Identification of versican as an isolectin B4-binding glycoprotein from mammalian spinal cord tissue Oliver Bogen1, Mathias Dreger1,*, Clemens Gillen2, Wolfgang Schroder2 and Ferdinand Hucho1 ă Freie Universitat Berlin, Institut fur Chemie-Biochemie, Thielallee, Berlin, Germany ă ă Research and Development Grunenthal GmbH, Aachen, Germany ă Keywords IB4; versican; nonpeptidergic C-fibers; extracellular matrix; neuropathic pain Correspondence Freie Universitat Berlin, Institut fur ă ă Chemie-Biochemie, Thielallee 63, 14195 Berlin, Germany Fax: +49 3083 853753 Tel: +49 3083 855545 E-mail: hucho@chemie.fu-berlin.de *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; nociceptor, primary sensory neuron that is activated by stimuli capable of causing tissue damage Nociceptors are specialized nerve fibers that transmit noxious pain stimuli to the dorsal horn of the spinal cord A subset of nociceptors, the nonpeptidergic 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 suggest that a splice variant of the extracellular matrix proteoglycan versican is the IB4-reactive glycoconjugate associated with these nociceptors We isolated (by subcellular fractionation and IB4 affinity chromatography) a glycoconjugate 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 unambiguously 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 antiversican 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 phenomena such as hyperalgesia and allodynia (Received November 2004, revised 11 December 2004, accepted 21 December 2004) doi:10.1111/j.1742-4658.2005.04543.x Noxius stimuli are detected by specialized sets of primary 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 subpopulation of these nociceptors express a cell-surface glycoconjugate that can be labeled by the plant isolectin B4 (IB4) from Griffonia simplicifolia [2] Owing to the fact Abbreviations DRG, dorsal root ganglia; ECL, enhanced chemiluminescence; GDNF, glial cell line-derived neurotrophic factor; GHAP, glial hyaluronatebinding protein; IB4, isolectin B4; NaCl ⁄ Pi, phosphate-buffered saline; PSD, post source decay; NaCl ⁄ Tris, Tris-buffered saline 1090 FEBS Journal 272 (2005) 1090–1102 ª 2005 FEBS O Bogen et al 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 polypeptide 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 containing a terminal a-d-galactoside The binding depends on the presence of Ca2+ 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] Concomitantly, 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 neuronal survival factor, has been shown to prevent the loss of IB4-positive fibers caused by axotomy [8] Simultaneously 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-glycosylation) Similarly, a reversal of the changes caused by injury could be a result of the survival and outgrowth of IB4-positive fibers, of an increased expression 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 transmission However, the investigation of its functional role was impossible because its identity remained obscure Here we describe experiments leading to the identification of a molecule containing the IB4-binding epitope We show that it is a protein which is enriched in a membrane preparation obtained from spinal cord tissue By means of biotinylated IB4 and Streptavidin– agarose we extracted a macromolecule from a ‘light FEBS Journal 272 (2005) 1090–1102 ª 2005 FEBS IB4-binding versican in spinal cord tissue membrane’ fraction which was identified by MALDITOF peptide mass fingerprinting, partial post source decay (PSD) sequencing and further experimental evidence We propose that the extracellular matrix protein 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 preparations We fractionated pig spinal cord tissue by density-gradient centrifugation and analysed the fractions 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 fractions The apparent molecular mass of this component was > 250 kDa (Fig 1) Fig 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 electrophoretically transferred to a nitrocellulose membrane The blot was developed with IB4-peroxidase (IB4-PO) Lane 1, marker; lane 2, homogenate; lane 3, low-speed supernatant; lane 4, low-speed pellet; 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 (light membranes) and (synaptosomes) 1091 IB4-binding versican in spinal cord tissue 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-dgalactopyranosyl 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 IB4peroxidase (IB4-PO) assay, in a dose-dependent manner 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- O Bogen et al 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 capacity dramatically (a 2-min incubation was sufficient to degrade the IB4-binding