© 1999 American Heart Association, Inc.
Vascular Biology |
Versican/PG-M Isoforms in Vascular Smooth Muscle Cells
From the Department of Pathology (J.M.L., K.R.B., E.D.K., S.M.S., T.N.W.), University of Washington, Seattle; and the Department of Pharmacology (P.M.), New York University Medical Center, New York. Present address of P.M., Department of Cell Biology, New York University Medical Center, New York, NY 10016.
Correspondence to Joan M. Lemire, Department of Pathology, University of Washington, 1959 NE Pacific Street HSB E-508, Box 357470, Seattle, WA 98195. E-mail joanlemi{at}u.washington.edu
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Abstract—The expression of increased amounts of proteoglycans in the extracellular matrix may play a role in vascular stenosis and lipid retention. The large chondroitin sulfate proteoglycan versican is synthesized by vascular smooth muscle cells (SMCs), accumulates during human atherosclerosis and restenosis, and has been shown to bind LDLs. We recently demonstrated that adult rat aortic SMCs express several versican mRNAs. Four versican splice variants, V0, V1, V2, and V3, have recently been described, which differ dramatically in length. These variants differ in the extent of modification by glycosaminoglycan chains, and V3 may lack glycosaminoglycan chains. In this study, we characterized versican RNAs from rat SMCs by cloning, sequencing, and hybridization with domain-specific probes. DNA sequence was obtained for the V3 isoform, and for a truncated V0 isoform. By hybridization of polyadenylated RNA with domain-specific probes, we determined that the V0, V1, and V3 isoforms are present in vascular SMCs. We confirmed the presence of the V3 isoform in polyadenylated RNA and in RT-PCR products by hybridization with an oligonucleotide that spans the splice junction between the hyaluronan-binding domain and the epidermal growth factor-like domain. In addition, a novel splice variant was cloned by PCR amplification from both rat and human SMC RNA. This appears to be an incompletely spliced variant, retaining the final intron. PCR analysis shows that this intron can be retained in both V1 and V3 isoforms. The predicted translation product of this variant would have a different carboxy-terminus than previously described versican isoforms.
Key Words: proteoglycan-M • proteoglycan • splicing • vascular smooth muscle • unspliced
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The major proteoglycan present in the interstitial matrix of vascular smooth muscle cells (SMCs) is a large chondroitin sulfate (CS) proteoglycan that forms aggregates with the glycosaminoglycan hyaluronan.1 This proteoglycan has been identified by immunohistochemistry, immunoblotting, and cloning experiments as versican.2 Versican, also known as PG-M, is a member of a gene family that also includes aggrecan, the predominant proteoglycan of cartilage, and 2 brain proteoglycans, neurocan and brevican.3 These proteoglycans bind hyaluronan via the amino-terminal globular domain (G1) comprising an immunoglobulin-like domain and a proteoglycan tandem repeat. The selectin-like domain (G3) at the carboxy-terminus is composed of epidermal growth factor (EGF)-like, lectin-like, and complement-regulatory protein (CRP)-like domains.3 The G3 domain of versican has been shown to bind tenascin-R, as well as several carbohydrates.4 5 6 Between the globular domains is a larger extended region bearing CS chains. These CS chains are believed to serve an antiadhesive function, and their hygroscopic properties make versican a large space-filling molecule that may resist the compression of pulsing blood.1 7 8
The central glycosaminoglycan (GAG)-attachment
domains of versican are encoded by exons that can undergo differential
splicing.9 10 The core proteins translated from the splice
variants are predicted to differ greatly in length and in the number of
CS chains attached.11 12 13 These variants are known as V0,
which contains both 
GAG and ßGAG exons, V1, containing the ßGAG
exon, V2, having the GAG exon, and V3, consisting only of the
globular domains.
We have previously shown that the V1 isoform is expressed by monkey and human vascular SMCs in vitro and in vivo.2 We recently showed that several RNA bands from total rat adult SMC RNA hybridize at high stringency to versican cDNA probes14 and thus appear to be products of the versican gene. These RNAs could include known and previously undescribed splice variants, or those using different initiation or termination sites. Identification of protein domains that may be differentially regulated should allow us to ask more specific questions about the function of those domains, and to develop tools (eg, antibodies, recombinant expression for specific domains) to answer those questions.
