Báo cáo khoa học: Identification of versican as an isolectin B4-binding glycoprotein from mammalian spinal cord tissue pptx

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

IB4; versican; nonpeptidergic C-fibers;
extracellular matrix; neuropathic pain
Correspondence
Freie Universita
¨
t Berlin, Institut fu
¨
r
Chemie-Biochemie, Thielallee 63, 14195
Berlin, Germany
Fax: +49 3083 853753
Tel: +49 3083 855545
E-mail:
*Present address
University Laboratory of Physiology, Parks
Road, Oxford OX1 3PT, UK
Glossary
Allodynia, sensation of pain caused by stimuli
that are normally innocuous; cross-excitation,
non-synaptic depolarization of dorsal root
ganglia neurons in response to excitation of
neighbouring neurons; hyperalgesia,
increased responsiveness of nociceptors
upon noxious stimulation; neuropathic pain,
pain initiated or caused by a primary lesion or
dysfunction in the nervous system; nocicep-
tor, primary sensory neuron that is activated
by stimuli capable of causing tissue damage.
(Received 4 November 2004, revised 11
December 2004, accepted 21 December

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

reactivity is primarily used as an anatomical marker for
these C-fibers. The lectin consists of four identical poly-
peptide chains, and the homotetramer has an apparent
molecular mass of 114 kDa. Purification initially was
based on its affinity to the disaccharide melibiose [4].
IB4 binds selectively to a carbohydrate epitope contain-
ing a terminal a-d-galactoside. The binding depends on
the presence of Ca
2+
ions. Because of the specificity for
nonpeptidergic C-fibers, the possibility exists that the
IB4-reactive glycoconjugate itself could participate in
nociception. Indeed, the IB4-positive subpopulation of
nociceptors appears to play a distinct role in certain
pain transmission paradigms. Spinal nerve ligation
leads to hyperalgesia and allodynia [5,6]. Concomit-
antly, IB4-reactivity depletion was observed in the
affected dorsal root ganglia (DRG) and in lamina II of
the spinal cord dorsal horn [7]. On the other hand, glial
cell line-derived neurotrophic factor (GDNF), a neur-
onal survival factor, has been shown to prevent the loss
of IB4-positive fibers caused by axotomy [8]. Simulta-
neously it prevents the development of hyperalgesia
and allodynia [9]. From these observations, a potential
therapeutic role of GDNF in states of neuropathic pain
was deduced.
The mechanism of IB4-reactivity downregulation is
unclear. It could be caused by neuronal cell death or
altered gene expression. A third possibility would be a
change in the post-translational modification (de-glyco-

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

Enrichment of IB4-binding activity via subcellular
fractionation
The central terminals of almost all nonpeptidergic
C-fibers terminate in the substantia gelatinosa of the
dorsal horn where they are connected to dorsal horn
neurons. This region is known to contain high levels of
IB4-binding activity. Various authors have suggested
that the IB4 target molecule is a transmembrane- or a
plasma membrane-associated glycoprotein [12,13]. In
our attempts to characterize the IB4-binding molecule,
we therefore focussed on neuronal membrane prepara-
tions. We fractionated pig spinal cord tissue by
density-gradient centrifugation and analysed the frac-
tions by PAGE and blotting, using an IB4-peroxidase
conjugate to detect the IB4-binding molecule. We
found that one predominant IB4-binding entity was
strongly enriched in the light membrane (probably
containing axonal membranes) and synaptosomal frac-
tions. The apparent molecular mass of this component
was > 250 kDa (Fig. 1).
Fig. 1. Isolectin B4 (IB4)-binding activity is enriched in the light
membranes and synaptosome preparation. Thirty micrograms of
protein from different fractions of the synaptosome preparation
were separated by SDS ⁄ PAGE [7.5% (w ⁄ v) gel] and electrophoreti-
cally transferred to a nitrocellulose membrane. The blot was devel-
oped with IB4-peroxidase (IB4-PO). Lane 1, marker; lane 2,
homogenate; lane 3, low-speed supernatant; lane 4, low-speed pel-
let; lane 5, high-speed supernatant; lane 6, high-speed pellet; lane
7, myelin; lane 8, light membranes; lane 9, synaptosomes; lane 10,
mitochondria. The highest IB4 reactivity (Arrow) is found in lanes 8

