From the Divisions of Cardiovascular Research and
¶ Neurosciences, The Hospital for Sick Children and the
§ Department of Laboratory Medicine and Pathobiology,
University of Toronto, Toronto, Ontario M5G 1X8, Canada
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ABSTRACT |
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Our previous studies showed immunological and
functional similarities, as well as partial sequence homology, between
the enzymatically inactive alternatively spliced variant of human
-galactosidase (S-gal) and the 67-kDa elastin/laminin-binding
protein (EBP) from sheep. To define the genetic origin of the EBP
further, a full-length human S-gal cDNA clone was constructed and
subjected to in vitro transcription/translation. The
cDNA was also transfected into COS-1 cells and into the
EBP-deficient smooth muscle cells (SMC) from sheep ductus arteriosus
(DA). In vitro translation yielded an unglycosylated form
of the S-gal protein, which immunoreacted with anti-
-galactosidase
antibodies and bound to elastin and laminin affinity columns. S-gal
cDNA transfections into COS-1 and DA SMC increased expression of a
67-kDa protein that immunolocalized intracellularly and to the cell
surface and, when extracted from the cells, bound to elastin. The
S-gal-transfected cells displayed increased adherence to
elastin-covered dishes, consistent with the cell surface distribution
of the newly produced S-gal-encoded protein. Transfection of DA SMC
additionally corrected their impaired elastic fiber assembly. These
results conclusively identify the 67-kDa splice variant of
-galactosidase as EBP.
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INTRODUCTION |
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Virtually all cell types, including tumor cells, interact with the
extracellular matrix (ECM)1
during certain stages of their development. Such contacts may be
strictly adhesive or can transduce signals from the ECM to the
intracellular machinery (1). These significant cell matrix interactions
are mediated through specialized cell surface receptors (2, 3).
Interactions between cells and elastin are mediated by a non-integrin
cell surface receptor complex consisting of three protein subunits
(4-6). Two of these subunits (61- and 55-kDa subunits) are cell
membrane-associated proteins that immobilize the third, a 67-kDa
peripheral subunit called the elastin-binding protein (EBP). The EBP
binds predominantly to the repeating VGVAPG hydrophobic domains on
elastin, but it may also bind to other similar hydrophobic domains on
elastin (7), and to the LGTIPG sequence on laminin (5, 8). Moreover,
the EBP also interacts with moieties containing -galactosugars
through a separate "lectin-like" binding domain. However, binding
of
-galactosugar-bearing moieties to the lectin domain of the EBP
causes such conformational changes in the 67-kDa protein that it loses
its affinity for elastin and separates from the other subunits of the
elastin receptor. Thus, the EBP can be shed from the cell surface by
interactions with galactosugars (galactose, lactose) or with
N-acetylgalactosamine-containing glycosaminoglycans
(chondroitin sulfate, dermatan sulfate), which bind to its lectin
site (9-11). The EBP appears to be directly involved in the generation
of intracellular signal transmission after contact with its matrix
ligands (6). Binding of elastin-derived peptides to the EBP, when
present on the cell surface, resulted in a rapid and transient increase
in free intracellular calcium (12, 13), whereas displacement of EBP
with either galactose or lactose prevented such an influx. It has been
suggested that the elastin receptor-mediated signal transduction
involves the G1 protein and the chain activation of
phospholipase C and phosphokinase C (14-16). In elastin-producing
cells, the EBP was also detected in endosomal compartments and proven
to act as a recyclable shuttle protein that binds tropoelastin
intracellularly and facilitates its secretion and assembly into elastic
fibers (17, 18).
We found that SMC from intimal cushions of the fetal ductus arteriosus
(DA) (a shunt between the fetal aorta and the pulmonary artery, which,
in preparation for closure shortly after birth, develops intimal
"cushions" as a result of massive migration of medial SMC into the
subendothelium; see Ref. 19), as well as SMC from atherosclerotic
vascular lesions are deficient in EBP (9). This indicated that the lack
of this protein may be responsible for SMC detachment from the elastin
matrix, hence linking it to transformation of these cells to a
synthetic migratory phenotype that underlies vascular thickening and
lumenal narrowing common in atherosclerosis (9, 10). Moreover, we have
determined that an unoccupied cell surface EBP interferes with binding
of interleukin-1 (IL-1
) to its cell surface receptor on arterial SMC and modifies the response of the vascular cells to the endogenous and exogenous cytokine (20, 21).
