strains was carried out using the procedures described in the Matchmaker Two-Hybrid system user manual
(CLONTECH Laboratories Inc., Palo Alto, CA). For
our studies, a bait was constructed by cloning in the LexA plasmid the
C-terminal domain of EMILIN-1 (gC1q-1), generated by PCR amplifications
using 1 ng of pCEpu-EMILIN template (26), 10 pM each primer
(see below), one unit of Taq polymerase (Promega Corp.), 0.2 mM each of the four deoxynucleotide triphosphates
(Pharmacia Ultrapure; Amersham Pharmacia Biotech), in a final
volume of 100 µl of 1× Promega PCR buffer (Promega Corp.). The
primers used were as follows: sense,
5'-GGGAATTCGCACCAGCAGCCCCTGTG-3'; and antisense,
5'-CCCTCGAGCTACGCGTGTTCAAGCTCTGG-3'. The underlined bases
correspond to appended EcoRI (sense) and XhoI
(antisense) restriction enzyme recognition sites plus two additional
protective nucleotides. The PCR fragments were digested with the
appropriate restriction enzymes, ligated overnight in pLexA vector, in
frame with the DNA-binding domain, and transformed in the
Escherichia coli competent DH5
strain.
Ampicillin-resistant colonies were screened for the presence of the PCR
fragment by restriction analysis of their plasmids. The nucleotide
sequences of plasmids carrying the insert, as determined by restriction analysis, were performed by automatic sequencing, and a selected plasmid was used as a bait in the library screening. To screen for
interacting proteins the EGY48 cells were sequentially transformed with
the bait and with a human kidney Matchmaker cDNA library (CLONTECH) using the LiAc method. Clones were
examined for transcriptional activation of reporter genes Leu- and
-galactosidase indicating interaction between bait/binding domain
and library/activation domain constructs. Only clones meeting all
standard two-hybrid specificity tests were considered as positive.
These tests included absence of an interaction between the target
construct and p53 and pLaminC-negative control constructs and the
inability of colonies containing the target construct alone, or the
target and the bait vectors without any insert, to pass Leu
and
-galactosidase assay. Positive clones were sequenced.
gt10 vector
(CLONTECH). Successive rounds of screening of a
human aorta cDNA library in the
gt10 vector with the most 5'-end
clones resulted in the isolation of overlapping clones comprising the
full-length cDNA of EMILIN-2. The sequences were performed using
the Big Dye terminator cycle sequencing kit and a model 310 DNA
sequencing system (PerkinElmer Life Sciences). 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-2 gene (see below).
-D-galactopyranoside for 3 h at
37 °C. The culture was then 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 sodium
chloride) at 5 volumes per gram of wet weight. The samples were
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 6His-tagged recombinant fragment was performed
by affinity chromatography on nickel-nitrilotriacetic acid resin
(Qiagen) under native conditions. The recombinant protein was eluted
from the affinity column in sonication buffer, pH 6.0, containing 10%
glycerol and 0.2 M imidazole. BALB/c mice were
immunized with the recombinant gC1q-2 fragment, and hybridomas that
reacted with the antigen in enzyme-linked immunosorbent assay assay
were selected and subcloned twice before using.
RESULTS
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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
-galactosidase
assay, 7 clones of 20 positive colonies were selected and identified as
representing the same clone by DNA sequencing analysis. The interaction
was specific, because neither the LexA DNA-binding domain-gC1q hybrid
interacted with the unfused pB42-activating domain, nor did a
pB42-activating domain-unrelated hybrid clone from the library with the
unfused LexA-binding domain (data not shown). Fusing the original bait
(gC1q-1) into the pB42-activating domain vector and the target protein
into the LexA-binding domain vector also gave a positive result. One
clone, EBP-1, was then further characterized. The EBP-1 cDNA
isolated contained 1000 nucleotides and encoded an in frame 192-amino
acid-long open reading frame with high homology to the C-terminal end
of EMILIN, including part of the collagenic region and the entire gC1q
domain. The stop codon is followed by a quite long 3'-untranslated
region. To obtain the full-length open reading frame, the cDNA was
extended by screening a human aorta library in
gt10. Five partly
overlapping cDNA clones were sequentially isolated using initially
the EBP-1 fragment as the probe and then probes derived from subclones
at the 5'-end of each successively isolated clone (Fig.
