Versican V2 Is a Major Extracellular Matrix Component
of the Mature Bovine Brain*
Michael
Schmalfeldt
,
María T.
Dours-Zimmermann
,
Kaspar H.
Winterhalter§, and
Dieter R.
Zimmermann
¶
From the
Institute of Clinical Pathology, Department
of Pathology, University of Zürich, 8091 Zürich, and
the § Laboratory of Biochemistry I, Federal Institute of
Technology, 8092 Zürich, Switzerland
 |
ABSTRACT |
We have isolated and characterized the
proteoglycan isoforms of versican from bovine brain extracts. Our
approach included (i) cDNA cloning and sequencing of the entire
open reading frame encoding the bovine versican splice variants; (ii)
preparation of antibodies against bovine versican using recombinant
core protein fragments and synthetic peptides; (iii) isolation of
versican isoforms by ammonium sulfate precipitation followed by anion
exchange and hyaluronan affinity chromatography; and (iv)
characterization by SDS-polyacrylamide gel electrophoresis and
Coomassie Blue staining or immunoblotting. Our results demonstrate that
versican V2 is, together with brevican, a major
component of the mature brain extracellular matrix. Versicans
V0 and V1 are only present in relatively small
amounts. Versican V2 migrates after chondroitinase ABC
digestion with an apparent molecular mass of about 400 kDa, whereas it
barely enters a 4-15% polyacrylamide gel without the enzyme
treatment. The 400-kDa product is recognized by antibodies against the
glycosaminoglycan-
domain and against synthetic NH2- and
COOH-terminal peptides. Our preparations contain no major proteolytic
products of versican, e.g. hyaluronectin or glial hyaluronate-binding protein. Having biochemical quantities of versican
V2 available will allow us to test its putative modulatory role in neuronal cell adhesion and axonal growth.
 |
INTRODUCTION |
The term hyalectans (or lecticans) defines a
family of large hyaluronan-binding proteoglycans (1, 2) whose members
currently include versican (3), aggrecan (4), neurocan (5), and brevican (6). Hyalectans share highly similar domain structures at
either end of the core protein, whereas the chondroitin
sulfate-carrying middle portions are clearly distinct. The homologous
NH2-terminal regions consist of an Ig loop and a tandem
repeat element that interacts with hyaluronan (7-9). The COOH-terminal
globular structure includes epidermal growth factor-like repeats, a
C-type lectin domain, and a sushi (or complement regulatory protein)
element. Recombinantly expressed C-type lectin domains of hyalectans
interact specifically with tenascin-R (10) and tenascin-C (11). The binding is mediated by a calcium-dependent protein-protein
interaction.
Alternative splicing and transcription termination add greatly to the
structural diversity of hyalectans. Brevican, for instance, exists
either as a secreted or glycosylphosphatidylinositol-anchored molecule as a result of alternative transcription termination (12, 13).
Variable usage of exons encoding the epidermal growth factor-like
elements and the sushi domain lead to several aggrecan isoforms (14,
15), and alternative splicing of human (16) and mouse (17, 18) versican
transcripts may generate up to four versican core protein variants. The
differently spliced exons encode the central glycosaminoglycan-carrying
GAG1-
and GAG-
domains
(19).
All known hyalectans are temporally expressed in the central nervous
system (2, 20, 21). Neurocan and aggrecan appear transiently during
early developmental stages (22, 23) and are replaced later by brevican
and versican (24-26), the major postnatal hyalectans in brain tissues.
Neurocan and brevican are restricted to the central nervous system,
whereas aggrecan is expressed predominantly in cartilage. The longest
versican splice variants (V0 and V1) display an
even wider distribution and are present in a number of mesenchymal (27)
and epithelial (28) tissues and in cultures of endothelial cells
(18).
In vitro experiments suggest that hyalectans play a
modulatory role in cell-cell and cell-matrix interactions and hence may participate in the nervous system in the control of axon growth and
guidance. Inhibitory activity on neuronal cell adhesion and neurite
outgrowth has been described for neurocan (29) and for brevican (24).
In addition, aggrecan from nasal cartilage is a potent inhibitor of
neurite outgrowth (30). Because brain and cartilage aggrecan differ
largely in the extent of post-translational modifications (31), this
finding still needs to be confirmed with the more relevant
brain-derived aggrecan. Evidence that hyalectans may also be involved
in vivo in the control of axonal growth and guidance is
provided by our recent observation of the transient versican
V0/V1 expression in tissues that act as
barriers during the development of the peripheral nervous system
(32).
Although versicans V0 and V1 are also present
in the central nervous system (27), there are indications that a third
splice variant, versican V2, is the major isoform in adult
brain. The assumption is based on results from reverse
transcriptase-PCR (16) and Northern blot experiments (18) as well as on
comparative immunohistochemical studies (26). In contrast to the other
versican splice variants, versican V2 transcripts are only
detectable in the central nervous system (16). On the protein level, a
versican-like proteoglycan (25) and several small molecular size
hyaluronan-binding proteins (33, 34) have been identified in brain
extracts. Partial peptide sequences suggest that these components are
derived from versican V1 by a proteolytic process (35). No
biochemical data are currently available to confirm the presence of the
versican V2 proteoglycan in the central nervous system.