Our previous studies aimed at detailed characterization of the EBP have
established that the EBP isolated from sheep SMC displays immunological, functional, and partial sequence homology to the 67-kDa
alternatively spliced variant of human -galactosidase described by
Morreau and colleagues (22) as a protein with undefined function. This
form of
-galactosidase arises through the splicing out of two
non-contiguous protein-encoding regions in such a way that the first
deletion introduces a frameshift, which is restored by the deletion of
a second region. The net result of these deletions is a shortened
protein with the introduction of a 32-amino acid sequence unique to the
alternatively spliced form of
-galactosidase (Fig. 1A).
Due to the loss of protein domains encoded by the spliced out exons 3, 4 (region 1), and 6 (region 2), S-gal does not display any enzymatic
activity of
-galactosidase and is not targeted to the lysosomes.
Furthermore, we have established that the unique domain of S-gal,
encoded by the frameshift-generated sequence, contains an
elastin/laminin-binding motif (11).
Our further studies showed that an antibody (anti-S-gal) made to a
synthetic oligopeptide corresponding to the elastin/laminin-binding motif of human S-gal showed an identical immunolocalization to the cell
surfaces and extracellular elastic fibers as the antibody to EBP (11).
Moreover, anti-S-gal co-localized intracellularly with tropoelastin
(17). Affinity chromatography of human placenta extract, as well as
extracts from sheep aorta SMC on immobilized elastin and laminin,
demonstrated that the 67-kDa protein-bound to these affinity columns
could be eluted by lactose or -D-galactolactone and
immunoreacted with a panel of antibodies recognizing the spliced variant of
-galactosidase (anti-S-gal, anti-C-gal, and anti-P-gal) and with BCZ (anti-EBP) antibody raised to bovine EBP. Thus, in addition to extensive homology, a similar molecular weight, and immunological cross-reactivity, this enzymatically inactive form of
-galactosidase also shows elastin- and laminin-binding properties that are identical to those of EBP (11). These data suggested that
these proteins are similar and, in fact, may be identical in the same
species.
Since the sheep -gal gene has not yet been characterized, the
conclusive proof of the genetic origin of the EBP remained to be
established. Therefore, the major objective of the present study was to
provide evidence of the identity between S-gal and EBP. Thus, the
full-length cDNA clone encoding the alternatively spliced variant
of human
-galactosidase was constructed and used in a series of
in vitro transcription/translation experiments and
transfected into COS-1 cells, which express very low levels of
-gal
and S-gal (24), and into the EBP-deficient sheep DA SMC (9) to
overexpress the S-gal-encoded protein and to determine whether its
structural and functional features will match those of the 67-kDa
EBP.
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EXPERIMENTAL PROCEDURES |
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Materials--
Kits, chemicals, DNA, and reagents were obtained
as follows. The pBluescript SK+ was obtained from
Stratagene (La Jolla, CA). The luciferase construct (TKSL) was kindly
provided by Dr. Harry Elsholtz (University of Toronto, Toronto, Ontario
(ON), Canada). The plasmid pSVL SV40 was obtained from Pharmacia
Biotech Inc. (Baie d'Urfe, Quebec, Canada). All restriction enzymes
were from New England Biolabs (Mississauga, ON, Canada). Gel
purification was conducted using the Geneclean II Kit from BIO 101 Inc.
(Vista, CA). DNA sequencing was done with the Sequenase version 2.0 DNA
sequencing kit from Amersham Canada Ltd. (Oakville, ON, Canada).
In vitro transcription and translation kits were supplied by
Promega (Madison, WI). [35S]Methionine (1200 Ci/mmol) was
obtained from ICN Radiochemicals (Irvine, CA). Radioactive signals were
enhanced using Amplify from Amersham Canada Ltd. (Oakville, ON,
Canada). Kodak X-Omat AR film was used for autoradiography (Eastman
Kodak Co.). Bovine ligamentum nuchae insoluble elastin, -elastin,
and the polyclonal antibody to bovine tropoelastin (23), were purchased
from Elastin Products Co., Inc. (Owensville, MI). Soluble laminin
isolated from Engelbreth-Holm-Swarm tumor and anti-laminin antibody
were obtained from Sigma. Collagen type I (Vitrogen 100®)
was from Collagen Biomaterials (Palo Alto, CA). Lactose, proteinase inhibitors (EDTA, phenylmethylslfonyl fluoride, and leupeptin), Protein
A-Sepharose beads, bovine albumin, and all reagent grade chemicals were
purchased from Sigma.