1, bottom). A composite
nucleotide sequence of about 3900 base pairs was then obtained from the
overlapping clones. A data bank search indicated that this novel
protein (provisionally called EBP), has not been identified previously,
and several partially overlapping expressed sequence tag entries showed
a good match with different regions of the human EMILIN-1 transcript
(20). However, expressed sequence tag clones harbored a total of 26 mismatches as compared with EBP-1, including base replacements
resulting in amino acid substitutions and single base insertions or
deletions. Independent sequencing of BAC clones confirmed the present
sequence. The complete coding sequence and the deduced amino acid
sequence and the complete 3'-untranslated region of the novel protein
(GenBankTM accession number AF270513) is shown in
(Fig. 2).
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Fig. 1.
A schematic diagram with the cloning strategy
and the domain structure of human EMILIN-2. The various types of
clones (striped and open boxes) used in
determining the sequence are shown. SP indicates the signal
peptide. The different domains are designated according to Bork and
Koonin (48). CC, coiled-coil domain; COL,
collagenous domain; C1q, gC1q-like domain; LZ,
leucine zipper. EMI indicates the novel domain recently
described in EMILIN family members (21). PR,
proline-rich. Bars in the COL domain of EMILIN-2 refer to
imperfections in the triple helix. Cysteines and potential
glycosylation sites are indicated by closed and open
circles, respectively. The diagram of EMILIN-1 is shown for direct
comparison.
1 (Ala; X value
of 0.268) and +1 (Gly; Y value of 0.804) of the present
sequence. Therefore, residues
30/
1 correspond most likely to the
signal peptide as it agrees with the classical consensus sequence (29)
and ends with a consensus signal cleavage site (30). Thus, the mature protein consists of 1023 amino acids, with a calculated molecular mass
of 112 kDa, and a statistical pI of 3.8. It contains 8 potential N-glycosylation sites and 20 cysteines with a number of them
clustered as doublets, separated by none or two residues, that
could be involved in intramolecular disulfide bonding.
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Fig. 2.
Nucleotide and predicted amino acid sequence
of human EMILIN-2. 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 UAA stop codon is indicated by an
asterisk. 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
coiled-coil sequences with the residues in the a and
d position are marked by a dot and by Greek
letters; the glycines (G) of the collagenous domain are
shown within triangles; the prolines of the proline-rich
region are indicated by reverse type; and the C1q-like
C-terminal domain is boxed.
helices (Fig.
4, top), also suggest that
EMILIN-2 might form extended homoassociations, as was determined to be
the case for EMILIN-1 (26). Finally, one striking difference
between the two members was the finding that in EMILIN-2 the collagenic
region is preceded by a sequence, absent in EMILIN-1, characterized by
an unusually high proline content. This novel proline-rich
55-residue-long sequence, in which the proline content exceeds 41%
(compared with an 8-12% of proline content in the EMI and gC1q
domains and a 2-4% in the coiled-coil regions), might be implicated
in additional protein-protein interactions.
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Fig. 3.
Sequence comparison. A, EMI
domain. B, gC1q domain. Identical/similar residues are
indicated in reverse type. The locations of the strands
according to the crystal structure of the gC1q-like domain of
ACRO-30/Adipo Q (22) are indicated above the sequence by
arrows and capital letters. The insertion of 10 residues shared between EMILIN-1 and EMILIN-2 is
double-underlined. ACRP-30/Adipo Q is shown here as
representing a prototype gC1q-containing member of the
superfamily.
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Fig. 4.
Sequences of the intron:exon junctions
of the human EMILIN-2 gene. Translated sequences are given in
uppercase letters; intron sequences are given in
lowercase letters. Amino acids encoded near and at splice
junctions are indicated in one-letter code above their
codons. Exon and intron sizes are also shown. Consensus sequences of
the splice acceptor and donor sites are in bold. Splice site
consensus sequences are shown at the bottom. y,
pyrimidine; n, any nucleotide. Ph1 and
Ph2 indicate introns that interrupt a codon triplet after
the first or after the second nucleotide, respectively. UTR,
untranslated region.
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Fig. 5.
Schematic representation of the human
EMILIN-2 gene. A schematic diagram of the EMILIN-1 gene, as
reported in Ref. 31, is shown at the top. Broken
lines connect the exons of EMILIN-1 with the corresponding exons
of EMILIN-2 that are numbered from 1 to 8. Numbers above the
various exons and introns refer to their length in base pairs. The
various domains corresponding to the exons are indicated as in Fig. 1.
PR, proline-rich; SP, signal peptide;
col, collagenous domain.
