Neurocan Is a Heparin Binding Proteoglycan

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Biochemical and Biophysical Research Communications 272, 449–455 (2000)

doi:10.1006/bbrc.2000.2823, available online at http://www.idealibrary.com on

eurocan Is a Heparin Binding Proteoglycan

ang Feng,* Irene Arnold-Ammer,† and Uwe Rauch*,1

Department of Experimental Pathology, Lund University, 221 85 Lund, Sweden; and †Department of Protein Chemistry,ax Planck Institute for Biochemistry, 82152 Martinsried, Germany

eceived May 5, 2000

also be expected from the central region of brevican,isc

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Neurocan and brevican are related chondroitin sul-ate proteoglycans which are mainly expressed in theentral nervous system. Neurocan and the secretedrevican variant are composed of globular N-terminalyaluronan binding domains, central O-linked oligo-accharide attachment regions, and globular C-ter-inal domains. Interaction studies of mouse brain

roteoglycans revealed that neurocan, but not brevi-an, was retained on a heparin affinity matrix. Also aecombinantly produced C-terminal fragment of neu-ocan, expressed by HEK 293 cells, was retained by theeparin affinity matrix. The substitution of this frag-ent with a chondroitin sulfate chain did not inhibit

inding to the heparin affinity matrix at physiologicalaCl concentrations, but decreased the NaCl concen-

ration necessary for elution. Two potential conse-uences of the heparin binding ability of neurocan aren enforcement of the interaction with other heparininding molecules and a directed secretion by polar-zed cells. © 2000 Academic Press

Key Words: chondroitin sulfate proteoglycan; neuro-an; heparin; brevican; tenascin-C, MDCK cells.

Neurocan and brevican are members of the hyalec-ican family of chondroitin sulfate proteoglycans.ther members of this family are aggrecan and versi-

an (1). These molecules share homologous N-terminallobular domains, which are able to bind to hyaluro-an. With the exception of a GPI-linked brevican spliceariant (2) they also share homologous C-terminallobular domains, containing C-type lectin modules.he attachment sites for glycosaminoglycan chains are

ocated in their central regions (1). Carbohydrate anal-sis and rotary shadowing electron micrographs of pu-ified neurocan isolated from rat brain indicate thatumerous O-linked oligosaccharides are attached to itsentral region, and that this protein region has aucin-like character (3–5). A mucin-like character can

1 To whom correspondence should be addressed at Department ofxperimental Pathology, Lund University Hospital, Solvegatan 25,2185 Lund. Fax: 146 46 158202. E-mail: [email protected].

449

ndicated by its amino acid composition and the ob-erved differences between the apparent and the cal-ulated Mr of the core protein (6).Brevican is specifically (7) and neurocan predomi-

antly (4, 8–10) expressed in the central nervous sys-em. In rodent brain neurocan is one of the majorhondroitin sulfate proteoglycans present during peri-atal development, whereas brevican is mainly ex-ressed in mature brain (11). In the cerebellum wherehe expression and distribution of both molecules haseen analysed, both mRNAs can be found predomi-antly in the granular layer (12, 13). Also immunohis-ochemically brevican could almost exclusively bebserved in the cerebellar granular layer (14). Inontrast, immunohistochemically neurocan was foundo be enriched in the molecular layer (15), althoughhis layer shows much lower levels of neurocan mRNAhan the granule layer (12). The type of cells responsi-le for the expression of the major, secreted form ofrevican is controversial. The view that neuroglialheets forming astrocytes are mainly responsible forrevican expression (14) has been questioned by in situybridisation studies with specific probes for the se-reted and the GPI-linked form of brevican, which in-icated a neuronal expression of the secreted brevicanorm (13). The neurocan in situ hybridisation patternnd the presence of neurocan before definitive astro-ytes become evident implicates an expression of neu-ocan by neurons (12, 16). However, an expression ofeurocan by astrocytes can be observed in vitro, andfter brain injury (16, 17).The function of neurocan and brevican, as the func-

ion of chondroitin sulfate proteoglycans in general inrain is elusive. In certain assay systems brain derivedeurocan and brevican molecules have been shown to

nhibit outgrows of neurites from neuronal cells (14,8), and therefore they could have a role in axonalathfinding. Due to their interactions with hyaluronannd tenascins, oligomeric glycoproteins, they have theotential to participate in the organization of the ex-racellular matrix. Molecules which have been showno interact with neurocan, but have not been shown to