(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-

cular-weight IB4-binding molecule therefore is a glyco-
protein.
Isolation of the IB4-binding glycoprotein
by affinity chromatography
SDS was found to be the most effective detergent for
extracting the IB4-binding glycoconjugate from light
membranes or synaptosomes (data not shown). More-
over, we found that IB4 reactivity, as detected by
Western blotting, was not affected by the presence of
SDS but was strongly dependent on Ca
2+
ions [2,4].
For these reasons we tried to isolate the IB4-binding
glycoconjugate from SDS-solubilized pig spinal cord
light membranes by means of biotinylated IB4 and
Streptavidin–agarose in the presence of Ca
2+
ions.
Binding in the presence of 0.1 mm CaCl
2
and elution
Fig. 2. Competition with melibiose. Thirty micrograms of synapto-
somal protein was electrophoresed on SDS ⁄ PAGE [7.5% (w ⁄ v) gel]
and blotted to nitrocellulose. The membrane was stained with
Ponceau S [0.1% (w ⁄ v) Ponceau S in 5% (v ⁄ v) acetic acid] and the
lanes were separated from each other by cutting the blotting mem-
brane into strips using a scalpel. The strips were transferred to a
strip-box and blocked overnight with 1% BSA in NaCl ⁄ Tris (TBS).
The strips were incubated for 1.5 h at room temperature with
isolectin B4-peroxidase (IB4-PO) (1 : 500) in NaCl ⁄ Tris containing

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)

gel] and blotted to nitrocellulose. (A) Coomassie Brilliant Blue-
stained gel: lane 1, native light membranes; lane 2, light mem-
branes after a 2 min incubation with proteinase K; lane 3, light
membranes after a 5 min incubation with proteinase K; lane 4, light
membranes after a 15 min incubation with proteinase K; lane 5,
marker. (B) Isolectin B4-peroxidase (IB4-PO)-developed Western
blot (the arrow indicates IB4-binding activity): lane 1, native light
membranes; lane 2, light membranes after a 2 min incubation with
proteinase K; lane 3, light membranes after a 5 min incubation with
proteinase K; lane 4, light membranes after a 15 min incubation
with proteinase K; lane 5, marker.
IB4-binding versican in spinal cord tissue O. Bogen et al.
1092 FEBS Journal 272 (2005) 1090–1102 ª 2005 FEBS
from IB4 with NaCl ⁄ P
i
(PBS) containing 2 mm EDTA
yielded a protein fraction that was analysed by
SDS ⁄ PAGE, Coomassie Blue staining and blotting
using IB4 peroxidase (Fig. 4). Only one IB4-binding
component was detected. This was strongly enriched in
the EDTA-eluate (Fig. 4, lanes 6 and 8). Both the lec-
tin blot with IB4 peroxidase (Fig. 4, lane 6) and the
protein stain with Coomassie Blue, respectively (Fig. 4,
lane 8), showed one predominant band corresponding
to an apparent molecular mass of > 250 kDa.
Identification of the > 250 kDa glycoprotein
To identify the isolated glycoprotein, it was digested
in-gel with trypsin and analysed by MALDI-MS
(Fig. 5). The protein detected is the versican splice
variant, V2 (database entry AAA67565; 23 tryptic pep-

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].

To confirm that versican is the IB4-binding glyco-
protein, we stripped the Western blot shown in
Fig. 7A and developed it with IB4 peroxidase
(Fig. 7B). The same band of > 250 kDa became vis-
ible. The 66 kDa band, probably representing GHAP,
did not show up in the stripped and IB4-peroxidase
developed blot, obviously because the IB4-binding epi-
tope is not located within the N-terminal portion of
versican.
To address the posibility that the laminin b2 chain
as well as neurofilament proteins, which have been
recently reported to bind IB4 [16], account for the IB4
reactivity that we observed within the fractions of por-
cine spinal cord and especially within the hyaluroni-
dase-released fraction, we tested the respective fraction
for anti-neurofilament and anti-laminin immunoreac-
tivity. As positive controls for the immunoreactivity
for these proteins, we used a neurofilament preparation
from porcine spinal cord and commercially available
laminin 1 from the Englebreth Holm-Swarm sarcoma,
which is known to bind IB4 and to contain both IB4-
binding b-chains [22]. As shown in lane 3 of Fig. 8,
neither neurofilament proteins nor laminin were detec-
ted within the protein fraction released from light
membranes by hyaluronidase treatment. However, the
typical IB4-reactive signal that we demonstrated to be
assignable to versican was well detected. Thus, this
IB4-reactive glycoprotein is neither a neurofilament
protein nor laminin, in agreement with our other
Fig. 4. Enrichment of the isolectin B4 (IB4)-binding glycoconjugate