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Fig. 6.
mRNA expression. Expression of
EMILIN-1 and EMILIN-2 using a human multiple tissue blot is shown at
the top. Corresponding tissues are indicated at the
bottom. The two blots were exposed for the same length of
time.
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Fig. 7.
Double-labeling immunostaining of EMILIN-1
and EMILIN-2. Human leiomyosarcoma cells were grown
in vitro for four days, fixed, and incubated with anti
EMILIN-1 (left) or anti EMILIN-2 (right)
antibodies. On the center panel with the merged images the
partial colocalization is indicated by the yellow
staining.
DISCUSSION
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
-helices. Although the
sequence homology between the EMILINs is negligible in this domain as
is the relative position of the heptad repeats, the overall propensity
to form coiled-coil structures (34, 35) is similarly high in both
molecules (20, 27).
1 chain (GenBankTM). The
precise chromosomal mapping of EMILIN-2 is not possible yet, because
that chromosomal region is still ill defined, but the EMILIN-2 gene is
very likely between the markers D18S476 and D18S481 right upstream of
the KIAA0249 gene.
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Fig. 8.
Schematic representation of the protomers of
EMILIN-1 and EMILIN-2: potential interaction models between these two
members.
The gC1q domain-containing molecules assemble to quaternary structures composed of multimers of several polypeptides reaching sizes of several millions of daltons. Both in vivo (15) and in EMILIN-1-transfected 293-EBNA cells (26) EMILIN-1 is present as large molecular aggregates. The closely related multimerin platelet protein similarly forms large multimers (46). These polymers are apparently because of intermolecular S-S bonds, because both EMILIN-1 and multimerin migrate as a trimeric protomer of about 500 kDa under reducing conditions in SDS gels. EMILIN-2 also has a number of cysteines that might potentially be involved in intermolecular S-S bonds.
Recombinant EMILIN-1 promoted cell adhesion of a number of hematopoietic and nonhematopoietic cell lines (27). In addition, a proadhesive function was also associated with the isolated recombinant and native gC1q-1 domain (20) suggesting that at least part of the cell binding activity could reside in this domain. Among the numerous members of the C1q/tumor necrosis factor superfamily, a cell adhesive function had been reported previously for the gC1q domain of the complement C1q, as well (47). It will be a matter of further studies to investigate whether EMILIN-2 and/or its gC1q-2 domain are endowed with a similar proadhesive function.
The data on tissue and developmental expression of EMILIN-2,
although still very preliminary, allow some considerations to be drawn.
The prominent expression in the fetal heart and the drastic reduction
in the adult heart suggest that EMILIN-2 might be involved in or
promote the development of heart chambers. On the contrary, the
striking reverse pattern observed in the lung, i.e. low expression in the fetus and high
expression in the adult, indicates a potential role of EMILIN-2 in the
physiology of respiration. More in depth studies and a comparative
analysis of EMILIN expression in the developing mouse are necessary and
should help elucidate the role played by these molecules. Finally,
although formal ultrastructural proof that also EMILIN-2 is located at
the elastin-microfibril interface is still lacking, the finding that
the tissue distribution of EMILIN-1 and EMILIN-2 is only partly
overlapping supports the notion that EMILINs contribute to the
compositional and maybe functional heterogeneity of ECM structures.
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ACKNOWLEDGEMENTS |
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We thank Francesco Bucciotti for excellent technical assistance and Dr. Paola Spessotto for performing immunofluorescence staining.
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FOOTNOTES |
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* This work was supported by Grants E 704 and E 1256 from Telethon, Ministero dell'Università e della Ricerca Scientifica e Tecnologica-Cofin 1998 and 1999, and Fondo Dipartimentale.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: Divisione di Oncologia Sperimentale, Centro di Riferimento Oncologico, 33081 Aviano, Italy. Tel.: 0039-0434-659-365; Fax: 0039-0434-659-428; E-mail: acolombatti@cro.it.
Published, JBC Papers in Press, January 16, 2001, DOI 10.1074/jbc.M011591200
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ABBREVIATIONS |
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The abbreviations used are: ECM, extracellular matrix; gC1q, globular C1q-like domain; PCR, polymerase chain reaction; bp, base pair; EBP, EMILIN-binding protein.