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Methods |
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Cell Culture and RNA Isolation
Rat SMCs were cultured in modified Waymouth's medium containing 5% calf serum, as previously described,15 and harvested on reaching confluence. The rat SMC lines WKY3M-22, WKY12-22, SD3M-23, and SD12 were previously established and characterized.14 15 16 17 18 Human aortic SMC line RR 87-80 (gift of Dr Russell Ross) was established from a 2- to 3-month-old child who died of sudden infant death syndrome.19 Human SMCs were cultured in DMEM containing 10% FCS. After culture in DMEM containing 1% human plasma-derived serum for 48 hours, cells were stimulated with 10 ng/mL platelet-derived growth factor for 24 hours. Total RNA was isolated as previously described.20 Polyadenylated [poly(A)+] RNA was isolated by oligo(dT) chromatography (Stratagene).
Electrophoresis, Northern, and Southern Transfer
Fifteen micrograms of total RNA or 10 µg of
poly(A)+ RNA was separated on 0.8% agarose gels
containing formaldehyde.21 RNA band sizes were determined
in comparison with an RNA ladder (Life Technologies) probed with
radiolabeled 
HindIII DNA. RNA in the gels was subjected
to limited alkaline hydrolysis, transferred to Zetaprobe (Bio-Rad)
membrane, and immobilized by UV
cross-linking.21 DNA was separated in agarose gels
buffered with Tris- borate–EDTA (TBE), depurinated for 10 minutes in
0.25 mol/L HCl, transferred to Zetaprobe,21 and
immobilized by UV cross-linking.
Preparing and Screening cDNA Library
RNA was isolated from confluent cultures of the rat SMC cell
line WKY3 M-22, and poly(A)+ RNA was isolated. A
cDNA library [primed with oligo(dT)] in ZAPII (Stratagene) was
prepared. Plaques (3x105) were screened for
clones that hybridized to both PCR1 and PCR4 versican
cDNAs,2 which contain sequences that are present in
all known versican splice variants (Figure 1
); 3 clones (rVa, rVd, and rVe) that
hybridized with both probes, and 1 (rVb) that hybridized with only the
3' probe, were rescued from the ZAPII clones (protocol
supplied by Stratagene), subjected to restriction mapping and
sequenced.
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Probes and Hybridization
Monkey versican PCR1 and human versican PCR4 cDNAs2
were isolated from vector DNA by restriction digestion before labeling.
An EcoRI/SacI fragment representing
nucleotides 1 to 631 of rat versican plasmid rVb (see
below) was used as a probe for the GAG domain. A
PstI/EcoRI fragment representing
nucleotides 1315 to 2148 of rVb was used as a probe for the
ßGAG domain. Probes for the 5'- and 3'-domains of versican were
prepared by PCR, using rat versican cDNAs as templates. Reaction
solutions were as described by the supplier (Perkin Elmer). For the
hyaluronan-binding region (HABR) probe, primers 16B' and 17B'
were used to amplify the second proteoglycan tandem repeat (see
Table, for
oligonucleotide sequences).
Oligonucleotides were synthesized by Operon, Life
Technologies, or National Biosciences. Plasmid rVd (10 ng/mL) template
was amplified for 30 cycles (94°C for 1 minute, 55°C for 1 minute,
and 72°C for 1 minute). For the G3 domain probe, primers 21VE5
and 24VL3 (see the Table) were used to amplify most of the
EGF-like and lectin-like domains. Plasmid rVa (10 ng/mL) was amplified
for 30 cycles (94°C for 1 minute, 43°C for 1 minute, and 72°C for
1 minute). After PCR, primers were removed from the desired product
by using a Centricon 50 filter apparatus (Millipore).