molecule) The high-molecular-weight IB4-binding molecule therefore is a glycoprotein 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) Moreover, we found that IB4 reactivity, as detected by Western blotting, was not affected by the presence of SDS but was strongly dependent on Ca2+ 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 Ca2+ ions Binding in the presence of 0.1 mm CaCl2 and elution A Fig Competition with melibiose Thirty micrograms of synaptosomal 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 membrane 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 mM CaCl2, 0.1 mM MnCl2, and 0.1 mM MgCl2, and increasing concentrations of melibiose (lane 2, without melibiose; lane 3, 10 lM; lane 4, 50 lM; lane 5, 100 lM; lane 6, 250 lM; lane 7, 500 lM; lane 8, mM; lane 9, mM melibiose) Lanes and 10, marker The IB4 reactivity (Arrow) decreases with increasing melibiose concentration 1092 B Fig The isolectin B4 (IB4)-binding molecule is proteinaceous Forty micrograms of protein from the light membrane fraction was combined with Proteinase K (0.1 mgỈmL)1) in 100 mM NaxHxPO4, pH 8, and incubated for different time-periods at 37 °C The digestion was stopped by adding 4· sample buffer and 10-min incubation at 95 °C Samples were separated on SDS ⁄ PAGE [7.5% (w ⁄ v) gel] and blotted to nitrocellulose (A) Coomassie Brilliant Bluestained gel: lane 1, native light membranes; lane 2, light membranes after a incubation with proteinase K; lane 3, light membranes after a incubation with proteinase K; lane 4, light membranes after a 15 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 incubation with proteinase K; lane 3, light membranes after a incubation with proteinase K; lane 4, light membranes after a 15 incubation with proteinase K; lane 5, marker FEBS Journal 272 (2005) 1090–1102 ª 2005 FEBS O Bogen et al IB4-binding versican in spinal cord tissue 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 Fig Enrichment of the isolectin B4 (IB4)-binding glycoconjugate with biotinylated IB4 and Streptavidin–agarose IB4-bound proteins were specifically eluted by Ca2+ withdrawal with NaCl ⁄ Pi (PBS) containing mM EDTA, 0.5% (w ⁄ v) SDS (lane and lane 8) Nonspecifically bound proteins were eluted with 4· sample buffer (lanes 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 (IB4PO), as described above Lanes 8–10 of the gel were stained with Coomassie Brilliant Blue R250 Lane 1, marker; lane 2, 15 lL of supernatant of the extracted light membranes; lane 3, 15 lg of protein 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 eluted under Ca2+ withdrawal; lane 7, half of all proteins eluted with 4· sample buffer; lane 8, half of all proteins eluted under Ca2+ withdrawal; lane 9, half of all proteins eluted with 4· sample buffer; lane 10, marker from IB4 with NaCl ⁄ Pi (PBS) containing 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 and 8) Both the lectin 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 peptides covering 12% of the full-length protein; see also Scheme 1) This result was confirmed by PSD sequencing of two selected peptides, which perfectly matched with sequences of the pig versican according to database entry AAF19155.1 (Fig 6) The data bank search indicated versican as the only significant match Laminin and the light- and mediumsized subunits of neurofilaments, which had been FEBS Journal 272 (2005) 1090–1102 ª 2005 FEBS It is known that the association of versican with the plasma membrane is mediated via binding to hyaluronan [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 synaptosome preparation with hyaluronidase We subsequently analysed the extract by Western blotting by using a mAb, anti-(glial hyaluronate-binding protein) (anti-GHAP), which is known to detect all splice variants 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 glycoprotein, we stripped the Western blot shown in Fig 7A and developed it with IB4 peroxidase (Fig 7B) The same band of > 250 kDa became visible The 66 kDa band, probably representing GHAP, did not show up in the stripped and IB4-peroxidase developed blot, obviously because the IB4-binding epitope 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 porcine spinal cord and especially within the hyaluronidase-released fraction, we tested the respective fraction for anti-neurofilament and anti-laminin immunoreactivity As positive controls for the immunoreactivity for these proteins, we used a neurofilament preparation from porcine spinal cord and commercially available laminin from the Englebreth Holm-Swarm sarcoma, which is known to bind IB4 and to contain both IB4binding b-chains [22] As shown in lane of Fig 8, neither neurofilament proteins nor laminin were detected 