Identification of versican as an isolectin B4-binding
glycoprotein from mammalian spinal cord tissue
Oliver Bogen
1
, Mathias Dreger
1,
*, Clemens Gillen
2
, Wolfgang Schro
¨
der
2
and Ferdinand Hucho
1
1 Freie Universita
¨
t Berlin, Institut fu
¨
r Chemie-Biochemie, Thielallee, Berlin, Germany
2 Research and Development Gru
¨
nenthal GmbH, Aachen, Germany
Noxius stimuli are detected by specialized sets of pri-
mary afferent neurons, the nociceptors. All nociceptors
are represented by C-fibers and Ad-fibers, neurons with
small- to medium-sized cell bodies and unmyelinated or
lightly myelinated axons, respectively [1]. A subpopu-
lation of these nociceptors express a cell-surface glyco-
conjugate that can be labeled by the plant isolectin B4
(IB4) from Griffonia simplicifolia [2]. Owing to the fact
Keywords

2004)
doi:10.1111/j.1742-4658.2005.04543.x
Nociceptors are specialized nerve fibers that transmit noxious pain stimuli
to the dorsal horn of the spinal cord. A subset of nociceptors, the nonpepti-
dergic C-fibers, is characterized by its reactivity for the plant isolectin B4
(IB4) from Griffonia simplicifolia. The molecular nature of the IB4-reactive
glycoconjugate, although used as a neuroanatomical marker for more than
a decade, has remained unknown. We here present data which strongly sug-
gest that a splice variant of the extracellular matrix proteoglycan versican is
the IB4-reactive glycoconjugate associated with these nociceptors. We isola-
ted (by subcellular fractionation and IB4 affinity chromatography) a glyco-
conjugate from porcine spinal cord tissue that migrated in SDS ⁄ PAGE as a
single distinct protein band at an apparent molecular mass of > 250 kDa.
By using MALDI-TOF ⁄ TOF MS, we identified this glycoconjugate unam-
biguously as a V2-like variant of versican. Moreover, we demonstrate that
the IB4-reactive glycoconjugate and the versican variant can be co-released
from spinal cord membranes by hyaluronidase, and that the IB4-reactive
glycoconjugate and the versican variant can be co-precipitated by an anti-
versican immunoglobulin and perfectly co-migrate in SDS ⁄ PAGE. Our
findings shed new light on the role of the extracellular matrix, which is
thought to be involved in plastic changes underlying pain-related phenom-
ena such as hyperalgesia and allodynia.
Abbreviations
DRG, dorsal root ganglia; ECL, enhanced chemiluminescence; GDNF, glial cell line-derived neurotrophic factor; GHAP, glial hyaluronate-
binding protein; IB4, isolectin B4; NaCl ⁄ P
i
, phosphate-buffered saline; PSD, post source decay; NaCl ⁄ Tris, Tris-buffered saline.
1090 FEBS Journal 272 (2005) 1090–1102 ª 2005 FEBS
that they apparently lack neuropeptide storage vesicles,
they are called nonpeptidergic C-fibers [3]. The IB4

sylation). Similarly, a reversal of the changes caused
by injury could be a result of the survival and out-
growth of IB4-positive fibers, of an increased expres-
sion of the glycoconjugate, or of an increased
concentration of the IB4-binding epitope caused by
post-translational modification (glycosylation). It has
also been observed that upon nerve injury, axons of
the surviving IB4-positive neurons in the DRG may
sprout and form so-called perineuronal ring-shaped
structures around the larger diameter A-fibers [10].
This effect was interpreted as an anatomical basis for
the cross-excitation phenomenon that may underlie
allodynia [11]. All of these reports suggest that the
IB4-reactive molecule may be important in pain trans-
mission. However, the investigation of its functional
role was impossible because its identity remained
obscure.
Here we describe experiments leading to the identifi-
cation of a molecule containing the IB4-binding epi-
tope. We show that it is a protein which is enriched in
a membrane preparation obtained from spinal cord tis-
sue. By means of biotinylated IB4 and Streptavidin–
agarose we extracted a macromolecule from a ‘light
membrane’ fraction which was identified by MALDI-
TOF peptide mass fingerprinting, partial post source
decay (PSD) sequencing and further experimental evi-
dence. We propose that the extracellular matrix pro-
tein versican is an IB4-binding molecule in nerve
tissue.
Results