Restriction fragments and PCR products were labeled by random
priming in the presence of [-32P]dCTP (ICN)
and hybridized to northern and Southern blots as previously
described.22
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Oligonucleotide probes 19VSPLH (human) and 18VSPLR
(rat) (see the Table
) were designed to be antisense to the
putative splice junction between the HABR and the first EGF-like
domain. Labeled oligonucleotides were prepared by using
polynucleotide kinase (Pharmacia) and
[
-32P]ATP,21 and hybridized as
previously described.15 Posthybridization washes were 2x5
minutes and 1x20 minutes in 2xSSPE 3.47 mmol/L (0.1%)
SDS at 42°C and 1x20 minutes 0.6xSSPE 3.47 mmol/L
(0.1%) SDS at 45°C (1xSSPE=0.15 mol/L NaCl, 0.2 mol/L
NaH2PO4, and 0.02 mol/L
Na4EDTA). Under these conditions, the
oligonucleotides would hybridize to molecules where
both halves of the splice junction are adjacent to each other; the rat
V3 clones (including rVa, rVd, and rVe) hybridized to the probe, but
the larger clone, rVb, corresponding to most of the V0 isoform, did not
(not shown).
RT-PCR Analysis
Total RNA was reverse-transcribed by using Superscript II (Life
Technologies). Rat and human SMC total RNAs (0.56 µg and 1 µg,
respectively) were reverse-transcribed by using random hexamer primers.
Versican sequences were amplified as previously
described,14 using either a kit from Perkin-Elmer or Taq
DNA polymerase from Stratagene, nucleotides from Life
Technologies, and primers as indicated in Results. Products were
analyzed on TBE gels, stained with ethidium bromide,
photographed, and transferred to Zetaprobe membrane as described
above.
Cloning of RT-PCR Products
Primers 27L and 10V were used to amplify reverse-transcribed rat
and human SMC RNA. RT-PCR products were cloned by using the TA
cloning kit from Invitrogen and sequenced.
DNA Sequencing and Sequence Analysis
DNA sequence analysis was performed by several methods,
ie, by an automated method with fluorescently tagged
terminators (Taq Dyedeoxy Terminator Cycle Sequencing Kit, Applied
Biosystems) and gel analysis on an Applied Biosystems 373A DNA
sequencer, or by using [35S]
-thio-dCTP and a Sequenase 2.0 kit from Life
Technologies, or a Sequitherm Cycle Sequencing Kit (Epicentre). "Long
Ranger" gel solutions (JT Baker) were used for analysis of
35S-labeled reactions, and dried gels were
exposed to X-Omat AR or Biomax film (Eastman Kodak). Sequence was
compiled by using GCG software.23
The size of the predicted translation product was determined by using GCG software,23 and the position of signal cleavage was predicted by using Signal P V1.1 software.24 O-linked polysaccharide addition was predicted by using the NetOGlyc V2.0 software.25 26 27
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Results |
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Cloning of V3, a Small Versican Isoform
To determine which versican protein coding regions are included in each of the versican RNAs expressed by adult rat smooth muscle cells, rat SMC cDNAs containing 5'- and 3'-versican domains were cloned (see Methods). Three small clones, rVa, rVd, and rVe, were subjected to restriction mapping and sequence analysis (see Figure 2A
and 2C; Genbank Accession No.
AF072892). The high degree of protein sequence identity between these
molecules and mouse (96%) and human (91%) versican/PG-M amino- and
carboxy-terminal globular domains28 supports their
identification as rat versican clones. The regions of homology included
amino-terminal HABR, and the entire G3 domain, that is, the EGF-like,
lectin-like, and CRP-like regions through the translation stop. The
HABR is immediately apposed to the first EGF-like domain and no
sequences with homology to either of the alternately spliced exons
(GAG or ßGAG) that encode the attachment sites for
glycosaminoglycans are present. The length of
the assembled sequence is 2465 bp. Zako et al12
have demonstrated the existence of this isoform in mice and humans and
refer to it as V3.
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Indicates the site where
A partial V0 isoform clone, rVb, was also sequenced (Figure 2B;
Genbank Accession No. AF062402). rVb contains part of the GAG
domain, the ßGAG domain, and the carboxy-terminal globular
domain.