-Minimal essential medium (
-MEM), fetal
calf serum, and other tissue culture reagents were obtained from Life
Technologies, Inc. (Burlington, ON, Canada). Assays for total protein
concentration were done using the Bio-Rad protein assay kit. Luciferin
for the luciferase assay was from Sigma. All SDS-PAGE reagents were
purchased from Bio-Rad. The Immobilon P transfer membranes were
supplied by Millipore Ltd. (Mississauga, ON). Affinity-purified
polyclonal antibodies: anti-S-gal (raised to a synthetic peptide
mimicking the elastin/laminin-binding sequence, present in the unique,
frameshift-encoded fragment of the 67-kDa spliced variant of
-gal)
and anti-C-gal (raised to a synthetic peptide reflecting the C-terminal
end of the
-gal precursor), as well as anti-P-gal (an
affinity-purified polyclonal antibody to the human
-galactosidase
precursor), were produced in our laboratories (11, 24). The cDNA
clone of
-gal in pGEM-3Z and the COS-1 cells have been described
previously (25). The BCZ monoclonal antibody to the bovine 67-kDa
elastin-binding protein (26) was from Dr. R. P. Mecham (Washington
University, St. Louis, MO). A monoclonal antibody to fibronectin
(mAb1940) was purchased by Chemicon (Temecula, CA). Species and
type-specific secondary antibodies, donkey anti-rabbit F(ab') and goat
anti-mouse antibodies conjugated to horseradish peroxidase, and the
enhanced chemiluminescence (ECL) Western blotting detection kit were
obtained from Amersham Canada Ltd. (Oakville, ON, Canada). Fluorescein
isothiocyanate-conjugated goat anti-mouse and goat anti-rabbit
secondary antibodies were obtained from ICN Immunobiologicals (Lisle,
IL).
Construction of the Full-length S-gal cDNA Clone-- To construct the full-length alternatively spliced cDNA clone (1986 bp) reflecting the sequence described by Morreau and colleagues (22), poly(A)+ mRNA was isolated from cultured normal human skin fibroblasts using a Quick Prep mRNA purification kit from Pharmacia. This mRNA (500 ng) was reverse-transcribed using random hexamers and superscript reverse transcriptase (Life Technologies, Inc.). To isolate overlapping fragments of the cDNA, two polymerase chain reactions (PCR) were carried out. The 5' portion of the cDNA (357 bp) was amplified using the primers 5'-GGTGGTCATGCCGGGGTTCCT-3' and 5'-ATGTTGCTGCCTGCACTGTT-3'. The primers 5'-CCATCCAGACATTACCTGGC-3' and 5'-CCCTCACACATTCCAGGTGGT-3' were used to amplify the 3' fragment of the cDNA (1598 bp). The reactions were carried out on a Perkin-Elmer thermal cycler using an annealing temperature of 55 °C. The fragments were gel-purified and ligated into the EcoRV site of pBluescript SK+. The respective fragments contained a 115-bp overlapping sequence at their respective 3' and 5' ends (see Fig. 1B). In addition, the absence of the initial 27 bp located at the 5' end of the 5' fragment and 119 bp at the 3' end of the 3' fragment was detected (attributed to primer positioning in the initial PCR reaction). Final assembly of the full-length clone eliminated the overlapping segment by employing a common PvuII site found in the overlapping region between the two portions of S-gal. Complete double digestion of the 5' clone with the restriction enzymes KpnI and PvuII yielded a 316-bp fragment representing the 5' segment (i.e.. 5' to the PvuII site) of S-gal, which included an additional 57 bp of vector at its 5' end. The KpnI digestion created a 3' overhang, which was blunt-ended by using Pfu DNA polymerase. The 316-bp 5' fragment and a PvuII-digested 3' clone were then both agarose gel-purified, gene-cleaned, and ligated. The ligation products were transformed into bacterial cells (27) and grown on LB-AMP plates. Restriction digests with XhoI and PvuII confirmed the correct orientation of the short 5' segment of S-gal in the new construct.
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In Vitro Transcription/Translation of the S-gal cDNA
Clone--
In vitro transcription/translation was done in
accordance to the protocols provided by Promega. S-gal cDNA (5 µg) in pGEM-3Z was linearized (digested with XbaI), and
in vitro transcription was conducted. This was followed by
in vitro translation using 2 µl of RNA substrate in a
nuclease-treated rabbit reticulocyte lysate (minus microsomal membranes
and protease inhibitors) in the presence of 0.8 mCi/ml
[35S]methionine ([35S]Met). The translation
mix (minus mRNA) was used as control. In vitro
transcription/translation of the cDNA clone encoding the wild type
-galactosidase precursor (24) was also conducted for comparison. The
supernatants were directly analyzed by SDS-polyacrylamide gel
electrophoresis (SDS-PAGE), followed by autoradiography to detect for
the presence of [35S]Met-labeled reaction products and to
compare molecular size. The reaction products were further
characterized using immunoprecipitation with antibodies recognizing
-gal, S-gal, and EBP, and then by elastin and laminin affinity
columns.