(light membranes) and 9 (synaptosomes).
O. Bogen et al. IB4-binding versican in spinal cord tissue
FEBS Journal 272 (2005) 1090–1102 ª 2005 FEBS 1091
Proof of the IB4-binding specificity
The lectin IB4 binds selectively to oligosaccharides
containing a terminal a-d-galactopyranosyl group.
There are two options to prove the specificity of IB4
binding. The first is destruction of the IB4 eptitope by
enzymatic treatment with a-galactosidase [14,15] and
the second is by competing with IB4 binding using
an appropriate sugar homologue. Melibiose, an a-d-
galactopyranosyl glucoside, is known to be bound by
IB4 [4]. We therefore used melibiose to analyse the
binding specifity. As shown in Fig. 2, this disaccharide
competes with IB4 binding, as detected by the IB4-
peroxidase (IB4-PO) assay, in a dose-dependent man-
ner. Analysis of IB4 reactivity after a-galactosidase
treatment of light membranes and synaptosomes gave
a consistent result (data not shown). No IB4 binding
was detectable after enzymatic treatment.
The IB4-binding glycoconjugate is a protein
In principle, the IB4-binding oligosaccharide can be
bound to proteins, lipids, or polymeric glycans. In
order to analyse the nature of the IB4-binding mole-
cule, we incubated light membranes with proteinase K,
which is known to digest the majority of proteins,
leaving only oligopeptides behind. As shown in Fig. 3,
proteinase K treatment reduced the IB4-binding capa-
city dramatically (a 2-min incubation was sufficient
to degrade the IB4-binding molecule). The high-mole-

0.1 m
M CaCl
2
, 0.1 mM MnCl
2
, and 0.1 mM MgCl
2
, and increasing
concentrations of melibiose (lane 2, without melibiose; lane 3,
10 l
M; lane 4, 50 l M; lane 5, 100 lM; lane 6, 250 lM; lane 7,
500 l
M; lane 8, 1 mM; lane 9, 2 mM melibiose). Lanes 1 and 10,
marker. The IB4 reactivity (Arrow) decreases with increasing melibi-
ose concentration.
AB
Fig. 3. The isolectin B4 (IB4)-binding molecule is proteinaceous.
Forty micrograms of protein from the light membrane fraction was
combined with Proteinase K (0.1 mgÆmL
)1
) in 100 mM Na
x
H
x
PO
4
,
pH 8, and incubated for different time-periods at 37 °C. The diges-
tion was stopped by adding 4· sample buffer and 10-min incuba-
tion at 95 °C. Samples were separated on SDS ⁄ PAGE [7.5% (w ⁄ v)

tides covering 12% of the full-length protein; see also
Scheme 1). This result was confirmed by PSD sequen-
cing of two selected peptides, which perfectly matched
with sequences of the pig versican according to data-
base entry AAF19155.1 (Fig. 6).
The data bank search indicated versican as the only
significant match. Laminin and the light- and medium-
sized subunits of neurofilaments, which had been
previously reported to be IB4-binding molecules [16],
were not supported by our peptide mass fingerprint.
Versican and IB4-binding activity are co-enriched
by subcellular fractionation of spinal cord tissue
It is known that the association of versican with the
plasma membrane is mediated via binding to hyaluro-
nan [17]. Hyaluronan is a polymeric glycan which can
be specifically digested with hyaluronidase [18]. In
order to analyse whether versican, like the IB4-binding
activity, is enriched in the same subcellular fractions,
we treated the insoluble part of each fraction of a syn-
aptosome preparation with hyaluronidase. We subse-
quently analysed the extract by Western blotting by
using a mAb, anti-(glial hyaluronate-binding protein)
(anti-GHAP), which is known to detect all splice vari-
ants of versican [19]. As shown in Fig. 7A, versican
was detected in nearly all fractions, but is – like the
IB4-binding glycoprotein – strongly enriched in the
light membranes (see also Fig. 1). Additional signals,
detected with anti-GHAP, of around 66 kDa probably
represent GHAP itself, the N-terminal part of versican
[19–21].