To close this gap, we have now determined the complete cDNA
sequence of bovine versican, we have prepared several domain-specific antibodies via immunization with recombinant core protein fragments or
synthetic peptides, and we have purified and characterized the
different versican isoforms from bovine brain tissues.
 |
EXPERIMENTAL PROCEDURES |
Tissues--
Bovine tissues were obtained from the local
abattoir. The tissues were immediately placed on ice and processed
within 1 h postmortem.
PCR Cloning and Sequencing--
Total RNA was isolated from
bovine forebrain using a RNeasy total RNA isolation kit (Qiagen,
Hilden, Germany). 1 µg of total RNA was reverse transcribed with a
first-strand cDNA synthesis kit (Boehringer Mannheim, Germany) and
random hexamer primers (total volume 20 µl). Either 1 µl of this
reverse transcription reaction or 100 ng of bovine genomic DNA was used
as the template in different PCRs. Primer sequences and cycling
conditions are listed in Table I. Initial
cDNA fragments were obtained by PCR with guessmer primers that were
based on comparisons of chick (36), mouse (18), and human versican (3,
16) cDNA sequences. The PCR products were either sequenced directly
or cloned into a pGEM-T vector using an A/T cloning kit (Promega,
Madison, WI). All sequences were determined in both directions on an
Applied Biosystems 373 DNA Sequencer using dye terminator chemistry.
View this table:
[in this window]
[in a new window]
|
Table I
PCR primers and conditions
(*): Guessmer primers. ( ): Initial denaturation 94 °C 2 min;
final extension 72 °C (68 °C, respectively) 7 min. (§): After 15 cycles, 20 s step extension/cycle. T: Standard PCR with
Taq polymerase (PCR Core Kit, Boehringer Mannheim). E: Long
PCR (Expand Long Template PCR Kit, Boehringer Mannheim). H: PCR with
proofreading (Expand High Fidelity PCR Kit, Boehringer Mannheim).
|
|
Northern Blotting--
Northern blots using 1.5 µg of
poly(A)+ RNA from bovine forebrain were prepared as
described previously (28). Digoxigenin-labeled riboprobes covering
bases 1-916 (numbers refer to bovine versican V0
cDNA), 2842-4092, and 9552-10291, respectively, were used for hybridization.
Preparation of Polyclonal Antibodies--
All polyclonal
antibodies were prepared by immunizing New Zealand White rabbits with
either histidine-tagged recombinant core protein fragments
(GAG-
I: amino acids 366-769, GAG-
II:
991-1335, and GAG-
: 1340-1613, all relative to versican
V0) or with NH2- and COOH-terminal peptides
(LQKVNMEKSPPVKGS and KHDHRWSRRWQESRR, respectively) linked to keyhole
limpet hemocyanin (Pierce). Both peptides were supplemented with a
COOH-terminal cysteine residue to allow efficient coupling via
m-maleimido benzoyl-N-hydroxysulfosuccinimide ester (Pierce). Fusion proteins were prepared as described elsewhere (28).
All antisera were purified by affinity chromatography. Antisera against
GAG-
I, GAG-
II, and GAG-
were
preabsorbed on bacterial control extract columns followed by
purification on corresponding fusion protein columns (NHS-activated
HiTrap matrix, Amersham Pharmacia Biotech). Antibodies against the
NH2- and COOH-terminal ends of versican were affinity
purified by binding to the corresponding peptides immobilized on
UltraLink iodoacetyl columns (Pierce).
Isolation and Characterization of Versican from Bovine
Brain--
300 g of bovine forebrain was homogenized in a Waring
blender using 4 volumes of extraction buffer containing 0.5 M NaCl, 50 mM Tris, pH 7.5, 25 mM
EDTA, 0.5% Nonidet P-40, 1 mM
4-(2-aminoethyl)-benzenesulfonylfluoride hydrochloride (Pefabloc SC;
Pentapharm, Basel, Switzerland), 1 µg/ml pepstatin, and 1 µg/ml
leupeptin (buffer modified from Ref. 37). The homogenate was stirred
for 5 h at 4 °C and subsequently centrifuged at 100,000 × g for 1 h. Proteins were differentially precipitated
from the supernatant by the addition of solid ammonium sulfate. After
an initial step at 30% saturation, versican-containing fractions were
precipitated by increasing the ammonium sulfate concentration in the
supernatant to 60%. All precipitates were allowed to form overnight at
4 °C and were collected by centrifugation at 27,000 × g for 45 min. The 60% precipitate was resuspended in buffer
A (6 M urea, 50 mM Tris, 10 mM
EDTA, pH 6) and batch absorbed to Q-Sepharose FF (Amersham Pharmacia
Biotech) at 4 °C overnight. After column packing, the resin was
washed with buffer A containing 0.3 M NaCl, and elution was
achieved by applying a NaCl gradient from 0.3 to 2 M.