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REFERENCES |
---|
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---|
1. | Sakai, L. Y., Keene, D. R., and Engvall, E. (1986) J. Cell Biol. 103, 2499-2509[Abstract] |
2. | Zhang, H., Apfelroth, S. D., Hu, W., Davis, E. E., Sanguineti, C., Bonadio, J., Mecham, R. P., and Ramirez, F. (1994) J. Cell Biol. 124, 855-863[Abstract] |
3. | Henderson, M., Polewski, R., Fanning, J. C., and Gibson, M. A. (1996) J. Histochem. Cytochem. 44, 1389-1397[Abstract] |
4. |
Gibson, M. A.,
Hatzinikolas, G.,
Kumaratilake, J. S.,
Sandberg, L. B.,
Nicholl, J. K.,
Sutherland, G. R.,
and Cleary, E. G.
(1996)
J. Biol. Chem.
271,
1096-1103 |
5. | Abrams, W. R., Ma, R. I., Kucich, U., Bashir, M. M., Decker, S., Tsipouras, P., McPherson, J. D., Wasmuth, J. J., and Rosenbloom, J. (1995) Genomics 26, 47-54[CrossRef][Medline] [Order article via Infotrieve] |
6. | Zhao, Z., Lee, C.-C., Jiralerspong, S., Juyal, R. C., Lu, F., Baldini, A., Greenberg, F., Caskey, C. T., and Patel, P. I. (1995) Hum. Mol. Gen. 4, 589-597[Abstract] |
7. | Kanzaki, T., Olofsson, A., Moren, A., Werntedt, C., Hellman, U. K., Claesson-Welsh, L., and Heldin, C. H. (1990) Cell 6, 1051-1061 |
8. | Gibson, M. A., Hatzinikolas, G., Davis, E. C., Baker, E., Sutherland, G. R., and Mecham, R. P. (1995) Mol. Cell. Biol. 15, 6932-6942[Abstract] |
9. |
Yin, W.,
Smiley, E.,
Flanders, K. C.,
and Sporn, M. B.
(1995)
J. Biol. Chem.
270,
10147-10160 |
10. |
Saharinen, J.,
Taipale, J.,
Monni, O.,
and Keski-Oja, J.
(1998)
J. Biol. Chem.
273,
18459-18469 |
11. |
Roak, E. F.,
Keene, D. R.,
Haudenschild, C. C.,
Godyna, S.,
Little, C. D.,
and Argraves, W. S.
(1995)
J. Histochem. Cytochem.
43,
401-411 |
12. |
Reinhardt, D. P.
(1996)
J. Biol. Chem.
271,
19489-19496 |
13. |
Raghunath, M.,
Tschodrich-Rotter, M.,
Sasaki, T.,
Meuli, M.,
Chu, M.-L.,
and Timpl, R.
(1999)
J. Invest. Dermatol.
112,
97-101 |
14. | Bressan, G. M., Daga-Gordini, D., Colombatti, A., Castellani, I., Marigo, V., and Volpin, D. (1993) J. Cell Biol. 121, 201-212[Abstract] |
15. |
Bressan, G. M.,
Castellani, I.,
Colombatti, A.,
and Volpin, D.
(1983)
J. Biol. Chem.
258,
13262-13267 |
16. |
Colombatti, A.,
Bonaldo, P.,
Volpin, D.,
and Bressan, G. M.
(1988)
J. Biol. Chem.
263,
17534-17540 |
17. | Colombatti, A., Bressan, G. M., Castellani, I., and Volpin, D. (1985) J. Cell Biol. 100, 18-26[Abstract] |
18. | Colombatti, A., Bressan, G. M., Volpin, D., and Castellani, I. (1985) Collagen Relat. Res. 5, 181-191 |
19. | Colombatti, A., Poletti, A., Bressan, G. M., Carbone, A., and Volpin, D. (1987) Collagen Relat. Res. 7, 259-275 |
20. |
Doliana, R.,
Mongiat, M.,
Bucciotti, F.,
Giacomello, E.,
Deutzmann, R.,
Volpin, D.,
Bressan, G. M.,
and Colombatti, A.
(1999)
J. Biol. Chem.
274,
16773-16781 |
21. | Doliana, R., Bot, S., Bonaldo, P., and Colombatti, A. (2000) FEBS Lett. 484, 164-168[CrossRef][Medline] [Order article via Infotrieve] |
22. | Shapiro, L., and Scherer, P. E. (1998) Curr. Biol. 8, 335-338[Medline] [Order article via Infotrieve] |
23. | Sage, H., Pritzl, P., and Bornstein, P. (1980) Biochemistry 19, 5747-5755[Medline] [Order article via Infotrieve] |
24. | Kittleberger, R., Davis, P. F., and Greenhill, N. S. (1989) Biochem. Biophys. Res. Commun. 159, 414-419[Medline] [Order article via Infotrieve] |
25. | Barber, R. E., and Kwan, A. P. (1996) Biochem. J. 320, 479-485[Medline] [Order article via Infotrieve] |
26. |
Mongiat, M.,
Mungiguerra, G.,
Bot, S.,
Mucignat, M.-T.,
Giacomello, E.,
Doliana, R.,
and Colombatti, A.