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 1093 IB4-binding versican in spinal cord tissue O Bogen et al Fig 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) experiments which demonstrate that the glycoprotein is versican The IB4-binding molecule is co-immunoprecipitated 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 precipitate (compare lanes and 9) The apparent molecular 1094 mass of the glycoprotein was again > 250 kDa The inverse experiment – precipitation with IB4-biotin and Streptavidin–agarose and detection with the antiversican 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 FEBS Journal 272 (2005) 1090–1102 ª 2005 FEBS O Bogen et al IB4-binding versican in spinal cord tissue Scheme 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 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 describe a first step towards elucidation of possible function 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 relative molecular mass (> 250 kDa) which was identified by MALDI-MS (peptide mass fingerprinting and PSD sequencing of two peptides) as versican As an additional criterion for the specificity of the binding to the affinity matrix, we used the Ca2+ 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 significantly 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 fraction that bound in a Ca2+-dependent manner to IB4 Moreover, although neurofilaments and laminin were detectable within the light membrane and synaptosome FEBS Journal 272 (2005) 1090–1102 ª 2005 FEBS 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 IB4reactive 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 fraction, 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 present in the fraction released from light membranes by hyaluronidase treatment Taken together, these data provide firm evidence that versican is the first unambiguously identified protein (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 information? What role might versican play with respect to the properties of C-fibre type nociceptors? 1095 IB4-binding versican in spinal cord tissue O Bogen et al [Abs Int * 1000] 4.50 4.25 LATVGELQAAWR 4.00 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 -0.25 200 400 600 800 1000 1200 m/z [Abs Int * 1000] 7.0 NGFDQCDYGWLLDASVR 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 Fig 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 porcine versican, according to database entry AAF19155.1 1096 FEBS Journal 272 (2005) 1090–1102 ª 2005 FEBS O Bogen et al IB4-binding versican in spinal cord tissue A B Fig 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 synaptosome 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 NaxHxPO4, pH 5.3 and homogenized 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 h at 37 °C Fifteen micrograms of the concentrated (3 kDa cut-off microconcentrators) protein 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; : 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 incubation at 40 °C with 2% (w ⁄ v) SDS, 10 mM b-mercaptoethanol in 62.5 mM 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, mitochondria; lane 10, marker The arrow indicates the IB4-reactive signals FEBS Journal 272 (2005) 1090–1102 ª 2005 FEBS Fig Neither laminin nor neurofilaments account for the isolectin B4 (IB4) reactivity in the protein fraction released from light membranes by hyaluronidase Left, proteins of a neurofilament preparation (lane 2), hyaluronidase-released light membrane proteins (lane3), and commercially available laminin (lane 4) were separated 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)], antilaminin 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 was readily detected by using laminin-specific antibody, and the b1 and b2 chains stained positive also for IB4 reactivity (lane 4) Fig 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 lg of light membranes; lane 3, 10 lg of extracted light membranes; lane 4, 10 lg of hyaluronidase extract; lane 5, lg of nonprecipitated proteins; lane 6, lg of protein from washing step 1; lane 7, 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 Versican is an extracellullar matrix proteoglycan of the chondroitin sulphate proteoglycan subfamily of versican, brevican, aggrecan and neurocan Four different splice variants of versican are currently known [21,23–25] and additional isoforms may exist [26] The 1097 IB4-binding versican in spinal cord tissue 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 tissues [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 differentiation in vitro by a different isoform, versican V1 [31] Intriguingly, versican shares the structural features 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 molecules may occur This