with biotinylated IB4 and Streptavidin–agarose. IB4-bound proteins
were specifically eluted by Ca
2+
withdrawal with NaCl ⁄ P
i
(PBS)
containing 2 m
M EDTA, 0.5% (w ⁄ v) SDS (lane 6 and lane 8). Non-
specifically bound proteins were eluted with 4· sample buffer
(lanes 7 and 9). All fractions were concentrated by using 30 kDa
cutoff microconcentrators and electrophoretically separated by
SDS ⁄ PAGE [7.5% (w ⁄ v) gel]. Lanes 1–7 of the gel were blotted
onto nitrocellulose and developed with isolectin B4-peroxidase (IB4-
PO), as described above. Lanes 8–10 of the gel were stained with
Coomassie Brilliant Blue R250. Lane 1, marker; lane 2, 15 lLof
supernatant of the extracted light membranes; lane 3, 15 lg of pro-
tein of the light membranes after extraction with SDS; lane 4,
15 lg of protein of the extracted (but not precipitated) proteins;
lane 5, combined washing fractions; lane 6, half of all proteins elu-
ted under Ca
2+
withdrawal; lane 7, half of all proteins eluted with
4· sample buffer; lane 8, half of all proteins eluted under Ca
2+
with-
drawal; lane 9, half of all proteins eluted with 4· sample buffer;
lane 10, marker.
O. Bogen et al. IB4-binding versican in spinal cord tissue
FEBS Journal 272 (2005) 1090–1102 ª 2005 FEBS 1093
experiments which demonstrate that the glycoprotein is

this was, of course, to provide insight into the special
role of these fibers in pain transmission. Here we des-
cribe a first step towards elucidation of possible func-
tion. We propose that the extracellular matrix protein,
versican, is the glycoconjugate targeted by IB4. The
evidence presented includes the following, namely that
the IB4-binding molecule is a protein enriched during
subcellular fractionation in synaptosomal and light
(axonal) membrane fractions. Affinity chromatography
using biotinylated IB4 and streptavidin agarose beads
extracted from pig spinal cord a protein of high relat-
ive molecular mass (> 250 kDa) which was identified
by MALDI-MS (peptide mass fingerprinting and PSD
sequencing of two peptides) as versican. As an addi-
tional criterion for the specificity of the binding to the
affinity matrix, we used the Ca
2+
dependence of the
binding of the glycoconjugate(s) to the lectin. Only one
protein, namely the V2-like variant of versican, was
recovered in this way from the affinity matrix. No
other protein matched the peptide mass spectrum sig-
nificantly. In particular, the light and medium subunits
of the neurofilament triad, as well as laminin b2, which
were recently proposed to be IB4-binding entities in
DRGs [16], could not be detected in the protein frac-
tion that bound in a Ca
2+
-dependent manner to IB4.
Moreover, although neurofilaments and laminin were

Scheme 1. Amino acid sequence of the
human versican splice isoform V2 (database
entry AAA67565.1): Peptides of the isolectin
B4 (IB4)-positive porcine versican that also
matched the human versican V2 are shown
in bold, peptides identified by post source
decay (PSD) are in bold and underlined.
Note that three of the matched peptides
correspond to the glycosaminoglycan (GAG)
a-domain (amino acids 348–1335). Peptides
that correspond to the GAG b-domain were
not detected.
O. Bogen et al. IB4-binding versican in spinal cord tissue
FEBS Journal 272 (2005) 1090–1102 ª 2005 FEBS 1095
[Abs. Int. * 1000]
4.50
4.25
4.00
LATVGELQAAWR
NGFDQCDYGWLLDASVR
3.75
3.50
3.25
3.00
2.75
2.50
2.25
2.00
1.75
1.50