0006-291X/00 $35.00Copyright © 2000 by Academic PressAll rights of reproduction in any form reserved.

interact with brevican, are NCAM (18), tenascin-C (19,2wtcnriptwbfmip

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Vol. 272, No. 2, 2000 BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS

0), amphoterin (21), HB-GAM (21) and bFGF (22),hich are all heparin binding molecules. A direct in-

eraction of neurocan or of any other molecule carryinghondroitin sulfate chains with heparin, however, hasever been reported. To investigate the ability of neu-ocan, brevican or other brain derived proteoglycans tonteract with heparin, the fraction of soluble brainroteoglycans was analysed for this ability. Whereashe bulk of the brain proteoglycans did not interactith heparin, neurocan could be identified as a heparininding molecule. This ability might have implicationsor the distribution and interactions of neurocan andight help to understand observations made in bind-

ng studies with tenascin-C and expression studies inolarized cells.

ATERIALS AND METHODS

Construction of expression vectors, transfections, and cell culture.he preparation of construct D925 and the characterisation of thexpression products in HEK293 cells has been described previously5). The coding sequences of the rat neurocan (4) and rat brevicanDNA (2) were linked to the BM40 signal peptide (23). Neurocan,revican and neurocan fragment D925 expressing MDCK II cellskindly provided by Dr. Kai Simons, Heidelberg) were produced bytable transfection with constructs in expression vectors with a CMVromotor and neomycin resistence gene, identified by the analysis ofhe medium by SDS-polyacrylamidgelelectrophoresis (SDS–PAGE)nd immunoblotting. The cells were maintained as described (5) inMEM/F12 medium (Gibco) supplemented with 10 mM Hepes, pH.2 and 10% FCS (Gibco).

Antibodies. Antiserum against neurocan has been preparedgainst native neurocan isolated from 7day rat brain by 1D1-mmunaffinity chromatography (3). For booster injections recombi-ant rat neurocan produced by mammalian cells and purified byD1-immunaffinity chromatography (5) was used.Antiserum against brevican, which was raised against recombi-

ant rat brevican produced in human embryonic kidney (HEK) 293ells, and purified from conditioned medium by standard biochemicalrocedures, was provided by Dr. Rupert Timpl, Martinsried.

Proteoglycan isolation. Brains of mice between 3 and 4 weeks ofge were homogenized in 150 mM NaCl, 50 mM sodium phosphate,H 7.4, 5 mM NEM, 5 mM Benzamidine, 1 mM PMSF.The homogenates were centrifuged for 30 min at 18,000 rpm in an

S34 rotor and the soluble fraction was passed through a 0.45 mmlter. The filtrate was applied to DEAE-Sephacel (Pharmacia). Theesin was washed with 250 mM NaCl, and eluted with 1 M NaCl. Theluent was dialysed against 150 mM NaCl with 20 mM Tris/HCl, pH.4. From this material 1 ml samples were applied to the Hi-trapeparin affinity column (Pharmacia). Proteoglycan fractions wereade 0.1% Triton and precipitated with 1/5 volume of 55% trichlo-

oacetic acid (TCA), and the precipitated material washed with ice-old acetone. D925 samples for the heparin affinity chromatographyere concentrated from serum-free medium by Centricon 10 (Ami-

on) centrifugation. 1 ml fractions for Western blotting were made.1% Triton X-100 and precipitated with 1/5 volume of 55% TCA.