Versican-containing fractions, identified on a slot blot with
anti-GAG-
II polyclonal antibodies, were pooled and
dialyzed extensively against 0.5 M NaCl, 20 mM Tris, 10 mM EDTA, pH 8 (buffer B). Subsequently,
hyaluronan-EAH-Sepharose, prepared by coupling hyaluronan (Sigma)
to EAH-Sepharose (Amersham Pharmacia Biotech) through cross-linking
with
N-(3-dimethylaminopropyl)-N'-ethylcarbodiimide hydrochloride (Fluka, Buchs, Switzerland) (38), was added to the
dialyzed fractions. The batch absorption was performed at 4 °C for
20 h. After packing the resin into a column, a washing gradient
from 0.5 to 2 M NaCl in buffer B was applied, and elution was achieved with 4 M guanidine-HCl, 20 mM
Tris, 10 mM EDTA, pH 8. Versican-containing fractions
(identified as described above) were pooled, concentrated in a
Biomax-100 spin concentrator (Millipore, Bedford, MA), and dialyzed
against 40 mM NaOAc, pH 8, 10 mM EDTA. 100-µl
aliquots were digested with 1 milliunit of carrier-free chondroitinase
ABC (Seikagaku, Tokyo, Japan) in 40 mM NaOAc, 10 mM EDTA, 1 mM
4-(2-aminoethyl)-benzenesulfonylfluoride hydrochloride, pH 8 at
37 °C for 7 h or overnight.
Electrophoresis and Immunoblotting--
Isolated fractions were
analyzed by SDS-PAGE on 4-15% gels (Bio-Rad) and Coomassie Blue
stained with a colloidal blue staining kit (Novex, San Diego, CA).
For immunoblots, samples were separated under reducing and denaturing
conditions on 4-15% Phast polyacrylamide gels (Amersham Pharmacia
Biotech) and blotted onto Nytran NY13 membranes (Schleicher & Schüll, Dassel, Germany) by diffusion transfer at 70 °C for 20 min. Binding of the first antibody (dilution 1:500-1:1000) was done
overnight followed by detection with alkaline phosphatase-conjugated anti-rabbit IgG antibodies and color reaction with nitro blue tetrazolium/5-bromo-4-chloro-3-indolyl phosphate (all from
Promega).
 |
RESULTS |
cDNA Cloning of Bovine Versican--
The starting points for
the cloning of the entire bovine versican cDNA sequence were short
cDNA stretches amplified from total bovine brain RNA with reverse
transcriptase-PCR and guessmer primers. These guessmer primers were
derived from versican cDNA sequences that were highly conserved
among the human, mouse, and chicken homologs and lay close to the
alternative splice junctions (Fig. 1,
primer pairs a*/b* and c*/d*). In this way we obtained bovine cDNA
fragments encoding parts of the hyaluronan binding region and the
COOH-terminal domains. Based on these sequences, we generated primers
(e/f) for a long reverse transcriptase-PCR, which yielded a 3.2-kb
amplification product extending over the entire GAG-
-encoding portion of versican V2 and a short PCR fragment, which
originated from the versican V3 transcript. No
amplification product derived from the V1 splice variant
was obtained in this reaction (expected size 5.5 kb). We therefore
amplified two small versican V1 cDNA fragments that
crossed over the splice junctions into the GAG-
-encoding sequence.
For each of these two reverse transcriptase-PCRs we used one perfect
match and one guessmer primer (primer pairs: e/k* and l*/f,
respectively). Because the GAG-
domain is encoded by a single exon
in the human and the mouse genome, we then tried to amplify the
remaining bovine GAG-
sequence in a long PCR using genomic DNA as
template. The 5.1-kb fragment we obtained corresponded to the expected
size. Finally, the portions including the translation initiation or
translation termination sequences were each amplified with a perfect
match and a guessmer primer (g* or j*), coined from species-conserved
5'- and 3'-untranslated regions, respectively.

View larger version (20K):
[in this window]
[in a new window]
|
Fig. 1.
PCR primers and products. The
localization of the primers used for the PCR cloning approach of the
different bovine versican splice forms is depicted in the upper
panel. Primers marked with an asterisk are guessmer
primers based on versican sequence comparisons of human, mouse, and
chicken. Resulting PCR products were analyzed by electrophoresis on 1%
agarose gels. HABR, hyaluronan binding region.
E/L/S, epidermal growth factor-like domains/c-type lectin
domains/sushi elements.
|
|
Using this strategy, we PCR cloned overlapping cDNA fragments that
covered the entire coding region of bovine versican corresponding to a
3,361-amino acid core protein in bovine versican V0, 2,380 amino acids in V1, 1,623 amino acids in V2, and
642 amino acids in V3 (all exclusive of the 20-amino acid
secretory signal sequence). The cDNA and deduced amino acid
sequences of the four splice variants of bovine versican are not shown
in this paper but are available from the GenBank data base.
Expression of Versican in Adult Bovine Brain--
From our PCR
results we could conclude that mRNAs of all versican splice
variants (V0-V3) are present in bovine brain
tissue. However, no quantitative assertion could be made based on these experiments. We therefore prepared Northern blots with probes that
covered portions encoding the NH2-terminal region, the
GAG-
domain, or the COOH-terminal portion of versican (Fig.
2). All three probes detected a single
band about 6.5 kb long corresponding to the expected size of the
V2 splice variant. Because no signals were detected in the
range predicted for the mRNAs of the V0, V1, and V3 isoforms, we concluded that these
transcripts remained below the detection limit of the Northern
blot.

View larger version (12K):
[in this window]
[in a new window]
|
Fig. 2.