(2000)
J. Biol. Chem.
275,
25471-25480 |
27. | Colombatti, A., Doliana, R., Bot, S., Canton, A., Mongiat, M., Mungiguerra, G., Paron-Cilli, S., and Spessotto, P. (2000) Matrix Biol. 19, 289-301[CrossRef][Medline] [Order article via Infotrieve] |
28. | Kozak, M. (1989) J. Cell Biol. 108, 229-241[Abstract] |
29. | Nielsen, H., Engelbrecht, J., Brunak, S., and von Hejne, G. (1997) Protein Eng. 10, 1-6[Abstract] |
30. | Perlman, D., and Halvorson, H. O. (1983) J. Mol. Biol. 167, 391-409[Medline] [Order article via Infotrieve] |
31. |
Doliana, R.,
Canton, A.,
Bucciotti, F.,
Mongiat, M.,
Bonaldo, P.,
and Colombatti, A.
(2000)
J. Biol. Chem.
275,
785-792 |
32. | Mount, S. M. (1982) Nucleic Acids Res. 10, 459-472[Abstract] |
33. | Polgar, J., Magnenat, E., Wells, T. N., and Clemetson, K. J. (1998) Thromb. Haemost. 80, 645-648[Medline] [Order article via Infotrieve] |
34. | Berger, B., Wilson, D. B., Wolf, E., Tonchev, T., Milla, M., and Kim, P. S. (1995) Proc. Natl. Acad. Sci. U. S. A. 92, 8259-8263[Abstract] |
35. |
Wolf, E.,
Kim, P. S.,
and Berger, B.
(1997)
Protein Sci.
6,
1179-1189 |
36. | Ervasti, J. M., and Campbell, K. P. (1991) Cell 66, 1121-1131[Medline] [Order article via Infotrieve] |
37. | Pereira, L., D'Alessio, M., Ramirez, F., Lynch, J. R., Sykes, B., Pangilinan, T., and Bonadio, J. (1993) Hum. Mol. Genet. 2, 961-968[Abstract] |
38. | Corson, G. M., Chalberg, S. C., Dietz, H. C., Charbonneau, N. L., and Sakai, L. Y. (1993) Genomics 17, 476-484[CrossRef][Medline] [Order article via Infotrieve] |
39. | Schuppan, D., Timpl, R., and Glanville, R. W. (1980) FEBS Lett. 115, 297-300[CrossRef][Medline] [Order article via Infotrieve] |
40. |
Chan, D.,
Weng, Y. M.,
Hocking, A. M.,
Golub, S.,
McQuillan, D. J.,
and Bateman, J. F.
(1996)
J. Biol. Chem.
271,
13566-13572 |
41. |
Frischholtz, S.,
Beier, F.,
Girkontaite, I.,
Wagner, K.,
Poschl, E.,
Turnay, J.,
Mayer, U.,
and von der Mark, K.
(1998)
J. Biol. Chem.
273,
4547-4555 |
42. | Rosenblum, N. D. (1996) Biochem. Biophys. Res. Commun. 227, 205-210[CrossRef][Medline] [Order article via Infotrieve] |
43. |
Illidge, C.,
Kielty, C.,
and Shuttleworth, A.
(1998)
J. Biol. Chem.
273,
22091-22095 |
44. | Reid, K. B. (1989) Behring Inst. Mitt. 8-19 |
45. | Smith, K. F., Haris, P. I., Chapman, D., Reid, K. B. M., and Perkins, S. J. (1994) Biochem. J. 301, 249-256[Medline] [Order article via Infotrieve] |
46. | Hayward, C. P. M., Warkentin, T. E., Horsewood, P., and Kelton, J. G. (1991) Blood 77, 2556-2560[Abstract] |
47. | Nicholson-Weller, A., and Klickstein, L. B. Curr. Opin. Immunol. 11, 42-46 |
48. | Bork, P., and Koonin, E. V. (1996) Curr. Opin. Struct. Biol. 6, 366-375[CrossRef][Medline] [Order article via Infotrieve] |