Immunoprecipitation-- Immunoprecipitation (IP) of the [35S]Met-labeled translation product was assessed with our well characterized anti-S-gal, anti-C-gal (11, 25), and anti-P-gal antibodies (24). The preimmune rabbit serum was used as a control. Briefly, the supernatants from the in vitro translation reactions (one volume) were mixed with two volumes of IP buffer (10 mM Tris-buffered saline, pH 7.4, containing 0.2% bovine serum albumin, 0.3% Nonidet P-40, 0.3% sodium deoxycholate, and 0.02% sodium azide) and precleaned by incubation for 1 h at 4 °C with normal rabbit serum (2 µg/µl) and then, for 30 min, with two volumes of Protein A-Sepharose beads (0.1 ml of beads/ml of IP buffer) to remove any nonspecific immunocomplexes. The supernatants were then incubated at 4 °C with either antibody (2 µg/µl) for 2 h and subsequently with two volumes of Protein A beads for 1 h. The beads were washed three times in IP buffer by centrifugation, and then the final pellets were resuspended in SDS sample buffer with 20 mM DTT, boiled for 5 min, resolved by 10% SDS-PAGE, and detected by autoradiography.
Elastin/Laminin Affinity Columns-- To assess the ECM-binding properties of the [35S]Met-labeled in vitro translation product, one volume of the incubation product was mixed with two volumes of IP buffer containing a 1 mg/ml suspension of each of powdered insoluble elastin, insoluble collagen type I, and soluble laminin, and incubated for 30 min at 4 °C. The slurry of insoluble elastin and collagen was then pelleted by centrifugation. The soluble laminin was immunoprecipitated using consecutive incubations with 2 µg/ml polyclonal anti-laminin antibody and two volumes of Protein A-Sepharose beads, as described above. The resulting pellets containing the affinity-bound ligands were rinsed in IP buffer, resolved by SDS-PAGE, and analyzed by autoradiography as above.
Transfections of COS-1 Cells with the Full-length S-gal cDNA
and Characterization of the S-gal-encoded Protein
Product--
Transient transfections of the S-gal cDNA clone into
COS-1 cells, which express low levels of -gal and S-gal (24), were conducted in accordance with a DEAE-dextran protocol (28) modified to
include adenovirus addition. The full-length S-gal cDNA was excised
from pGEM-3Z using SalI digestion and ligated into the mammalian expression vector pSVL SV40 previously digested with XhoI. A series of digestions with the site-specific
restriction enzymes (SmaI and DraIII) confirmed
frame and orientation of the S-gal clone. COS-1 cells were plated
evenly in the 100-mm dishes (10 × 106 cells/dish) and
in 20-mm dishes containing coverslips (1 × 106/dish)
and maintained in
-MEM supplemented with 1%
antibiotics/antimycotics and 10% (v/v) fetal calf serum for 2 days
before transfections were carried out. The transfection mixture
consisted of 5% (0.25 ml) adenovirus deletion-325 (approximately
2 × 108 plaque-forming units/ml), 80% (4 ml)
serum-free
-MEM, 8 µg of S-gal cDNA in pSVL, 8 µg of TKSL,
10% (0.5 ml)
-MEM + 10% fetal calf serum, and 1.6% (80 µl)
DEAE-dextran (5 mg/ml). Control cultures (no DNA, vector alone, wild
type
-gal alone; each including TKSL) were treated in the same
manner. The cells were then harvested from 72-h cultures, and a
luciferase assay was conducted to monitor transfection efficiency (29).
The production of the S-gal encoded protein in control and
S-gal-transfected cells was assessed by immunohistochemistry and
immunoblotting, while its elastin-binding capability was tested by
affinity chromatography and an in vitro adhesion assay.