Northern blot analysis of
poly(A)+ RNA from bovine brain. Versican mRNA was
detected with antisense riboprobes that included the coding sequence of
the NH2- or COOH-terminal globular domains or the GAG-
encoding region.
|
|
Identification of Versican Isoforms in Protein Extracts from Bovine
Brain--
Based on the cDNA sequence, we prepared polyclonal
antibodies against different parts of the versican core protein (Fig.
3). Some of the antigens chosen for
immunization are present in all four versican isoforms
(NH2- and COOH-terminal peptides), and others are only
included in the splice variants V0 and V2
(fragments GAG-
I and GAG-
II) or in
V0 and V1 (fragment GAG-
), respectively. In
immunoblotting experiments with bovine brain extracts, the polyclonal
antibodies specific for the GAG-
domain detected a predominant core
protein band of approximately 400 kDa after chondroitinase ABC
digestion (Fig. 4). This band was also
weakly visible before the enzyme treatment, probably reflecting
unprocessed versican core protein from the intracellular pool. An
almost identical picture was obtained with antibodies directed against
the NH2-terminal versican peptide, whereas antibodies
specific for the GAG-
domain did not recognize the 400-kDa
component. In contrast, GAG-
-specific antibodies detected two larger
core proteins, which migrated well above the 400-kDa marker protein.
Because these two bands were barely visible on blots developed with the
polyclonal antibodies against the NH2-terminal peptide we
concluded that these two core proteins must be present in minute
amounts and are only detectable as a result of the high
immunoreactivity of the anti-GAG-
antibodies. Finally, a minor band
migrating at around 80 kDa was recognized by the
NH2-terminal peptide antibodies. The mobility of this band was unaffected by chondroitinase ABC treatment.

View larger version (13K):
[in this window]
[in a new window]
|
Fig. 3.
Localization of the fragments and peptides
used for raising polyclonal antibodies. The splice
variant-specific fragments I and II (both
found in V0 and V2), and (present in
V0 and V1) were overexpressed as
histidine-tagged fusion proteins in a bacterial expression system. The
two peptides N and C contain the 15 most
NH2- and COOH-terminal amino acids, respectively, which are
present in all four splice forms. HABR, hyaluronan binding
region. E/L/S, epidermal growth factor-like domains/C-type
lectin domains/sushi elements.
|
|

View larger version (49K):
[in this window]
[in a new window]
|
Fig. 4.
Immunoblot analysis of bovine brain
extract. A protein extract from bovine brain was separated on a
4-15% SDS-Phastgel under reducing conditions before ( ) and after
(+) chondroitinase (Ch'ase) ABC digestion. Blots were
incubated with polyclonal antibodies against the
NH2-terminal peptide (N) and against the fusion
proteins GAG- ( II) and GAG- .
|
|
Isolation of GAG-containing Isoforms of Versican from Brain
Extracts--
Versicans were solubilized efficiently from brain
tissues with a detergent-containing high salt buffer, confirming
previous experiments by Yamagata and co-workers (37). Subsequent
extraction of the insoluble fraction with 4 M guanidine-HCl
yielded less than 5% additional material recognized by GAG-
- and
GAG-
-specific antibodies (data not shown). More than 90% of the
soluble immunoreactive material could be precipitated with ammonium
sulfate at saturation levels between 30 and 60% (data not shown).
Further purification of the versican proteoglycan isoforms could be
achieved by anion exchange chromatography on Q-Sepharose (Fig.
5), in which the versican-containing
fractions eluted in a broad peak between 0.7 and 1.2 M
sodium chloride. The subsequent affinity chromatography step on a
hyaluronan column demonstrated the high stability of the
versican-hyaluronan interaction, which could be disrupted only under
relatively harsh conditions (4 M guanidine-HCl).

View larger version (19K):
[in this window]
[in a new window]
|
Fig. 5.
Purification of versican isoforms with anion
exchange and hyaluronan affinity chromatography. Anion exchange
chromatography was performed under dissociative conditions using a
Q-Sepharose FF column (upper). Fractions that were positive
on a slot blot using the anti-GAG- II antibody were
pooled (solid bar) and subjected to affinity chromatography
on a hyaluronan column. During washing only every second fraction was
monitored (white gaps indicate fractions that were not
tested). Anti-GAG- II-positive fractions were pooled
(solid bar) and processed for SDS-PAGE analysis.
|
|
To monitor the purification process, aliquots of versican-containing
fractions were analyzed with SDS-PAGE (Fig.
6). The 400-kDa core protein component
observed by immunoblotting the crude brain extract became clearly
visible in the Coomassie Blue-stained gels after anion exchange
chromatography and was highly enriched after affinity purification on
the hyaluronan column. Without chondroitinase ABC treatment, this
component barely entered the gel, indicative of its proteoglycan
character. At least three other GAG-carrying components were present
after the Q-Sepharose chromatography step. Their core proteins either
migrated on SDS-PAGE as a double band around 150 kDa or as single band
slightly above the 84-kDa protein marker. None of these smaller size
bands displayed immunoreactivity with anti-versican antibodies (data
not shown), and they mostly disappeared after hyaluronan affinity
chromatography. The final proteoglycan preparation usually contained
50-100 µg of protein/100 g of brain tissue (wet weight).

View larger version (82K):
[in this window]
[in a new window]
|
Fig. 6.