)
+
¼ 1314.71 Da and of
(M + H
+
)
+
¼ 2015.91 Da that had been observed directly within the peptide mass fingerprint (Fig. 5) or after microfractionation of the tryptic
digest by using desalting of the peptides by C18-Zip Tips followed by sequential elution of the peptides by increasing concentrations of
organic solvent were analysed by PSD. Both fragment ion spectra were consistent with the proposed peptide sequences derived from por-
cine versican, according to database entry AAF19155.1.
IB4-binding versican in spinal cord tissue O. Bogen et al.
1096 FEBS Journal 272 (2005) 1090–1102 ª 2005 FEBS
Versican is an extracellullar matrix proteoglycan of
the chondroitin sulphate proteoglycan subfamily of
versican, brevican, aggrecan and neurocan. Four dif-
ferent splice variants of versican are currently known
[21,23–25] and additional isoforms may exist [26]. The
A
B
Fig. 7. Co-enrichment of versican and isolectin B4 (IB4) reactivity
by subcellular fractionation. (A) Subcellular fractions probed for
antibody to glial hyaluronate-binding protein (anti-GHAP) reactivity.
One milligram of protein from different fractions of a synapto-
some preparation was pelleted by ultracentrifugation at
436 000 g. The pellets were resuspended in protease inhibitor
and 0.15
M NaCl containing 0.05 M Na
x
H

chondria; lane 10, marker. The arrow indicates the IB4-reactive
signals.
Fig. 8. Neither laminin nor neurofilaments account for the isolectin
B4 (IB4) reactivity in the protein fraction released from light mem-
branes by hyaluronidase. Left, proteins of a neurofilament prepar-
ation (lane 2), hyaluronidase-released light membrane proteins
(lane3), and commercially available laminin 1 (lane 4) were separ-
ated by SDS ⁄ PAGE and visualized by Coomassie staining. Lane 1,
molecular mass marker. Right: proteins according to the gel shown
on the left were transferred to a nitrocellulose membrane and
probed for various immunoreactivities by Western blot or lectin blot
analysis. Light membrane hyaluronidase extract (lane 3) was probed
for IB4 reactivity [by using isolectin B4-peroxidase (IB4-PO)], anti-
laminin immunoreactivity (anti-L1), and anti-neurofilament NF-M
reactivity (anti-NF-M). Although IB4 reactivity is present (arrow), no
reactivity for laminin or NF-M was observed. NF-M was detected in
a neurofilament preparation (lane 2), but no IB4 reactivity was
observed in this preparation. Commercially available laminin 1 was
readily detected by using laminin-specific antibody, and the b1and
b2 chains stained positive also for IB4 reactivity (lane 4).
Fig. 9. Co-immunoprecipitation of versican and isolectin B4 (IB4)
reactivity. Western blot using isolectin B4-peroxidase (IB4-PO) for
detection of IB4 reactivity (arrow). Lane 1, marker; lane 2, 30 lgof
light membranes; lane 3, 10 lg of extracted light membranes; lane
4, 10 lg of hyaluronidase extract; lane 5, 5 lg of nonprecipitated
proteins; lane 6, 5 lg of protein from washing step 1; lane 7, 5 lg
of protein from washing step 2; lane 8, half volume of the eluted
proteins; lane 9, 30 lg of light membranes; lane 10, Marker.
O. Bogen et al. IB4-binding versican in spinal cord tissue
FEBS Journal 272 (2005) 1090–1102 ª 2005 FEBS 1097

the spinal cord [36]. We propose that this may also
apply to the IB4-positive versican variant.
There are indeed reports that versican can be a com-
ponent of perineuronal nets [37]. In the case of versican
V2, however, there has been no previous report of a
neuronal expression, but V2 expression has been
suggested to be assignable to oligodendrocytes and
Schwann cells [29,30]. This contrasts with immunohisto-
chemical data on the expression of IB4 reactivity in
the dorsal root ganglion, which appears, owing to the
unambiguous stain of neuronal cell bodies including the
Golgi apparatus, clearly neuronal [10,12]. Therefore,
an immunohistochemical stain for versican within the
spinal cord and within the DRG should resolve this
problem. In summary, we suggest that there is a versi-
can V2-like or V2-related versican variant that is
modified by IB4-reactive carbohydrates and that is syn-
thesized by neurons.
It is an exciting feature of potential high medical rele-
vance that the IB4-reactive moiety underlies dynamic
changes in experimental paradigms of neuropathic pain,
namely a loss of IB4 reactivity within the dorsal horn of
the spinal cord and within the DRG, that can be allevi-
ated or reversed by GDNF [7,8]. Moreover, nerve injury
can lead to the formation of IB4-reactive basket-like
structures that emanate from IB4-positive C-fibres and
that surround the cell bodies of large-diameter A-neu-
rons within the DRG [10]. As nerve injury renders DRG
neurons hyperexcitable, afferent impulses invading the
somata of A-neurons may initiate ectopic discharges in