protein family was thus also called ‘hyalectans’ or ‘lecticans’ and have been suggested 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 demonstrated the specific expression of differentially glycosylated variants of the same core protein by different subsets of neurons, even in some cases restricted 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 component 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 immunohistochemical 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 versican V2-like or V2-related versican variant that is modified by IB4-reactive carbohydrates and that is synthesized by neurons It is an exciting feature of potential high medical relevance 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 allevi1098 O Bogen et al 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-neurons 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 peptide fragments from two other important extracellular matrix proteins, laminin and fibronectin, inhibited hyperalgesia caused by prostaglandin E2 and epinephrine, 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 inflammatory hyperalgesia [38] The C-terminal domain of versican has been shown in pull-down and co-immunoprecipitation assays to bind to b1-integrin and to regulate 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 spinal cord (and probably also in the DRG) fuels a significant 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 FEBS Journal 272 (2005) 1090–1102 ª 2005 FEBS O Bogen et al 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 Development (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 °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, mm EDTA, 320 mm sucrose) containing a protease inhibitor cocktail (Roche Diagnostics) Homogenization was performed with a motor-driven glass-Teflon homogenizer (0.2 mm clearance) by 12 up-and-down strokes at 800 r.p.m The homogenate was centrifuged at 1000 g for 10 The supernatant (S1) was removed and placed on ice The pellet (P1) was resuspended in homogenization buffer and homogenized again as described above The homogenate was centrifuged at 1000 g for 10 The resulting pellet (P1¢, cell debris and nuclei) was discarded The supernatant (S1¢) was combined with supernatant S1 and centrifuged at 12 000 g for 15 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 The supernatant (S2¢) was discarded, the pellet (P2¢) was resuspended with 0.32 mm sucrose in 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 h The resulting subcellular fractions were harvested using a widened Pasteur pipette Myelin accumulated at the 0.32 ⁄ 0.85 m sucrose interface, light membranes at the 0.85 ⁄ 1.0 m sucrose interface, 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 ⁄ Pi (PBS) (137 mm NaCl, 2.7 mm KCl, mm Na2HPO4, 1.5 mm KH2PO4, pH 7.4), centrifuged at 12 000 g for 10 min, and recovered from the bottom of the tube with protease inhibitor containing NaCl ⁄ Pi The protein concentration 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% FEBS Journal 272 (2005) 1090–1102 ª 2005 FEBS IB4-binding versican in spinal cord tissue (w ⁄ v) SDS, 10% (v ⁄ v) glycerol, 5% (v ⁄ v) b-mercaptoethanol, 0.025% (w ⁄ v) Bromophenol blue], heated for 10 at 60 °C and electrophoresed on 7.5% (w ⁄ v) polyacrylamide 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 h at 1.5 mcm)2, with 47.9 mm Tris, 38.9 mm glycine, 0.038% (w ⁄ v) SDS and 20% (v ⁄ v) methanol] 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 IB4PO (Sigma, : 500) in NaCl ⁄ Tris containing 0.1 mm CaCl2, 0.1 mm MnCl2, and 0.1 mm MgCl2, and washed three times with NaCl ⁄ Tris-T [NaCl ⁄ Tris containing 0.1% (v ⁄ v) Tween 20] containing 0.1 mm CaCl2, 0.1 mm MnCl2, and 0.1 mm MgCl2 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 h at room temperature in 1% (w ⁄ v) SDS containing 0.1 mm CaCl2, 0.1 mm MnCl2, 0.1 mm MgCl2, and protease inhibitor-containing NaCl ⁄ Tris Extracted proteins were separated by centrifugation at 10 000 g for 10 min, combined with 25 lL (50 lg) of IB4-biotin (Sigma) and incubated for 16 h at °C under continuous rotation The SDS concentration was reduced to 0.5% by adding an equal volume of 0.1 mm CaCl2, 0.1 mm MnCl2, 0.1 mm MgCl2, and protease inhibitor containing NaCl ⁄ Tris IB4labelled proteins were affinity-bound by adding 750 lL of Streptavidin–agarose (Sigma) in 0.01 m NaxHxPO4, pH 7.2, containing 0.15 m NaCl and 0.02% (w ⁄ v) Na3N The sample was incubated under vigorous shaking for h at °C Beads were centrifuged and washed twice under vigorous shaking with 0.1 mm CaCl2, 0.1 mm MnCl2, 0.1 mm MgCl2 and protease inhibitor containing NaCl ⁄ Tris IB4-captured proteins were eluted from the beads by using 0.5% (w ⁄ v) SDS and mm EDTA containing