Analysis of the different steps of versican
isolation. Samples of the different isolation steps were separated
on 4-15% SDS-polyacrylamide gels under reducing conditions. (+) and
( ) indicate whether or not samples were digested with chondroitinase
(Ch'ase) ABC before loading. Gels were stained with
colloidal Coomassie Blue.
|
|
Characterization of the Isolated Versican Core Proteins--
The
proteoglycan fraction obtained by hyaluronan affinity chromatography
was characterized further by immunoblot analysis using the entire panel
of domain-specific polyclonal antibodies (Fig.
7). Compared with the immunoblotting
analysis of the crude brain extracts (Fig. 4), no major changes in the
relative amounts of the large molecular mass core proteins were
observed. Only the quantitatively minor 80-kDa component of the crude
brain extract had been lost during our purification procedure. Again,
the two large core proteins migrating around 500 kDa were recognized by the GAG-
-specific polyclonal antibodies and displayed very weak immunoreactivity with antibodies against the NH2- and the
COOH-terminal ends of versican. Because the upper band was also
apparent on both GAG-
-specific immunoblots, we could conclude that
these two components corresponded to the intact V0 and
V1 core proteins of bovine versican, respectively. Both of
these large versican splice variants had formerly evaded detection in
the less sensitive Coomassie Blue staining (Fig. 6). In contrast to
these quantitatively minor splice variants, the predominant 400-kDa
core protein band displayed no immunoreactivity with antibodies against
the GAG-
domain. It was, however, recognized by both anti-GAG-
antibodies and by antibodies directed against the NH2- and
COOH-terminal peptides. We therefore could conclude that the
predominant band in our brain extract preparation corresponded to the
complete versican V2 core protein. The intact versican
V2 carries several chondroitin sulfate side chains
preventing it from entering 4-15% Phastgels without chondroitinase
ABC digestion. Although the size of intact versican V2
cannot be assessed from the electrophoretic mobility, it seems likely
that the molecular mass exceeds 600 kDa.

View larger version (40K):
[in this window]
[in a new window]
|
Fig. 7.
Immunoblot analysis of the isolated versican
core protein isoforms. Bound fractions of the hyaluronan affinity
chromatography were separated on 4-15% SDS-Phastgels under reducing
conditions before ( ) and after (+) chondroitinase
(Ch'ase) ABC digestion. Blots were incubated with
antibodies against the NH2-terminal GAG-
( I and II), GAG- , and COOH-terminal
parts of the versican core proteins.
|
|
 |
DISCUSSION |
In our earlier immunohistochemical study of human brain tissues
(26) we observed a more abundant and widely spread staining with
GAG-
-specific antibodies than with antibodies recognizing the
GAG-
domain. Yet these antibodies did not allow a distinction between the versican isoforms V0 and V2, or
V0 and V1, respectively. We therefore could
only assume on the basis of the differential staining patterns that the
V2 isoform might be the predominant versican splice variant
in mature brain tissues. To prove this hypothesis, we initiated
experiments isolating hyalectans from brain extracts. Because autopsy
samples of human brain displayed various degrees of autolysis, we used
bovine brain as the tissue source. Unfortunately, our previously
prepared polyclonal antibodies against human (16, 28) and chick
versican (32) showed only weak or no cross-reactivity with the bovine
homolog. In consequence, a more elaborate approach including cDNA
cloning and preparation of recombinant core protein fragments had to be
used to prepare highly reactive antibodies specific for bovine
versican.
By comparing the cDNA-deduced primary structures of different
species homologs of versican (Table II),
we observed a very high amino acid sequence conservation in the
NH2-terminal and particularly in the COOH-terminal portion
of the core protein, whereas a significant drop in sequence similarity
was noted in the chondroitin sulfate-carrying GAG-
and GAG-
domains. This is not surprising because both highly conserved globular
ends of the core protein are involved in protein-mediated interactions with other extracellular ligands. In vitro, the
NH2-terminal region of versican binds to hyaluronan (8) and
possibly also to link protein (39). The recombinantly expressed C-type
lectin domain, which is localized in the COOH-terminal globule of
intact versican, interacts with the fibronectin type III repeats of
tenascin-R (10). In the chondroitin sulfate-carrying middle portion of the versican core proteins, we observed high sequence conservation in
the vicinity of serine-glycine pairs, which are likely to be recognized
by
-D-xylosyltransferase (40). Furthermore, the highly
acidic amino acid sequence present in a potential cystine loop at the
NH2-terminal end of the human GAG-
domain could also be
identified in bovine versican. The function of this core protein stretch is still unknown.
All versican isoforms are expressed in bovine brain. However, the
majority of the versican splice variants, namely the mRNAs encoding
versican V0, V1, or V3, are only
picked up by the very sensitive reverse transcriptase-PCR analysis. It
seems therefore likely that these splice forms are only present at very
low levels in mature brain tissues. The sole versican mRNA detected
in the Northern blot experiment corresponds to the V2
isoform. Human versican splice variants appear usually as double bands
on the Northern blot (16) because of multiple polyadenylation signals in the human versican gene (19). That the bovine versican
V2 transcript appears as a single band may indicate either
that the bovine gene only contains one polyadenylation signal or that
they are localized only a few bases apart. From our reverse
transcriptase-PCR and Northern blot experiments we did not obtain
evidence for the occurrence of an additional "plus" domain that is
present in chick versican (41), nor was there any indication for the
existence of a membrane-associated versican splice variant similar to
the glycosylphosphatidylinositol-anchored form of brevican (12).