[31]. Versican V2, however, exerted no such effects on
PC12 cell differentiation as compared to the V1 splice
variant.
Taken together, our finding that versican with most
similarity to versican V2 among the known versican
variants is the principal IB4-binding protein in the spi-
nal cord (and probably also in the DRG) fuels a signi-
ficant new aspect into the investigation of perineuronal
nets and provides long sought-after information in the
ongoing struggle to elucidate the molecular basis of
neuropathic pain.
Experimental procedures
All substances and biochemicals were of the highest purity
commercially available. The mAb anti-GHAP, developed
IB4-binding versican in spinal cord tissue O. Bogen et al.
1098 FEBS Journal 272 (2005) 1090–1102 ª 2005 FEBS
by R. A. Asher [18], was obtained from the Developmental
Studies Hybridoma Bank founded under the auspices of the
National Institute of Child Health and Human Develop-
ment (NICHD) and maintained by the University of Iowa
(Department of Biological Sciences, Iowa City, IA, USA).
Subcellular fractionation
Pig spinal cords were obtained from a local slaughterhouse,
separated from the meninges and taken to the laboratory in
liquid nitrogen. All procedures, including all centrifugation
steps, were carried out at 4 °C.
Preparation of synaptosomes was based on the method
established by Gray & Whittaker [40], with some minor
modifications: Briefly, frozen pieces of pig spinal cord were
homogenized in homogenization buffer (10 mm Hepes,

2
HPO
4
,
1.5 mm KH
2
PO
4
, pH 7.4), centrifuged at 12 000 g for
10 min, and recovered from the bottom of the tube with
protease inhibitor containing NaCl ⁄ P
i
. The protein concen-
tration was determined using the Bradford assay [41] with
BSA (type V; Pierce) as standard.
Western blot analysis of IB4-binding activity
Samples (30–40 lg of protein) were combined with sample
buffer [final concentration: 62.5 mm Tris ⁄ HCl, pH 6.8, 3%
(w ⁄ v) SDS, 10% (v ⁄ v) glycerol, 5% (v ⁄ v) b-mercaptoetha-
nol, 0.025% (w ⁄ v) Bromophenol blue], heated for 10 min
at 60 °C and electrophoresed on 7.5% (w ⁄ v) polyacryl-
amide gels in 25 mm Tris containing 192 mm glycine and
0.1% (w ⁄ v) SDS [42]. Proteins were electrophoretically
transferred to nitrocellulose by using the semidry method
[transfer time was 2 h at 1.5 mAÆcm
)2
, with 47.9 m m Tris,
38.9 mm glycine, 0.038% (w ⁄ v) SDS and 20% (v ⁄ v) meth-
anol]. Blots were blocked overnight with 1% (w ⁄ v) BSA in
NaCl ⁄ Tris (Tris-buffered saline; 20 mm Tris, 150 mm

and protease inhibitor-containing NaCl ⁄ Tris. Extracted
proteins were separated by centrifugation at 10 000 g for
10 min, combined with 25 l L (50 lg) of IB4-biotin (Sigma)
and incubated for 16 h at 4 °C under continuous rotation.
The SDS concentration was reduced to 0.5% by adding an
equal volume of 0.1 mm CaCl
2
, 0.1 mm MnCl
2
, 0.1 mm
MgCl
2
, and protease inhibitor containing NaCl ⁄ Tris. IB4-
labelled proteins were affinity-bound by adding 750 lLof
Streptavidin–agarose (Sigma) in 0.01 m Na
x
H
x
PO
4
, pH 7.2,
containing 0.15 m NaCl and 0.02% (w ⁄ v) Na
3
N. The sam-
ple was incubated under vigorous shaking for 3 h at 4 °C.
Beads were centrifuged and washed twice under vigorous
shaking with 0.1 mm CaCl
2
, 0.1 mm MnCl
2