NaCl ⁄ Pi 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 Brilliant 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 Daltonics, Leipzig, Germany) as matrix substances A Bruker 1099 IB4-binding versican in spinal cord tissue Reflex and a Bruker Ultaflex (Bruker Daltonics) mass spectrometer 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 single 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 nonredundant rodent (NCBInr) database entries Hyaluronidase extraction of subcellular fractions A total of mg of protein from each fraction of a synaptosome preparation was pelleted by centrifugation (30 min, °C, 436 000 g) The pellets were resuspended in protease inhibitor 0.15 m NaCl containing 0.05 m NaxHxPO4 (prepared from stock solutions of NaH2PO4 and Na2HPO4), 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 (Sigma) and incubated for h at 37 °C The extracted proteins were separated from the insoluble pellet by centrifugation (10 at 10 000 g) and concentrated in miroconcentraters with a molecular mass cut-off of kDa (Amicon) Fifteen micrograms of protein from each fraction was electrophoresed on SDS ⁄ PAGE [7.5% (w ⁄ v) gel] and blotted onto a nitrocellulose membrane The membrane was blocked by incubation at °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 each wash) and incubated for 1.5 h at room temperature with peroxidase-conjugated secondary anti-mouse immunoglobulin [Amersham Bioscience; : 1000 in 5% (v ⁄ v) nonfat milk containing NaCl ⁄ Tris-T] After washing with NaCl ⁄ Tris-T (three times for 10 each), the anti-GHAP immunoreactivity 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 °C Preparation of neurofilaments was based on the protocoll described by Hayes et al [44], with some minor modifica- 1100 O Bogen et al tions: Briefly, frozen pieces of pig spinal cord tissue were transferred to an equal volume of isotonic buffer (10 mm NaxHxPO4, pH 7.0, containing mm EDTA, mm EGTA, and 100 mm NaCl) supplemented with a protease inhibitor cocktail and homogenized by · s bursts in a Waring blender at top speed The homogenate was centrifuged at 17 500 g for 30 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 The supernatant (S2) was combined with supernatant S1, mixed with an equal volume of protease inhibitor containing harvest buffer (1.7 m sucrose, 10 mm NaxHxPO4, pH 7.0, containing mm EDTA and 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, m urea, 0.1% (v ⁄ v) 2-mercaptoethanol] and clarified by a h centrifugation at 100 000 g The solubilized (supernatant) proteins were immediately applied to a Mono Q column (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 mLỈmin)1, collecting mL fractions All collected fractions were concentrated and equilibrated with isotonic buffer 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 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 (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 antineurofilament immunoreactivity were blocked with 5% (w ⁄ v) nonfat milk in NaCl ⁄ Tris-T and probed with antilaminin immunoglobulin [Acris antibodies; : 500 in 5% (w ⁄ v) nonfat milk containing NaCl ⁄ Tris-T] or anti-NF-M immunoglobulin [Sigma; : 1000 in 5% (w ⁄ v) nonfat milk containing NaCl ⁄ Tris-T] Blots were washed with NaCl ⁄ Tris-T (three times for 10 each) and probed with the respective secondary, horseradish peroxidase-conjugated antibodies After washing with NaCl ⁄ Tris-T (three times for 10 each), immunoreactivities were visualized by using the ECL detection system FEBS Journal 272 (2005) 1090–1102 ª 2005 FEBS O Bogen et al 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 under vigorous shaking at °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 °C overnight The sepharose beads were washed twice by a 15 incubation under powerful shaking with 0.1 m Tris ⁄ HCl, pH 7.4, containing 0.2% (w ⁄ v) dodecylmaltosid, protease inhibitor and 0.15 m NaCl Bound proteins were eluted by incubation for 30 with sample buffer at room temperature All fractions were concentrated using microconcentrators with a molecular mass cut-off of kDa and analysed by Western blotting using IB4-PO Acknowledgements We thank 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[21,23–25] and additional isoforms may exist [26] The 1097 IB4-binding versican in spinal cord tissue versican variant V2 is the dominant splice variant of versican in neuronal tissue and the one... proteins; lane 6, lg of protein from washing step 1; lane 7, 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 Versican. .. Versican is an extracellullar matrix proteoglycan of the chondroitin sulphate proteoglycan subfamily of versican, brevican, aggrecan and neurocan Four different splice variants of versican are currently