Because the globular domains of hyalectans are highly similar, the
distinct sequences of the GAG-carrying middle portions are often used
to generate specific antibodies (8, 16, 28). Unfortunately, these
antibodies recognize only some of the splice variants, but none is able
to detect versican V3. We therefore looked for highly
variable sequence portions at either end of the versican core protein
and prepared anti-peptide antibodies. These antibodies recognize all
four versican isoforms and allow us to test whether the core proteins
are intact. Because a number of reports have described the isolation of
small versican fragments named glial hyaluronate-binding protein (34)
or hyaluronectin (33), the issue of proteolytic degradation has gained
particular interest. Aware of this fact, we used for the extraction a
detergent containing high salt buffer at neutral pH (37) supplemented with a mixture of protease inhibitors including the water-stable serine
protease inhibitor 4-(2-aminoethyl)-benzenesulfonylfluoride hydrochloride. As a result, we detected with our set of antibodies mainly the intact core proteins of versican V2,
V1, and V0 on immunoblots of bovine brain
extracts. The only lower molecular mass component we observed with
antibodies against the NH2-terminal end of versican
migrated in the range of 80 kDa. This is significantly larger than the
60 kDa found for glial hyaluronate-binding protein, which is possibly
derived from versican V1 by proteolytic cleavage with
metalloproteinases (35). In fact, it seems more likely that our 80-kDa
component corresponds to the V3 isoform of versican because
its size is in good agreement with the calculated molecular mass of
about 73 kDa for this splice variant. Unfortunately, from all of our
polyclonal antibodies against bovine versican the antibodies directed
against the COOH-terminal core peptide displayed by far the lowest
immunoreactivity, only weakly recognizing the most abundant versican
isoforms in the crude brain extracts. Hence, further experiments are
needed to prove finally that the 80-kDa polypeptide is indeed versican
V3.
The most abundant splice variant of mature brain tissues is versican
V2, as demonstrated by Northern and immunoblotting
experiments as well as by analyzing the isolated versican proteoglycan
isoforms in SDS-PAGE followed by Coomassie Blue staining. Versican
V2 is expressed exclusively in the central nervous system
(16), whereas most other tissues synthesize the larger V0
and V1 splice variants. On SDS-PAGE, the core proteins of
versican V2, V1, and V0 migrate after chondroitinase ABC digestion with a significantly higher relative
size than expected from the calculated molecular masses of 180, 260, and 368 kDa. This discrepancy may result in part from a high proportion
of N- and O-linked oligosaccharides covalently attached to the core protein and to poor SDS binding because of the
highly acidic primary structures (calculated pI values around 4).
At present we do not know how the 400-kDa versican V2 core
protein relates to versican isolated earlier from human brain tissue (25). This proteoglycan has a significantly smaller core protein of 345 kDa and seems to carry only a few short chondroitin sulfate side chains
with a total size of 20 kDa. However, because one of the peptide
sequences deduced from the core protein of this proteoglycan matches a
sequence within the GAG-
domain, it seems likely that this
proteoglycan is derived from the V1 splice variant through
a proteolytic process. Other only partly characterized proteoglycans
with core proteins in the size range of versican V2 are the
CAT-301 antigen (42) and the DSD-1-proteoglycan (43). Whether these
proteoglycans are identical or only similar to versican V2
will be the subject of further experiments.
Apart from versican V2 mature bovine brain tissues express
another major proteoglycan that belongs to the hyalectan family. Brevican (6), the smallest currently known proteoglycan with hyaluronan
binding capacity, exists in two alternatively spliced versions (12),
one secreted and one membrane-associated isoform. In our proteoglycan
preparation we identified core proteins in the range of about 150 and
85 kDa, matching the reported sizes of full-length brevican and its
COOH-terminal proteolytic product. After anion exchange chromatography
similar amounts of versican V2 and brevican were present in
Coomassie Blue-stained gels. In contrast, only trace amounts of
brevican could be detected after hyaluronan affinity chromatography,
which included stringent wash steps with high salt buffers. This
observation may indicate that brevican binds to hyaluronan with a
significantly lower affinity than versican.
The functional role that versican V2 and brevican play in
mature brain tissues is currently unknown. Hockfield et al.
(44) hypothesized that changes in the extracellular matrix composition during brain maturation lead to a stabilization of the synaptic structures. This could in part be achieved by the strengthening of
adhesive mechanisms and by inhibiting further neurite growth. Although
the loss of synaptic plasticity will certainly not only depend on
changes in hyalectan expression, it seems conceivable that matrices
rich in versican V2 and/or brevican act as inhibitors of
cell interactions and therefore reduce the formation of new synapses in
the mature central nervous system. Such an inhibitory activity of
brevican has been demonstrated recently by Yamada et al.