Immunostaining--
The presence and distribution of S-gal in
the control and S-gal-transfected cells were then compared 72 h
after transfection using immunostaining with anti-P-gal, anti-S-gal,
and anti-EBP (BCZ) antibodies. Cultures of transfected cells from each
experimental group (maintained in small dishes with coverslips) were
fixed at 20 °C in 100% methanol for 30 min, rinsed with water and
PBS, after which the separate coverslips were incubated for 1 h
with PBS-diluted primary antibodies (2 µg/µl) followed by a 1-h
incubation with the appropriate fluorescein isothiocyanate-conjugated
goat anti-rabbit or goat anti-mouse secondary antibodies. An additional 10-min incubation with propidium iodide (0.1 µg/ml) assured nuclear counterstaining. The coverslips were mounted on glass slides with elvanol and analyzed using an Olympus Vanox AH BT3 fluorescence microscope.
Western Blotting of COS-1 Cell Lysates--
72 h after
transfection, the cells maintained in 100-mm dishes were rinsed in PBS
and harvested by scraping. The pelleted cells were then lysed in 200 µl of PBS containing 0.25%
n-octyl--D-glucopyranoside, 0.1 M
lactose, 1 mM EDTA, 1 mM phenylmethylslfonyl
fluoride, 20 µM leupeptin, and freeze-thawed (10 times).
The insoluble remnants were pelleted by centrifugation (14,000 rpm/25
min), and the supernatants were resolved by 10% SDS-PAGE. 100 µg of
total protein was loaded into each well. The proteins were then
electrotransferred to Immobilon P membranes and analyzed by Western
blotting with 2 µg/ml anti-P-gal antibody, which also recognizes EBP
(11) followed by a donkey anti-rabbit F(ab')2 (horseradish
peroxidase-labeled) secondary antibody diluted 1:5000 and ECL.
Elastin Affinity Columns-- To assess the elastin-binding capability of the S-gal-encoded protein produced in COS-1 cells, equal amounts of lysates (200 µg of total protein) (same lysate as used in the Western blot above) were incubated with 5 mg of insoluble elastin (1 mg/ml in PBS) for 2 h at 4 °C. The elastin slurry was then pelleted by centrifugation, rinsed with PBS, and then resuspended in SDS sample buffer with 20 mM DTT. The elastin-bound proteins released to the buffer were resolved by 10% SDS-PAGE and analyzed by Western blotting using anti-P-gal antibody, as described above.
Adhesion Assay-- The elastin-binding capabilities of S-gal-transfected and control cells were tested in an in vitro assay using elastin coated wells on 96-well plates.
Cells transfected with vector alone, with wild typeTransfections of DA SMC with the Full-length S-gal cDNA-- Since SMC from the late gestation ductus arteriosus have been shown to be deficient in EBP and incapable of normal elastic fiber assembly (despite the normal production of tropoelastin) and attachment to elastic fibers (9), we speculated that effective transfection of these cells with the S-gal clone may confer EBP expression and restore a normal SMC phenotype.
Transient transfections (vector alone, wild type ![]() |
RESULTS |
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In Vitro Transcription/Translation of the S-gal cDNA Resulted in the Synthesis of an Elastin/Laminin-binding Protein-- In vitro transcription/translation of the full-length S-gal cDNA resulted in the synthesis of a protein with a molecular weight of approximately 60,000 (Fig. 2A), consistent with the primary sequence deduced by Morreau and colleagues (22). In addition to the 60-kDa [35S]Met-labeled reaction product, autoradiography also detected several lower molecular weight bands, probably representing degradation products and/or incomplete translation products. The control reaction (i.e. no DNA added) did not show any species corresponding to a labeled product encoded by the S-gal cDNA clone.
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The S-gal cDNA Clone Transfected into COS-1 Cells Increased
Expression of the Elastin-binding Protein--
Although the 60-kDa
S-gal cDNA-encoded product from in vitro translation
displayed both properties of S-gal and EBP, it was necessary to define
further if the S-gal protein would hold these properties after final
processing in these primate (COS-1) cells. To achieve this, we
transiently expressed the S-gal cDNA in COS-1 cells. A luciferase
assay confirmed successful transfection in all experimental groups.
Cells transfected with luciferase construct only displayed 3202 RLU,
vector-transfected cells 44,891 RLU, -gal-transfected cells 4049 RLU, and S-gal-transfected cells 8662 RLU.
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S-gal cDNA Transfections of DA SMC Increases Expression of EBP
and Affects Deposition of Extracellular Matrix--
A luciferase assay
indicated an efficient and rather uniform transfection in all
experimental groups. Immunostaining with anti-P-gal antibody of the
S-gal cDNA-transfected DA SMC cultures showed numerous cells with a
vividly increased expression of immunoreactive material, localized
predominantly to the cell surface. As depicted in Fig.