Structure of the V3 RNA
The Kozak29 rule predicts the initiation of
translation of rat versican at nucleotide 281, which is the
position equivalent to that of mouse and human
versican.11 30 The 3'-untranslated regions (3'-UTR) of the
3 rat versican V3 cDNAs differed in length (147, 176, and 220 bases)
(Figure 2C). This variation may reflect the
actual lengths of the template molecules, or merely the binding of the
oligo(dT) primer used for library synthesis to A-rich regions
present in the 3'-UTR, rather than to the poly(A) tail. A
polyadenylation consensus sequence is present only in the clone
with the longest 3'-UTR (rVa) at position 2435 (Figure 2C).
Predicted Translation Product of V3
The predicted translation product of the V3 sequence is a
74474 Da polypeptide. Cleavage of a signal sequence is predicted after
residue 20, and would result in a protein core of 72217 Da. As for the
mouse,11 an additional cysteine residue is present in
the HABR, in comparison with human or chicken versican. O-link
polysaccharide addition is predicted at threonine-51. The
translated sequence includes 5 potential sites (NXS or NXT) for
N-linked polysaccharide addition. CS chain addition could
potentially occur at the SG or GS sequences indicated in Figure 2C; however, none of these sites match the D/EXSG consensus
proposed by Bourdon.31 Thus, V3 is likely to be a small
glycoprotein and not a large proteoglycan.
Expression of V0 and V1 Versican Isoforms in Rat SMCs
All known versican isoforms contain the amino-terminal HABR and
the G3 domain at the carboxy-terminus. Polyadenylated RNA was
probed with PCR products amplified from those regions (Figure 3A). RNAs of 11 to 13 kb (smear), 9, 8.3,
7.7, 3.3, and 2.5 kb hybridized with both probes (Figure 3B, lanes 1 and 4). Thus, even the small RNAs contained amino- and
carboxy-terminal sequences, implying that they are not degradation
products of the larger bands. The 11- to 13-kb smear also
hybridized to the GAG and ßGAG probes [lanes 2 and 3' (a long
exposure of lane 3)], identifying those molecules as V0 isoform. This
variant has not been previously described in vascular SMCs. This
finding was confirmed by cloning a partial V0 cDNA (see above). The 9-,
8.3-, and 7.7-kb RNAs hybridized to the ßGAG (lane 3), but not the
GAG probes (lane 2), and thus are V1 isoform RNAs, in agreement with
our previous findings in human and monkey SMCs.2 No RNA
hybridized to GAG but not ßGAG probes, indicating that SMCs do not
express the V2 isoform. Each isoform is present as multiple RNA
sizes. It is possible that these molecules are differentially spliced
at other sites within the molecules. Mouse versican, however, has been
shown to have multiple transcription stop signals, and the 3 large
versican RNA bands (8, 9, and 10 kb), have been shown to vary in the
length of the 3'-UTR.9
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Expression of V3 in Rat SMCs
The 2 smallest versican RNA bands (3.3 and 2.5 kb) hybridized with
both amino- and carboxy-terminal probes, but not the GAG and ßGAG
probes (Figure 3B, lanes 1 through 4). Both bands also
hybridized with an oligonucleotide (18VSPLR, see the
Table) designed to be antisense to the junction between the amino- and
carboxy-terminal versican globular domains (Figure 3B, lane 5).
Because this junction is only present in the V3 isoform, both the
3.3- and 2.5-kb RNAs are V3 isoform RNA.
We further examined the expression of V3 in rat SMCs by RT-PCR
analysis, using primers for the HABR and lectin-like domains. A
band of the appropriate size (603 bp) was amplified from the V3 isoform
plasmids, and from reverse-transcribed RNA from 2 lines of adult rat
SMCs, but not from a rat pup SMC line (Figure 4B, left panel). This is in agreement
with our previous finding that rat pup SMC lines express little or no
versican RNA (of any size).14 Bands corresponding to the
larger, more abundant, versican isoforms were not detected, possibly
because of the difficulty in either reverse-transcribing or amplifying
across the large GAG-attachment exons. When a Southern blot of these
reactions was probed with the splice junction
oligonucleotide 18VSPLR, the
oligonucleotide hybridized to the 603-bp bands (Figure 4B, right panel), confirming that they are derived from the V3
isoform.