(24) in a neurite outgrowth assay, whereas functional studies with
versican V2 are just beginning. Now having biochemical quantities of versican V2 available will allow us to
explore potential roles of this brain-specific splice variant in
in vitro and in vivo model systems.
 |
ACKNOWLEDGEMENTS |
We thank Regula Städeli-Brodbeck and
Malek Ajmo for excellent technical assistance, Dr. Lloyd Vaughan for
advice and comments on the manuscript, Norbert Wey and Hannes Nef for
helping with the photographs, and Prof. Philipp U. Heitz for continued
support.
 |
FOOTNOTES |
*
This work was supported by grants from the Krebsliga des
Kantons Zürich and from the Lydia Hochstrasser Foundation (to
D. R. Z.).The costs of publication of this
article were defrayed in part by the
payment of page charges. The article
must therefore be hereby marked
"advertisement" in
accordance with 18 U.S.C. Section
1734 solely to indicate this fact.
The nucleotide sequence(s) reported in this paper has been submitted to the GenBankTM/EMBL Data Bank with accession number(s) AF060456, AF060457, AF060458, and AF060459 for versican V0, V1, V2, and V3,
respectively.
¶
To whom correspondence should be addressed: Dept. of
Pathology, Institute of Clinical Pathology, University of Zürich,
Schmelzbergstrasse 12, CH-8091 Zürich, Switzerland. Tel.:
41-1-255-3945 Fax: 41-1-255-4508 E-mail:
dieterzi{at}pathol.unizh.ch.
1
The abbreviations used are: GAG,
glycosaminoglycan; PCR, polymerase chain reaction; PAGE,
polyacrylamide gel electrophoresis; kb, kilobase(s).
 |
REFERENCES |
-
Iozzo, R. V.,
and Murdoch, A. D.
(1996)
FASEB J.
10,
598-614[Abstract/Free Full Text]
-
Ruoslahti, E.
(1996)
Glycobiology
6,
489-492[Abstract]
-
Zimmermann, D. R.,
and Ruoslahti, E.
(1989)
EMBO J.
8,
2975-2981[Abstract]
-
Doege, K. J.,
Sasaki, M.,
Kimura, T.,
and Yamada, Y.
(1991)
J. Biol. Chem.
266,
894-902[Abstract/Free Full Text]
-
Rauch, U.,
Karthikeyan, L.,
Maurel, P.,
Margolis, R. U.,
and Margolis, R. K.
(1992)
J. Biol. Chem.
267,
19536-19547[Abstract/Free Full Text]
-
Yamada, H.,
Watanabe, K.,
Shimonaka, M.,
and Yamaguchi, Y.
(1994)
J. Biol. Chem.
269,
10119-10126[Abstract/Free Full Text]
-
Heinegård, D.,
and Hascall, V. C.
(1974)
J. Biol. Chem.
249,
4250-4256[Abstract/Free Full Text]
-
LeBaron, R. G.,
Zimmermann, D. R.,
and Ruoslahti, E.
(1992)
J. Biol. Chem.
267,
10003-10010[Abstract/Free Full Text]
-
Rauch, U.,
Gao, P.,
Janetzko, A.,
Flaccus, A.,
Hilgenberg, L.,
Tekotte, H.,
Margolis, R. K.,
and Margolis, R. U.
(1991)
J. Biol. Chem.
266,
14785-14801[Abstract/Free Full Text]
-
Aspberg, A.,
Miura, R.,
Bourdoulous, S.,
Shimonaka, M.,
Heinegård, D.,
Schachner, M.,
Ruoslahti, E.,
and Yamaguchi, Y.
(1997)
Proc. Natl. Acad. Sci. U. S. A.
94,
10116-10121[Abstract/Free Full Text]
-
Rauch, U.,
Clement, A.,
Retzler, C.,
Fröhlich, L.,
Fässler, R.,
Göhring, W.,
and Faissner, A.
(1997)
J. Biol. Chem.
272,
26905-26912[Abstract/Free Full Text]
-
Seidenbecher, C. I.,
Richter, K.,
Rauch, U.,
Fässler, R.,
Garner, C. C.,
and Gundelfinger, E. D.
(1995)
J. Biol. Chem.
270,
27206-27212[Abstract/Free Full Text]
-
Rauch, U.,
Meyer, H.,
Brakebusch, C.,
Seidenbecher, C.,
Gundelfinger, E. D.,
Beier, D. R.,
and Fässler, R.
(1997)
Genomics
44,
15-21[CrossRef][Medline]
[Order article via Infotrieve]
-
Grover, J.,
and Roughley, P. J.
(1993)
Biochem. J.
291,
361-367[Medline]
[Order article via Infotrieve]
-
Fülöp, C.,
Walcz, E.,
Valyon, M.,
and Glant, T. T.
(1993)
J. Biol. Chem.
268,
17377-17383[Abstract/Free Full Text]
-
Dours-Zimmermann, M. T.,
and Zimmermann, D. R.
(1994)
J. Biol. Chem.
269,
32992-32998[Abstract/Free Full Text]
-
Zako, M.,
Shinomura, T.,
Ujita, M.,
Ito, K.,
and Kimata, K.
(1995)
J. Biol. Chem.
270,
3914-3918[Abstract/Free Full Text]
-
Ito, K.,
Shinomura, T.,
Zako, M.,
Ujita, M.,
and Kimata, K.
(1995)
J. Biol. Chem.
270,
958-965[Abstract/Free Full Text]
-
Naso, M. F.,
Zimmermann, D. R.,
and Iozzo, R. V.
(1994)
J. Biol. Chem.
269,
32999-33008[Abstract/Free Full Text]
-
Rauch, U.