5 (upper panel),
immunostaining with anti-P-gal antibody detected mostly intracellular
epitopes localized to the lysosomes and cisternas in cells transfected
with the wild type -gal clone, while cells transfected with the
S-gal clone expressed a cell surface localized protein immunoreactive
with the same antibody. These data were consistent with the fact that
the lysate of the S-gal-transfected cells demonstrated a substantial
increase in the expression of a 67-kDa protein immunoreactive with the
anti-S-gal antibody on a Western blot (Fig.
6). Densitometric assessments of the
67-kDa bands depicted in Fig. 6, additionally normalized to RLU
(reflecting the transfection efficiency), indicated an approximate
10-fold increase in expression, as compared with controls. Furthermore,
the S-gal-transfected DA SMC demonstrated a 5-fold increase in the
number of cells attached to elastin-coated plates (4 h after initial
plating), as compared with controls (Fig.
7). There was no statistically
significant difference in adhesion between vector-,
-gal-, and
S-gal-transfected cells when plated on plastic alone, albumin, or
collagen type I (data not shown). Most interestingly, the
immunocytochemistry of 7-day-old cultures revealed that
S-gal-transfected DA SMC were capable of normal elastic fiber assembly,
while the vector- (data not shown) and
-gal-transfected cells did
not deposit elastic fibers in the newly produced matrix (Fig. 5,
middle panel). Moreover, the extracellular matrix produced
by the S-gal-transfected DA SMC showed very little immunodetectable
fibronectin, while
-gal-transfected cells deposited an abundance of
fibronectin (Fig. 5, lower panel). This result is consistent
with the previously described mechanism implicating the cell surface
EBP as a blocker of the adjacent IL-1 receptor type I, which transduces
signals (evoked by endogenous IL-1
), leading to the up-regulation of
fibronectin synthesis and secretion in vascular SMC (20, 21).
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DISCUSSION |
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Despite comprehensive functional characterization of the
elastin-binding protein (EBP) (4-6, 9, 10), its structure has not been
determined. In 1993, after tryptic digestion of sheep EBP, we noted
(11) that in addition to several oligopeptides fully homologous to
human -galactosidase, a 14-amino acid sequence, obtained from
cyanogen bromide digestion, contained 50% direct homology (and an
additional three amino acids, which could be substituted without change
in net charge) with a unique sequence (encoded by the frameshifted exon
5) of the alternatively spliced catalytically inactive variant of human
-galactosidase (S-gal) originally described by Morreau et
al. (22) and confirmed by Yamamoto et al. (32).
Subsequent studies showed further immunological and functional
similarities between these two proteins (6, 11, 17, 18, 31). We have
shown that both extracts of human placenta and extracts of sheep
arterial SMC contained a 67-kDa protein which bound to elastin and
laminin affinity columns and could be eluted from those columns with
several galactosugars. These elastin/laminin-binding proteins, isolated
from both human and sheep tissues, were equally immunoreactive with
anti-P-gal, anti-C-gal, and anti-S-gal antibodies, as well as with the
monoclonal BCZ antibody raised to bovine EBP (11). In addition to
identical patterns of immunostaining assessed under light and electron
microscopy, we also found that anti-S-gal antibody co-localized with
intracellular tropoelastin and with extracellular elastic fibers in
ovine arteries (17, 18). Moreover, the treatment of cultured ovine
aortic SMC with anti-S-gal antibody (recognizing and blocking to
elastin-binding domain of S-gal) caused impaired assembly of elastic
fibers (18).
To demonstrate that S-gal and EBP are indeed the same protein, the cDNA clone for S-gal was required. Thus, the full-length clone was constructed after precise trimming and ligation of two separate and partially overlapping cDNA fragments pulled out from human fibroblast mRNA. Sequencing of the final PCR product showed that the resulting clone was in frame and error-free as compared with the described original S-gal cDNA (22).