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Versican V3 Is Expressed by Human SMCs
Although we have previously examined the expression of versican
RNA in human and monkey SMC total RNA, we have never detected the
smaller bands (<7 kb) seen in the rat. To determine whether these
forms were also present in human SMC RNA, we performed RT-PCR
analysis to amplify from the HABR to either the 3'-UTR or the
lectin-like domain (Figure 4A). Figure 4C shows that
bands of the predicted sizes for the V3 isoform (750 and 1177 bp) can
be reverse-transcribed from human SMC RNA (left panel, lanes 1 and
2). A PCR product of the appropriate size for V1, 383 bp,
was produced by using the same forward primer and a reverse primer for
the ßGAG exon (Figure 4C, left panel, lane 3). The HABR/EGF
splice junction oligonucleotide 19VSPLH (see the
Table) hybridized to the 1177- and 750-bp bands in lanes 1 and
2, but not to the 383-bp band (corresponding to V1) in lane 3,
confirming that the first 2 bands represent the human V3
isoform (Figure 4C, right panel).
Cloning of Splice Variant Encoding a Different
Carboxy-Terminus
To our knowledge, no differential splicing has been described
within the region encoding the carboxy-terminal globular domain of
versican. In a previous article, we uncovered preliminary evidence for
the existence of an uncharacterized versican splice
variant.14 By RT-PCR analysis using primers within
the carboxy-terminal region, we amplified 2 bands, where only 1 was
predicted. The primers used were within the lectin-like domain and
within the region after the CRP domain (Figure 5A). This latter region encodes the final
42 amino acids, and, together with the 3'-UTR, is encoded by the final
exon in both mouse and humans; we will refer to this region as the
"tail." In addition to the predicted 575-bp band, we amplified a
band of 
725 bp, suggesting the incorporation of additional unknown
exon sequences into versican (Figure 5B, lanes 1 and 2). Bands
of the same sizes were also amplified from human SMC RNA (Figure 5B, lanes 3 and 4).
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To determine whether these PCR products represented a
novel versican isoform, we cloned and sequenced the 725-bp bands
(Figure 6A and 6B; Genbank Accession Nos.
AF084544 and AF084545). The clones, rVint and hVint, contained
sequences identical to rat and human versican, respectively. They also
contained insertions of 145 bp (rat) and 144 bp (human) between the CRP
and tail sequences, indicating the existence of a new splice variant
(Figure 6B).
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We compared the structure of the new clones with the published
intron/exon structures of mouse and human versican. The CRP is encoded
on a single exon, 14, and the tail is part of exon 15. Intron 14,
between these exons, is very small in both species, and in humans its
size is specified as 144 bp.9 10 Thus, the new splice is
an "unspliced" variant, where the last intron is not removed. In
both the rat and human clones, consensus splice acceptor and splice
donor elements were present (see Figure 6A). The relative
abundance of the 725-bp band and the 575-bp band in the original RT-PCR
reaction suggested that we had not simply cloned a molecule in which
splicing was incomplete, as we might expect such molecules to be
relatively rare.
The predicted translation products of versican isoforms that retain
intron 14 terminate within the intron (Figure 6C). Both human
and rat translation products terminated after 16 amino acid
residues, and showed a high sequence identity (81%). This may be
compared with the 88% sequence identity between the rat and human tail
regions. The net result of including intron sequences within the coding
region of the carboxy-terminal globular domain would be to use this
16-amino acid region as the terminus, instead of the 42-amino acid
tail, predicted from all previous versican cDNAs.
To determine whether the unspliced intron is expressed in versican RNAs
containing or lacking the GAG attachment exons, RT-PCR was performed
(see Figure 7A). PCR products of the
appropriate size for V3 isoforms were amplified between the HABR primer
and reverse primers within intron 14, or spanning the junction of exons
14 and 15, showing that V3 forms may retain or splice out intron 14
(Figure 7B, top). No bands representing V0 or V1
isoforms were detected in those reactions. This was expected, as PCR
polymerization times (1 minute) were not long enough to amplify large
molecules. By using a primer within the ßGAG exon with either reverse
primer, we determined that V1 isoform can also retain or splice out
intron 14 (Figure 7B, bottom).