mass spectrometer, PSD spectra could be recorded in a sin-
gle step as a result of the use of the potential lift technology
(Bruker). The search engines profound and pepfrag
(available at http://prowl.rockefeller.edu/), or mascot
(available at http://www.matrixscience.com), were used to
match peptide mass fingerprints and fragment ion pattern
to National Center for Biotechnology Information non-
redundant rodent (NCBInr) database entries.
Hyaluronidase extraction of subcellular fractions
A total of 1 mg of protein from each fraction of a synapto-
some preparation was pelleted by centrifugation (30 min,
4 °C, 436 000 g ). The pellets were resuspended in protease
inhibitor 0.15 m NaCl containing 0.05 m Na
x
H
x
PO
4
(pre-
pared from stock solutions of NaH
2
PO
4
and Na
2
HPO
4
),
pH 5.3, and homogenized with a glass ⁄ glass homogenizer
(0.1 mm clearance). A total of 250 lg of protein from each

x
H
x
PO
4
, pH 7.0, containing 1 mm EDTA, 2 mm EGTA,
and 100 mm NaCl) supplemented with a protease inhibitor
cocktail and homogenized by 5 · 5 s bursts in a Waring
blender at top speed. The homogenate was centrifuged at
17 500 g for 30 min. The supernatant (S1) was removed
and placed on ice. The pellet was resuspended in an equal
volume of protease inhibitor containing isotonic buffer and
homogenized again, as described above. The homogenate
was centrifuged at 17 500 g for 30 min. The supernatant
(S2) was combined with supernatant S1, mixed with an
equal volume of protease inhibitor containing harvest buf-
fer (1.7 m sucrose, 10 mm Na
x
H
x
PO
4
, pH 7.0, containing
1mm EDTA and 1 mm dithiothreitol) and immediately
centrifuged at 77 000 g for 17 h. The supernatant was
discarded, gelatinous neurofilament-enriched pellets were
resuspended in start buffer [10 mm bis Tris ⁄ HCl, pH 6.8,
8 m urea, 0.1% (v ⁄ v) 2-mercaptoethanol] and clarified by a
1 h centrifugation at 100 000 g. The solubilized (superna-
tant) proteins were immediately applied to a Mono Q col-

respective secondary, horseradish peroxidase-conjugated
antibodies. After washing with NaCl ⁄ Tris-T (three times
for 10 min each), immunoreactivities were visualized by
using the ECL detection system.
IB4-binding versican in spinal cord tissue O. Bogen et al.
1100 FEBS Journal 272 (2005) 1090–1102 ª 2005 FEBS
Immunoprecipitation
The supernatant of 500 lg of hyaluronidase-treated light
membranes was equilibrated for immunoprecipitation by
adding an equal volume of 0.15 m NaCl containing 0.2 m
Tris ⁄ HCl, pH 7.4, supplemented by the protease inhibitor
cocktail. Five micrograms of anti-GHAP was added and
the mixture incubated for 30 min under vigorous shaking
at 4 °C. About 50 lg of protein G–sepharose (Amersham
Biosciences) was equilibrated by extensive washing with
protease inhibitor and 0.15 m NaCl containing 0.1 m
Tris ⁄ HCl, pH 7.4. Protein G–sepharose was added and
the mixture incubated under continuous rotation at 4 °C
overnight. The sepharose beads were washed twice by a
15 min incubation under powerful shaking with 0.1 m
Tris ⁄ HCl, pH 7.4, containing 0.2% (w ⁄ v) dodecylmalto-
sid, protease inhibitor and 0.15 m NaCl. Bound proteins
were eluted by incubation for 30 min with sample buffer
at room temperature. All fractions were concentrated
using microconcentrators with a molecular mass cut-off
of 3 kDa and analysed by Western blotting using
IB4-PO.
Acknowledgements
We thank Chandan Goswami and Lisa Muenter for
many helpful discussions, Arndt Asperger, Bruker