In attempting to determine if S-gal and EBP are really the same
protein, we undertook a series of studies to characterize the
properties of the S-gal-cDNA encoded protein and to determine the
degree to which it displayed the features attributed to date to EBP. As
a first step, we employed in vitro transcription/translation and obtained a protein of approximately 60,000 (Fig. 2), which corresponded well to the 60-kDa unglycosylated version of S-gal, as
deduced from its primary sequence described by Morreau et
al. (22). For comparison, we also tested the wild type -gal
cDNA clone. SDS-PAGE/autoradiography revealed that both proteins
were immunoprecipitable with polyclonal anti-P-gal antibody (which recognizes common epitopes in both spliced variants of
-gal), but
differed in their molecular size (Fig. 2C). The
S-gal-encoded protein had a molecular mass of 60 kDa, whereas the
76-kDa,
-gal-encoded product was consistent with the deduced size of
an unglycosylated and unprocessed precursor of
-gal (22). Moreover,
only the 60-kDa S-gal cDNA-encoded product immunoprecipitated with
an anti-S-gal antibody, raised to an unique sequence present in the
frameshift-generated domain of S-gal. In addition, the 60-kDa protein
produced by in vitro translation was also immunoprecipitable
with anti-C-gal antibody, which recognizes a C-terminal domain present
in the
-gal precursor and S-gal, but cleaved off from the mature
form of the active enzyme (Fig. 2B).
The most important test for the S-gal-encoded protein was its relation to EBP, as judged by its functional ability to bind to elastin and laminin. We demonstrated that the 60-kDa, S-gal-encoded product efficiently bound to these two matrix ligands but not to collagen type I (Fig. 2D). This result confirmed the tight protein-protein associative character of the elastin-S-gal and laminin-S-gal binding, suggested by our previous studies (11), and additionally showed that such an association does not require glycosylation or other post-translational modification of S-gal.
Since these data indicated that the S-gal produced is immunologically
the same as EBP, and has the ability to bind both elastin and laminin,
subsequent experiments were aimed at determining whether the protein,
when expressed in mammalian cells, displayed additional properties of
EBP. First, we tested whether transfection of the primate COS-1 cells
with the S-gal cDNA clone would increase expression of the fully
processed 67-kDa S-gal protein and then whether this overexpressed
protein would localize to the cell surface and serve as a functional
EBP by mediating interactions of the transfected cells with elastin.
Indeed, affinity chromatography confirmed a substantial increase in the
amount of the 67-kDa elastin-bound protein retained from extracts of
the S-gal-transfected COS-1 cells as compared with controls (Fig.
3B). The 67-kDa protein overexpressed by S-gal-transfected
COS-1 cells was immunoreactive with anti-P-gal antibody, which also
recognized the 85-kDa -gal precursor overexpressed in the
-gal-transfected cells, on Western blots (Fig. 3A). We
therefore concluded that the S-gal cDNA-encoded protein, which
resolves with an identical (67-kDa) molecular mass when produced in
COS-1 cells, likely undergoes post-translational modifications
comparable to the spliced variant of
-gal synthesized in normal
cells (22). It also has the elastin-binding capabilities identical to
EBP isolated from sheep vascular SMC or from human placenta (11).
Immunostaining with anti-P-gal, anti-S-gal, and BCZ antibodies (each
recognizes EBP) revealed that cultures of S-gal-transfected COS-1 cells
contained clusters of cells with increased expression of intracellular
and cell surface-localized immunoreactive protein, as compared with
controls. Moreover, patterns of immunostaining were identical to those
of EBP in normal aortic SMC (9, 11).
Although both -gal and S-gal contain identical signal peptides,
which targets these proteins to the endoplasmic reticulum, the cell
surface localization of the S-gal encoded protein indicates that this
variant, directly translated in COS-1 cells, does not contain the
putative lysosomal targeting domain (mannose 6-phosphate receptor-binding domain) present in the precursor of active
-gal. Since the exact location of this lysosomal targeting domain in the
precursor of active
-gal has not been conclusively determined, one
can speculate that such a critical aspect of this targeting requirement
must be supplied by one, or a combination, of the exons (exons 3, 4, or
6) that are spliced out in the S-gal variant. Our results affirm that
the newly produced S-gal proceeds through the secretory pathways of
transfected COS-1 cells, where it is prepared for its release to the
cell surface. Currently, we do not know whether the S-gal protein
overexpressed in COS-1 cells associates with the two other 55-kDa and
61-kDa subunits of the elastin receptor. Immobilization of S-gal on the
cell surface suggests, however, that such an association, which was
recently documented by double and triple immunostaining of vascular
SMC,2 is most probable. The
increased expression of the functional elastin-binding protein was
additionally confirmed by an adhesion assay, which showed significant
increases in the number of S-gal-transfected cells attached to
elastin-covered plates, as compared with controls (Fig. 4). An increase
in specific adherence to elastin (but not to plastic, albumin, and
collagen type I) documents that the newly produced S-gal-encoded
protein was transferred to the cell surface where it can play a role
previously ascribed to EBP, thereby mediating ligand-specific
cell/matrix interactions.