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Discussion |
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We have shown that splice variants of versican are expressed by rat vascular SMCs in vitro. In our previous study, we showed that the V1 isoform is expressed in arteries in vivo and in vitro.2 32 That study was completed before the discovery of versican splice variants and would not have detected the V3 isoform. In this study, we demonstrated that the V3 and V0 RNAs are also expressed by SMCs in vitro. We isolated 3 V3 cDNAs from an adult rat SMC cDNA library. We confirmed the existence of this splice variant in rat and human SMC RNA by RT-PCR, followed by hybridization to an oligonucleotide specific for this product. We also used this probe to detect V3 RNAs (3.3 and 2.5 kb) on northern blots.
In this study, we have asked only whether the V3 isoform is expressed
by SMCs in vitro. Although it is possible that V3 is only expressed by
cultured SMCs, which are known to dedifferentiated in culture, versican
V3 RNA has been detected in RNA isolated from several other tissues in
vivo. V3 cDNA was cloned originally by PCR from mouse END-D cell cDNA
library and a human cerebral cortex cDNA library.12 The V3
mRNA from these sources was shown to be 3 kb by northern
analysis.12 V3 RNAs have also been detected by PCR
from a mouse brain cDNA library, and human cerebral cortex, stomach,
and fetal liver cDNA libraries, and by RT-PCR of human brain and brain
tumor RNA.12 33 Of these, only the END-D cell RNA is
derived from cultured cells, and thus, V3 is expression is not
restricted to dedifferentiated cultures. Nevertheless, changes in the
pattern of splicing of other molecules have been shown to occur when
smooth muscle cells undergo phenotypic modulation in
culture.34 35 It will be important to determine which
versican splice variants are present in vivo and whether they
change during disease processes.
The difference between the V0, V1, and V3 versican isoforms expressed
by SMCs is in the presence or length of the central GAG attachment
domains. The V0 core is longer than the V1 core (370 kDa vs 262 kDa for
the human isoforms).13 30 V1 has 12 to 15 putative GAG
attachment sites whereas V0 has sites for an additional 5 to 8
chains.13 The rat V3 cDNA sequence predicts a 72 kDa core
lacking CS chains. Thus far, protein products have been
demonstrated for the V0, V1, and V2 forms only, using antibodies
specific for the GAG and ßGAG domains.13 36 37 It is
not known whether the V3 isoform has a protein product. Candidates
for the V3 product are 2 small brain glycoproteins,
glial hyaluronate-binding protein and hyaluronectin,38 39
that were shown by protein sequence analysis to be products
of the versican gene. These small hyaluronan-binding molecules appear
to be either proteolytic products or small splice variants of
versican.38 40 Glial hyaluronate-binding protein appears
to be a product of metalloproteinase digestion of the V1 isoform
because its carboxy-terminus matches sequence within the ßGAG exon
and is identical to the terminus generated by metalloproteinase
digestion of versican.41 Both glial hyaluronate-binding
protein and hyaluronectin are smaller than the predicted size of the V3
isoform. A protein product for the V3 isoform therefore has not yet
been demonstrated. In our recent studies, however, we have
overexpressed versican V3 RNA in rat SMCs and found alterations in cell
shape and adhesion (Lemire et al, unpublished data, 1998). Thus, if V3
encodes a functional protein, it may have significant effects on cell
phenotype.
The V0, V1, V2, and V3 isoforms have identical amino- and carboxy-termini. Functions shared by the 3 isoforms are likely to include hyaluronan binding via the amino-terminus, previously shown for the V1 isoform, and binding of tenascin-R, demonstrated for the lectin-like domain.4 6 42 Other ligands may interact with the carboxy-terminal globular region via the EGF-like, CRP-like, or tail regions. Versican isoforms may therefore act as bridges of different lengths,11 12 13 cross-linking matrix molecules.
The differences in the number of GAG attachment sites among the V0, V1, and V3 isoforms could also lead to significantly different roles in the vessel wall. The GAG chains are hygroscopic, and thus the V0 and V1 isoforms are large space-filling molecules that may contribute to vessel narrowing. The binding of LDL to versican's GAG chains may contribute to lipid deposition.43 If the factors responsible for regulating the differential splicing could be understood, it may be possible to control the relative amounts of large and small versican isoforms, and thus prevent some of the pathological effects of excess versican synthesis. Alternatively, it may be possible to target the larger isoforms for destruction via the induction of specific proteolysis.