F & Lai J (2003) Glial cell line-derived neurotrophic
factor normalizes neurochemical changes in injured dor-
sal root ganglion neurons and prevents the expression
of experimental neuropathic pain. Neuroscience 121,
815–824.
8 Bennett DL, Michael GJ, Ramachandran N, Munson
JB, Averill S, Yan Q, Mcmahon SB & Priestley JV
(1998) A distinct subgroup of small DRG cells express
GDNF receptor components and GDNF is protective
for these neurons after nerve injury. J Neurosci 18,
3059–3072.
9 Boucher TJ, Okuse K, Bennett DL, Munson JB, Wood
JN & Mcmahon SB (2000) Potent analgesic effects of
GDNF in neuropathic pain states. Science 290, 124–
127.
10 Li L & Zhou XF (2001) Pericellular Griffonia simplicifo-
lia I isolectin B4-binding ring structures in the dorsal
root ganglia following peripheral nerve injury in rats.
J Comp Neurol 439, 259–274.
11 Amir R & Devor M (2000) Functional cross-excitation
between afferent A- and C-neurons in the dorsal root
ganglia. Neuroscience 95, 189–195.
12 Streit WJ, Schulte BA, Balentine JD & Spicer SS (1986)
Evidence for glycoconjugate in nociceptive primary sen-
sory neurons and its origin from the Golgi complex.
Brain Res 377, 1–17.
13 Gerke MB & Plenderleith MB (2004) Ultrastructural ana-
lysis of the central terminals of primary sensory neurones
labelled by transganglionic transport of Bandeiraea sim-
plicifolia I-isolectin B4. Neuroscience 127, 165–175.

domains of the large fibroblast proteoglycan, versican.
EMBO J 8, 2975–2981.
22 Shibata S, Peters BP, Roberts DD, Goldstein IJ &
Liotta LA (1982) Isolation of laminin by affinity chro-
matography on immobilized Griffonia simplicifolia 1 lec-
tin. FEBS Lett 142, 194–198.
23 Dours-Zimmermann MT & Zimmermann DR (1994) A
novel glycosaminoglycan attachment domain identified
in two alternative splice variants of human versican.
J Biol Chem 269, 32992–32998.
24 Ito K, Shinomura T, Zako M, Ujita M & Kimata K
(1995) Multiple forms of mouse PG-M, a large chon-
droitin sulfate proteoglycan generated by alternative
splicing. J Biol Chem 270, 958–965.
25 Zako M, Shinomura T, Ujita M, Ito K & Kimata K
(1995) Expression of PG-M (V3), an alternatively
spliced form of PG-M without a chondroitin sulfate
attachment in region in mouse and human tissues.
J Biol Chem 270, 3914–3918.
26 Lemire JM, Braun KR, Maurel P, Kaplan ED,
Schwartz SM & Wight TN (1999) Versican ⁄ PG-M iso-
forms in vascular smooth muscle cells. Arterioscler
Thromb Vasc Biol 19, 1630–1639.
27 Wight TN (2002) Versican: a versatile extracellular mat-
rix proteoglycan in cell biology. Curr Opin Cell Biol 14,
617–623.
28 Schmalfeldt M, Dours-Zimmermann MT, Winterhalter
KH & Zimmermann DR (1998) Versican V2 is a major
extracellular matrix component of the mature bovine
brain. J Biol Chem 273, 15758–15764.

dence for the brevican–tenascin–R interaction: colocali-
zation in perineuronal nets suggests a physiological role
for the interaction in the adult rat brain. J Comp Neurol
410, 256–264.
38 Dina OA, Parada CA, Yeh J, Chen X, Mccarter GC &
Levine JD (2004) Integrin signaling in inflammatory
and neuropathic pain in the rat. Eur J Neurosci 19,
634–642.
39 Wu Y, Chen L, Zheng PS & Yang BB (2002) Beta
1-integrin-mediated glioma cell adhesion and free radi-
cal-induced apoptosis are regulated by binding to a
C-terminal domain of PG-M ⁄ versican. J Biol Chem 277,
12294–12301.
40 Gray EG & Whittaker VP (1962) The isolation of nerve
endings from brain: an electron-microscopic study of
cell fragments derived by homogenization and centrifu-
gation. J Anat 96, 79–88.
41 Bradford MM (1976) A rapid and sensitive method for
the quantitation of microgram quantities of protein util-
izing the principle of protein-dye binding. Anal Biochem
72, 248–254.
42 Laemmli UK (1970) Cleavage of structural proteins
during the assembly of the head of bacteriophage T4.
Nature 227, 680–685.
43 Shevchenko A, Wilm M, Vorm O & Mann M (1996)
Mass spectrometric sequencing of proteins silver-stained
polyacrylamide gels. Anal Chem 68 , 850–858.
44 Hayes NVL, Holmes FE, Roobol A, Carden MJ &
Baines AJ (1997) Proteins of the neuronal cytoplasmic
and membrane-associated cytoskeleton. In Neurochemis-