(1997)
Cell Tissue Res.
290,
349-356[CrossRef][Medline]
[Order article via Infotrieve]
-
Margolis, R. K.,
and Margolis, R. U.
(1993)
Experientia (Basel)
49,
429-446
-
Domowicz, M.,
Krueger, R. C.,
Li, H.,
Mangoura, D.,
Vertel, B. M.,
and Schwartz, N. B.
(1996)
Int. J. Dev. Neurosci.
14,
191-201[CrossRef][Medline]
[Order article via Infotrieve]
-
Meyer-Puttlitz, B.,
Milev, P.,
Junker, E.,
Zimmer, I.,
Margolis, R. U.,
and Margolis, R. K.
(1995)
J. Neurochem.
65,
2327-2337[Medline]
[Order article via Infotrieve]
-
Yamada, H.,
Fredette, B.,
Shitara, K.,
Hagihara, K.,
Miura, R.,
Ranscht, B.,
Stallcup, W. B.,
and Yamaguchi, Y.
(1997)
J. Neurosci.
17,
7784-7795[Abstract/Free Full Text]
-
Perides, G.,
Rahemtulla, F.,
Lane, W. S.,
Asher, R. A.,
and Bignami, A.
(1992)
J. Biol. Chem.
267,
23883-23887[Abstract/Free Full Text]
-
Paulus, W.,
Baur, I.,
Dours-Zimmermann, M. T.,
and Zimmermann, D. R.
(1996)
J. Neuropathol. Exp. Neurol.
55,
528-533[Medline]
[Order article via Infotrieve]
-
Bode-Lesniewska, B.,
Dours-Zimmermann, M. T.,
Odermatt, B. F.,
Briner, J.,
Heitz, P. U.,
and Zimmermann, D. R.
(1996)
J. Histochem. Cytochem.
44,
303-312[Abstract/Free Full Text]
-
Zimmermann, D. R.,
Dours-Zimmermann, M. T.,
Schubert, M.,
and Bruckner-Tuderman, L.
(1994)
J. Cell Biol.
124,
817-825[Abstract]
-
Friedlander, D. R.,
Milev, P.,
Karthikeyan, L.,
Margolis, R. K.,
Margolis, R. U.,
and Grumet, M.
(1994)
J. Cell Biol.
125,
669-680[Abstract]
-
Snow, D. M.,
Watanabe, M.,
Letourneau, P. C.,
and Silver, J.
(1991)
Development
113,
1473-1485[Abstract]
-
Li, H.,
Domowicz, M.,
Hennig, A.,
and Schwartz, N. B.
(1996)
Mol. Brain Res.
36,
309-321[CrossRef][Medline]
[Order article via Infotrieve]
-
Landolt, R. M.,
Vaughan, L.,
Winterhalter, K. H.,
and Zimmermann, D. R.
(1995)
Development
121,
2303-2312[Abstract/Free Full Text]
-
Delpech, B.,
and Halavent, C.
(1981)
J. Neurochem.
36,
855-859[Medline]
[Order article via Infotrieve]
-
Perides, G.,
Lane, W. S.,
Andrews, D.,
Dahl, D.,
and Bignami, A.
(1989)
J. Biol. Chem.
264,
5981-5987[Abstract/Free Full Text]
-
Perides, G.,
Asher, R. A.,
Lark, M. W.,
Lane, W. S.,
Robinson, R. A.,
and Bignami, A.
(1995)
Biochem. J.
312,
377-384[Medline]
[Order article via Infotrieve]
-
Shinomura, T.,
Nishida, Y.,
Ito, K.,
and Kimata, K.
(1993)
J. Biol. Chem.
268,
14461-14469[Abstract/Free Full Text]
-
Yamagata, M.,
Shinomura, T.,
and Kimata, K.
(1993)
Anat. Embryol.
187,
433-444[Medline]
[Order article via Infotrieve]
-
Tengblad, A.
(1979)
Biochim. Biophys. Acta
578,
281-289[Medline]
[Order article via Infotrieve]
-
Mörgelin, M.,
Paulsson, M.,
Malmström, A.,
and Heinegård, D.
(1989)
J. Biol. Chem.
264,
12080-12090[Abstract/Free Full Text]
-
Brinkmann, T.,
Weilke, C.,
and Kleesiek, K.
(1997)
J. Biol. Chem.
272,
11171-11175[Abstract/Free Full Text]
-
Zako, M.,
Shinomura, T.,
and Kimata, K.
(1997)
J. Biol. Chem.
272,
9325-9331[Abstract/Free Full Text]
-
Fryer, H. J. L.,
Kelly, G. M.,
Molinaro, L.,
and Hockfield, S.
(1992)
J. Biol. Chem.
267,
9874-9883[Abstract/Free Full Text]
-
Faissner, A.,
Clement, A.,
Lochter, A.,
Streit, A.,
Mandl, C.,
and Schachner, M.
(1994)
J. Cell Biol.
126,
783-799[Abstract]
-
Hockfield, S.,
Kalb, R. G.,
Zaremba, S.,
and Fryer, H.
(1990)
Cold Spring Harbor Symp. Quant. Biol.
55,
505-514[Medline]
[Order article via Infotrieve]
Copyright © 1998 by The American Society for Biochemistry and Molecular Biology, Inc.