When considering possible functions for the versican isoforms, it may be important to consider not only their individual effects on the cells or tissues, but also the effect of 1 variant on the function of another. Our northern analysis suggests that the V3 RNA may be relatively abundant in smooth muscle; when poly(A)+ RNA was probed with 5' and 3' sequences present in all versican isoforms, the V3 RNA forms were only somewhat less abundant than the V1 forms. The identical amino-termini and carboxy-termini of the V0, V1, and V3 isoforms may compete for ligands. For example, do hyaluronate–versican aggregates containing both the small V3 isoforms (lacking CS chains) and the larger isoforms (having many CS chains) have a looser structure with less resistance to compression, when compared with aggregate formed with only larger variants? Are the carboxy-terminal selectin-like domains of the small variant inaccessible to ligands when that variant is part of an hyaluronate–aggregate containing V0 or V1?
We have also cloned a new variant that encodes a putative versican form
having a different carboxy-terminus. We detected and cloned this
variant by RT-PCR from both human and rat SMC RNA (note added in proof:
while this article was in press, Perveen et al cloned the
intron-containing versican variant from human fibroblasts. Perveen R,
Hart-Holden N, Dixon MJ, Wiszniewski W, Fryer AE, Brunner HG, Pinkners
AJLH, van Beersum SEC, Black GCM. Refined genetic and physical
localization of the Wagner Disease (WGN1) locus and the genes CRTL1 and
CSPG2 to a 2- to 2.5-cM region of chromosome 5q14.3.
Genomics. 1999;57:219–226). This variant appears to be the
product of incomplete splicing, and indeed it is possible that it
represents a premature, nuclear RNA form. The relative
abundance of this molecule, in comparison with the completely spliced
form in RT-PCR experiments [Figures 5 and 7 and in
RT-PCR using fewer amplification cycles (data not shown)], suggests it
may be a functional mRNA. Similar "unspliced" or "optional"
introns have been described in a few cases. The final introns are
optional for the c-H-ras protooncogene, growth hormone, and 1 of the
somatostatin receptors.44 45 46 Retention of such introns
appears to require poor matches with the splice site consensus
sequences.47 In the case of the versican intron 14, the
consensus sequences within the intron match fairly well, but the
adjacent sequences in exon 14 and 15 match poorly. Versican exon 15
also contains purine-rich sequences similar to those that function as
splicing enhancers for optional introns.48
We have shown that versican intron 14 can be retained in both V1 and V3 variants. This unspliced form encodes a putative product lacking the 42 terminal residues of the original form and replacing those amino acids with a different 16-residue terminus. In both cases, the EGF-like, lectin-like, and CRP-like domains are retained. This is in agreement with previous studies that failed to find evidence for differential splicing of versican's G3 domains, but unlike the related molecule, aggrecan, which has splice forms lacking the EGF and CRP domains.49
Finally, what could be the role of the incompletely spliced isoform? It is possible that the carboxy-terminal tail may modify the binding properties of the adjacent CRP-like domain. The termini may alternatively have function independent of the proximate domain. The terminus of the form that splices out intron 14 (tail) has some (42% to 47%) homology to a family of intracellular protein tyrosine phosphatases.28 The region of homology does not correspond to the active site and is proximal to the GLGF repeats. In a similar manner, the terminus of the form that retains intron 14 has only low homology to any known protein, in this case to snake venoms.28 This homology is to the cysteine-rich region, which is hypothesized to modulate the activity of the disintegrin domain. Determining the functions of either of the 2 putative carboxy-termini will require further studies using antibodies and/or recombinant proteins.
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Acknowledgments |
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This work was supported by grants HL18645 to Dr Wight and HL03174 to Dr Schwartz. Some of the DNA sequence analysis was carried out at the University of Washington Molecular Pharmacology Facility.
Received August 20, 1998; accepted November 25, 1998.
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References |
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