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

versican.
The IB4-binding molecule is co-immunoprecipi-
tated with versican by an antibody to GHAP
The possibility exists that two or more molecular
species co-migrate in the >250 kDa electrophoretic
band. We therefore performed imunoprecipitation with
an anti-versican immunoglobulin and developed the
Western blot of the precipitate with IB4 peroxidase.
As shown in Fig. 9, only one IB4-binding glycoprotein
was detected. This was strongly enriched in the precipi-
tate (compare lanes 8 and 9). The apparent molecular
mass of the glycoprotein was again > 250 kDa. The
inverse experiment – precipitation with IB4-biotin
and Streptavidin–agarose and detection with the anti-
versican immunoglobulin – gave the corresponding
result (data not shown). This suggests that versican
and the IB4-binding entity are the same molecule.
Discussion
The IB4-binding epitope clearly is more than just an
anatomical marker for a subpopulation of nociceptive
C-fibers (see the Introduction and the references
therein). The aim of our study was to elucidate the
molecular identity of the glycoconjugate carrying the
Fig. 5. Identification of versican by MALDI-MS, peptide mass fingerprint. The affinity-purified protein (Fig. 4, lane 8, indicated by the question
mark) was digested in-gel with trypsin and tryptic peptides were analysed by MALDI-MS. Upper figure: peptide mass fingerprint; lower
figure, list of tryptic peptides that could be matched with peptides of versican V2 (database entry AAA67565.1).
IB4-binding versican in spinal cord tissue O. Bogen et al.
1094 FEBS Journal 272 (2005) 1090–1102 ª 2005 FEBS
terminal a-d-galactosyl moiety, which renders a subset
of nociceptive C-fibers IB4 positive. The idea behind

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

detectable within the light membrane and synaptosome
fractions of porcine spinal cord, they never stained
positive for IB4-reactivity in our hands and they were
distributed over the subcellular fractions in a pattern
that deviated from the distribution pattern of the IB4-
reactive glycoprotein (data not shown).
Binding of peroxidase-linked IB4 to the > 250 kDa
protein could be competitively prevented by melibiose,
an a-d-galactopyranoside-containing disaccharide, and
both IB4-peroxidase and an anti-versican (anti-GHAP)
immunoglobulin bound to this high-molecular-mass
protein. Immunoprecipitation with an anti-versican
immunoglobulin yielded both versican and IB4 affinity.
Moreover, hyaluronidase treatment of light membranes
not only released versican into the supernatant frac-
tion, it also released an IB4-positive molecule that
co-migrated identically with versican in SDS ⁄ PAGE.
Neither neurofilaments stained positive for IB4, nor
was the known IB4-positive protein laminin 1 present
in the fraction released from light membranes by hy-
aluronidase treatment.
Taken together, these data provide firm evidence
that versican is the first unambiguously identified pro-
tein (not necessarily the only one) accounting for the
IB4 stain in the spinal cord (and probably in DRGs)
described by anatomists.
What are the implications of our findings with
respect to C-fibre transmission of nociceptive informa-
tion? What role might versican play with respect to the
properties of C-fibre type nociceptors?

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

1.25
1.00
0.75
0.50
0.25
0.00
-0.25
[Abs. Int. * 1000]
7.0
6.5
6.0
5.5
5.0
4.5
4.0
3.5
3.0
2.5
2.0
1.5
1.0
0.5
200 400 600 800 1000
m/z
1200 1400 1600 1800 2000
200 400 600 800 1000
m/z
1200
Fig. 6. Post source decay (PSD) fragment ion spectra of two selected tryptic peptides: peptides of (M + H
+