Further studies with EBP-deficient DA SMC, which are characterized by
poor attachment to elastin, impaired elastic fiber assembly (despite
normal production of tropoelastin), and abundant fibronectin expression
(which stimulates their migratory capabilities in neointima) (9, 10,
20, 21), also confirmed the functional significance of S-gal
transfection. Only S-gal cDNA-transfected DA SMC (but not vector-
or -gal cDNA-transfected cells) displayed a substantial increase
in the expression of a 67-kDa protein immunoreactive with anti-S-gal
antibody (Fig. 6) and additionally demonstrated a significant increase
in attachment to elastin coated plates (Fig. 7). Furthermore, 7-day old
cultures of S-gal-transfected cells demonstrated focal accumulation of
elastic fibers, which were virtually not visible in control cultures of
DA SMC. Thus, the restoration of normal elastic fiber assembly by DA
SMC can be clearly linked to overexpression of the 67-kDa S-gal-encoded protein, indicating that this variant of
-gal plays the identical role as the previously described EBP, the presence of which is imperative for proper secretion and assembly of tropoelastin into elastic fibers (9, 17, 18). Interestingly, the increased cell surface
expression of the anti-P-gal-reactive protein in clusters of the
S-gal-transfected (but not
-gal-transfected) DA SMC coincided with a
substantial decrease in the pericellular deposition of fibronectin.
This effect seems to be consistent with the previously described
ability of the EBP to inhibit the IL-1
-dependent
autocrine/paracrine stimulation of fibronectin synthesis by blocking
the adjacent IL-1 receptor type I present on surface of vascular SMC
(21).
Thus, all the above described cellular effects linked to the increased expression of S-gal are entirely consistent with the previously described effects mediated by EBP (6, 9, 17, 18, 21, 31).
In summary, our results conclusively identify the S-gal-encoded protein as EBP, the protein that plays an important role in vascular development and deficiency of which may contribute to impaired elastic fiber assembly and the SMC detachment from the extracellular matrix and migration in vascular diseases. Moreover, we demonstrated the overexpression of functional EBP in S-gal-transfected vascular SMC deficient in EBP. This may lead to the development of new therapeutic strategies, based on local in vivo S-gal cDNA delivery to neointimal vascular SMC and aimed at reversing their migratory phenotype and attenuating the progressive process of neointimal formation, which complicate the long lasting effects of balloon angioplasty (endarterectomy) of the occluded atherosclerotic coronaries.
The identification of EBP as S-gal may also have a direct impact on the
understanding of the connective tissue and cardiovascular disorders
observed in some cases of GM1 gangliosidosis and all cases
of Morquio disease type B, which represent two primary
-galactosidase deficiency diseases (33). It seems quite probable
that multiple mutations, and especially missense mutations, described
in these diseases would alter function and distribution of both
-gal
and S-gal proteins. Further studies (already initiated in our
laboratory) will determine the respective roles of each protein in
these diseases.
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ACKNOWLEDGEMENTS |
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We thank Dr. Sunqu Zhang and C. C. Eric Yang for their excellent technical assistance and advice.
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FOOTNOTES |
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* This work was supported in part by Grants PG 12351 (to C. A. P.) and PG 13920 (to A. H.) from the Medical Research Council of Canada.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.
Career Investigator of the Heart and Stroke Foundation of
Ontario. To whom correspondence should be addressed: Div. of
Cardiovascular Research, The Hospital for Sick Children, 555 University
Ave., Toronto, Ontario M5G 1X8, Canada. Tel.: 416-813-5918; Fax:
416-813-7480; E-mail: alek.hinek{at}mailhub.sickkids.on.ca.
1
The abbreviations used are: ECM, extracellular
matrix; S-gal, spliced variant of -galactosidase; C-gal, C-terminal
end of the
-galactosidase precursor; P-gal, human
-galactosidase
precursor; EBP, elastin/laminin-binding protein;
-gal,
-galactosidase; DA, ductus arteriosus; SMC, smooth muscle cells;
-MEM,
-minimal essential medium; PAGE, polyacrylamide gel
electrophoresis; PCR, polymerase chain reaction; IP, immunoprecipitate;
DTT, dithiothreitol; PBS, phosphate-buffered saline; RLU, relative
light unit(s); IL-1, interleukin-1; bp, base pair(s); GM1,
Gal
(1-3)GalNAc
(1-4)Gal-[
(2-3)NeuAc-]
(1-4)Glc-
- 1-ceramide.
2 A. Hinek and J. W. Callahan, unpublished results.
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REFERENCES |
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