)
+
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

x
PO
4
, pH 5.3 and homo-
genized with a glass ⁄ glass homogenizer (0.1 mm clearence).
Two-hundred and fifty micrograms of each fraction was treated
with 50 units of hyaluronidase for 2 h at 37 °C. Fifteen micro-
grams of the concentrated (3 kDa cut-off microconcentrators) pro-
tein extract from each fraction was separated by SDS ⁄ PAGE
[7.5% (w ⁄ v) gel], electrophoretically blotted to nitrocellulose and
developed by using monoclonal anti-GHAP [12C5; 1 : 250 dilution
in NaCl ⁄ Tris (TBS) containing 5% (w ⁄ v) dry milk and 0.1% (w ⁄ v)
Tween 20]. Lane 1, homogenate; lane 2, low-speed supernatant;
lane 3, low-speed pellet; lane 4, high-speed supernatant; lane 5,
high-speed pellet; lane 6, myelin; lane 7, light membranes; lane
8, synaptosomes; lane 9, mitochondria; lane 10, marker. The top
arrow indicates the IB4-binding versican, the arrow at 66 kDa
indicates GHAP, the N-terminal portion of versican. (B) Blotted
proteins corresponding to Fig. 7A, probed for IB4-reactivity. The
Western blot from Fig. 7A was stripped by a 30 min incubation
at 40 °C with 2% (w ⁄ v) SDS, 10 m
M b-mercaptoethanol in
62.5 m
M Tris ⁄ HCl, pH 6.7 and washed extensively with NaCl ⁄
Tris. The membrane was blocked by overnight incubation in
NaCl ⁄ Tris containing 1% BSA and developed with IB4-PO, as
described in the Materials and methods. Lane 1, homogenate;
lane 2, low-speed supernatant; lane 3, low-speed pellet; lane 4,
high-speed supernatant; lane 5, high-speed pellet; lane 6, myelin;
lane 7, light membranes; lane 8, synaptosomes; lane 9, mito-

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

versican variant V2 is the dominant splice variant of
versican in neuronal tissue and the one that gave the
best match with our mass spectrometric data.
Versican isoforms are expressed in a variety of tis-
sues [27], including in the extracellular matrix of the
brain [28]. Known functional effects of versican in the
nervous system were reported to be the impairment of
axonal or neurite growth by versican V2 [29,30], and
the promotion of neurite outgrowth and neuronal dif-
ferentiation in vitro by a different isoform, versican V1
[31]. Intriguingly, versican shares the structural fea-
tures of the other chondroitin sulphate proteoglycans,
providing a binding module for the interaction with
hyaluronan and a C-type lectin domain though which
the interaction with other extracellular matrix mole-
cules may occur. This protein family was thus also
called ‘hyalectans’ or ‘lecticans’ and have been sugges-
ted to form ternary complexes with hyaluronan and
tenascin R [32]. These complexes contribute to the
molecular makeup of perineuronal nets, extracellular
matrix-based structures that surround neurons and
that create, e.g. barriers that shield the neurons from
the outside and also prevent axonal sprouting [33,34].
Notably, there are reports of neuronal synthesis of at
least one type of lectican, namely aggrecan [35,36].
Interestingly, in the latter study the authors even dem-
onstrated the specific expression of differentially gly-
cosylated variants of the same core protein by
different subsets of neurons, even in some cases restric-
ted to a particular lamina within the gray matter of

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

the surrounding C-fibers of these basket-like structures.
This cross-excitation phenomenon between A- and
C-fibers in the DRG is discussed as a candidate for the
development of allodynia in neuropathic pain [11]. Our
identification of versican is obviously remarkable in the
light of the data of Li & Zhou [10] who described the
above-mentioned basket-like structures that emanate
from C-fibres to surround A-cell bodies, because it is
obvious that these structures may be at least partially
made up of extracellular matrix proteins. Our findings
thus open the door for further investigations of these
phenomena.
There is another emerging set of evidence for the
important role of extracellular matrix molecules in
pain transmission. There was a recent report that pep-
tide fragments from two other important extracellular
matrix proteins, laminin and fibronectin, inhibited
hyperalgesia caused by prostaglandin E2 and epineph-
rine, respectively. Both extracellular matrix proteins
are involved in signalling through integrins, and mAbs
directed against the b1-integrin subunit, as well as a
knockdown of b1-integrin expression, inhibited inflam-
matory hyperalgesia [38]. The C-terminal domain of
versican has been shown in pull-down and co-immuno-
precipitation assays to bind to b1-integrin and to regu-
late glioma cell adhesion and free radical-induced
apoptosis [39]. Moreover, Wu et al. recently reported
that PC12 cell differentiation and neurite outgrowth
was dependent on integrin signalling and was blocked
by application of an anti-b1 integrin immunoglobulin

[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,

pH 7.4, 1 mm EDTA, 320 mm sucrose) containing a prote-
ase inhibitor cocktail (Roche Diagnostics). Homogenization
was performed with a motor-driven glass-Teflon homo-
genizer (0.2 mm clearance) by 12 up-and-down strokes at
800 r.p.m. The homogenate was centrifuged at 1000 g for
10 min. The supernatant (S1) was removed and placed on
ice. The pellet (P1) was resuspended in homogenization buf-
fer and homogenized again as described above. The homo-
genate was centrifuged at 1000 g for 10 min. The resulting
pellet (P1¢, cell debris and nuclei) was discarded. The super-
natant (S1¢) was combined with supernatant S1 and centri-
fuged at 12 000 g for 15 min. The supernatant (S2) was
discarded, the pellet (P2, crude membrane fraction) was
resuspended in homogenization buffer and homogenized
again with six up-and-down strokes at 800 r.p.m. using the
motor-driven glass-Teflon homogenizer. The homogenate
was centrifuged at 12 000 g for 20 min. The supernatant
(S2¢) was discarded, the pellet (P2¢) was resuspended with
0.32 mm sucrose in 5 mm Tris ⁄ HCl, pH 8.1, layered onto a
discontinuous sucrose gradient (1.2 ⁄ 1.0 ⁄ 0.85 m sucrose)
and centrifuged at 85 000 g for 2 h. The resulting subcellu-
lar fractions were harvested using a widened Pasteur pip-
ette. Myelin accumulated at the 0.32 ⁄ 0.85 m sucrose
interface, light membranes at the 0.85 ⁄ 1.0 m sucrose inter-
face, synaptosomes at the 1.0 ⁄ 1.2 m sucrose interface and
mitochondria at the bottom of the centrifugation tube. All
fractions were diluted to a final sucrose concentration of
less than 0.3 m with protease inhibitor-containing NaCl ⁄ P
i
(PBS) (137 mm NaCl, 2.7 mm KCl, 8 mm Na

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

NaCl), incubated for 1.5 h at room temperature with IB4-
PO (Sigma, 1 : 500) in NaCl ⁄ Tris containing 0.1 mm CaCl
2
,
0.1 mm MnCl
2
, and 0.1 mm MgCl
2
, and washed three times
with NaCl ⁄ Tris-T [NaCl ⁄ Tris containing 0.1% (v ⁄ v) Tween
20] containing 0.1 mm CaCl
2
, 0.1 mm MnCl
2
, and 0.1 mm
MgCl
2
. Lectin-reactivity was visualized by using the
enhanced chemiluminescence (ECL) detection system
(Amersham Biosciences).
Affinity chromatography
A 1.25 mg sample of freshly prepared light membranes was
extracted for 1 h at room temperature in 1% (w ⁄ v) SDS
containing 0.1 mm CaCl
2
, 0.1 mm MnCl
2
, 0.1 mm MgCl
2
,

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

, 0.1 mm MgCl
2
and protease inhibitor containing NaCl ⁄ Tris. IB4-captured
proteins were eluted from the beads by using 0.5% (w ⁄ v)
SDS and 2 mm EDTA containing NaCl ⁄ P
i
. Nonspecifically
bound proteins were eluted with 4· sample buffer according
to Laemmli [42]. All fractions were concentrated by using
micro concentrators with a molecular weight cut-off of
30 kDa (Amicon), electrophoresed by SDS ⁄ PAGE [7.5%
(w ⁄ v) gel] and visualized by staining with Coomassie Brilli-
ant Blue or blotted onto nitrocellulose and analysed with
IB4-PO, as described above.
MALDI-MS
The protein of interest was digested in-gel with trypsin
according to standard protocols [43]. The MALDI-MS
measurements were performed using dihydroxy benzoic acid
(Sigma) or a-cyano 4-hydroxy cinnamic acid (Bruker Dal-
tonics, Leipzig, Germany) as matrix substances. A Bruker
O. Bogen et al. IB4-binding versican in spinal cord tissue
FEBS Journal 272 (2005) 1090–1102 ª 2005 FEBS 1099
Reflex and a Bruker Ultaflex (Bruker Daltonics) mass spec-
trometer equipped with a nitrogen laser, a reflectron, pulsed
ion extraction and an ion gate were used to acquire peptide
mass fingerprint spectra as well as fragment ion spectra
obtained from PSD of selected precursor ions. With
the Bruker reflex mass spectrometer, PSD spectra were
acquired in several segments, and were assembled by using
the FAST method (Bruker Daltonics). With the Ultraflex

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 or mascot
(available at ), 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

fraction was combined with 50 units of hyaluronidase (Sig-
ma) and incubated for 2 h at 37 °C. The extracted proteins
were separated from the insoluble pellet by centrifugation
(10 min at 10 000 g) and concentrated in miroconcentraters
with a molecular mass cut-off of 3 kDa (Amicon). Fifteen
micrograms of protein from each fraction was electrophore-
sed on SDS ⁄ PAGE [7.5% (w ⁄ v) gel] and blotted onto a
nitrocellulose membrane. The membrane was blocked
by incubation at 4 °C with 5% (v ⁄ v) nonfat milk in
NaCl ⁄ Tris-T overnight and incubated for 1.5 h at room
temperature with the anti-GHAP immunoglobulin [1 : 250
in 5% (v ⁄ v) nonfat milk containing NaCl ⁄ Tris-T]. The blot
was washed with NaCl ⁄ Tris-T (three times for 10 min each
wash) and incubated for 1.5 h at room temperature with
peroxidase-conjugated secondary anti-mouse immunoglob-
ulin [Amersham Bioscience; 1 : 1000 in 5% (v ⁄ v) nonfat
milk containing NaCl ⁄ Tris-T]. After washing with NaCl ⁄
Tris-T (three times for 10 min each), the anti-GHAP immu-
noreactivity was visualized by using the ECL detection
system.
Neurofilament preparation
Pig spinal cords were obtained from a local slaughterhouse,
separated from meninges and taken to the laboratory in
liquid nitrogen. All procedures, including all centrifugation
steps, were carried out at 4 °C.
Preparation of neurofilaments was based on the protocoll
described by Hayes et al. [44], with some minor modifica-
tions: Briefly, frozen pieces of pig spinal cord tissue were
transferred to an equal volume of isotonic buffer (10 mm
Na

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-

umn (Amersham Biosciences), pre-equilibrated at room
temperature with start buffer, and bound proteins were
eluted following application of a 90 mL linear gradient
of 8–4 m urea and 0–500 mm NaCl at a flow rate of
0.5 mLÆ min
)1
, collecting 1 mL fractions. All collected frac-
tions were concentrated and equilibrated with isotonic buf-
fer using microconcentraters with a molecular mass cut-off
of 30 kDa. Protein concentration was determined by using
the Bradford assay [41] with BSA as standard. Individual
neurofilament polypeptides adjudged pure by SDS ⁄ PAGE
were pooled, supplemented with 1 mm dithiothreitol and
20% (w ⁄ w) glycerol, and stored at )80 °C.
Detection of laminin and neurofilament
immunoreactivities by western blotting
Five micrograms of combined neurofilament proteins,
10 lg of proteins resulting from hyaluronidase treatment of
light membranes, and 2.5 lg of laminin 1 (Acris antibodies)
were electrophoresed on 5–7.5% (w ⁄ v) polyacrylamide gels
and stained with Coomassie Brilliant Blue or blotted onto a
nitrocellulose membrane. IB4 reactivity was analysed as
described above. Blots detected for anti-laminin or anti-
neurofilament immunoreactivity were blocked with 5%
(w ⁄ v) nonfat milk in NaCl ⁄ Tris-T and probed with anti-
laminin immunoglobulin [Acris antibodies; 1 : 500 in 5%
(w ⁄ v) nonfat milk containing NaCl ⁄ Tris-T] or anti-NF-M
immunoglobulin [Sigma; 1 : 1000 in 5% (w ⁄ v) nonfat milk
containing NaCl ⁄ Tris-T]. Blots were washed with NaCl ⁄
Tris-T (three times for 10 min each) and probed with the

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

Daltonics, Leipzig, for taking the PSD spectra and
Hermann Bayer and Beatrice Petter for technical
assistance. Financial support was obtained from the
German BMBF, grant no. 01 GG 9818 ⁄ 0, the Deut-
sche Forschungsgemeinschaft DFG, SFB 515, and
from the Fonds der Chemischen Industrie.
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