Heparin affinity chromatography. Protein samples were chro-atographed with 0.1 ml/min on a 1 ml Hi-trap heparin affinity

olumn in 20 mM Tris/HCl buffer, pH 7.4, with a NaCl gradient from50 to 650 mM, starting 6 ml after application of the sample in 150M NaCl. Thereafter, the NaCl concentration was first raisedithin 9 ml from 150 to 300 ml, followed by a steeper gradient from

450

alculated based on the conductivity of the eluent entering the frac-ion collector. For the analysis of the mouse brain proteoglycans, fourml fractions were combined, to produce pools of unbound molecules

P1), molecule eluting below 180 mM NaCl (P2), molecule elutingetween 180 and 300 mM NaCl (P3), between 300 and 450 mM NaClP4), molecules eluting between 450 and 650 mM NaCl (P5), andolecules eluting at 650 mM NaCl (P6).

C-terminal neurocan domain affinity chromatography. The affin-ty chromatography has been described in detail (19). Briefly, the-terminal neurocan domain starting with amino acid threonine50, expressed in HEK 293 cells, was coupled to CNBr-activatedepharose. 50 mg of a tenascin-C fragment composed of the 4th andth FN III domain (TNfn4,5, provided by Dr. Andreas Faissner,eidelberg) expressed in bacteria, alone or in the presence of 400 mgeparin, was incubated over night with 2 ml of affinity matrix in aotal volume of 4 ml TBSTCM (10 mM Tris/HCl, pH 8, 150 mM NaCl,.05% (w/v) Tween 20, 2 mM CaCl2 and 2 mM MgCl2). The resin wasashed in a column with 5 volumes of TBSTCM (fractions 2–6), 3olumes TBSTCM with 25 mM EDTA (fractions 7–9) and 3 volumesBSTCM containing 25 mM EDTA and 1 M NaCl (fractions 10–12).00 ml aliquots were precipitated with TCA, separated on a 12%DS–PAGE and stained in the gel with Coomassie blue.

Transwell experiments. MDCK II cells surviving the selectionith G418 were grown on polycarbonate filters (0.4 mm pore size, 24m diameter) fitted within Transwell Chambers (Corning Costar)

nd cultured at 37°C, in 5% CO2 atmosphere and in DMEM 1 F12ontaining 10% FCS, 2 mM glutamine, 10 mM HEPES, penicillinnd streptomycin. The polarization of the monolayer was assessed byeasuring the electrical resistance between the apical and basolat-

ral compartments of the filter chamber using a Millicell-ERS in-trument (Millipore). Only filter cultures with an electrical resis-ance of at least 225 Vcm2 were used for experiments. Equivalentolumes of medium of the upper and the lower compartment of thelter cultures were precipitated with TCA, and analysed by Westernlotting. Secreted chondroitinase-treated neurocan and neurocanragment D925 were detected by Western blot with a monoclonalntibody 1D1 (3) recognizing an epitope within this C-terminal por-ion. Secreted brevican was detected by Western blot with a poly-lonal rabbit antiserum.

Analytical methods. SDS–PAGE was performed under non re-ucing conditions on 10% slab gels (24) and stained with Coomassielue (Serva) according to standard protocols. Western blots wereerformed by transfer of proteins separated by SDS–PAGE to sup-orted nitrocellulose (Bio-Rad) in Tris/glycine buffer containing 10%ethanol for 1 h at 100 V using the Bio-Rad mini gel system. The

lots were blocked with 1% bovine serum albumin, incubated withhe respective primary antibodies, and developed with alkaline phos-hatase conjugated secondary antibodies. Digestion with protease-ree chondroitinase ABC (Seikagaku) was carried out for 1 h at 37°Cn 100 mM Tris/HCl, pH 8.0, 30 mM sodium acetate using 1 mUnzyme per proteoglycan preparation.

ESULTS AND DISCUSSION

To evaluate whether neurocan, brevican or otherrain proteoglycans in their endogenous form in brainre able to interact with heparin, the fraction of solubleroteoglycans from brains of 3- to 4-week-old mice wassolated by DEAE-sephacel ion exchange chromatogra-hy and applied to a heparin affinity matrix underhysiological (150 mM NaCl and pH 7.4) conditions.ound material was eluted by raising the NaCl con-entration from 150 to 650 mM. After chondroitinasereatment of the material eluting between 300 and 500

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M NaCl, and SDS–PAGE analysis followed by Coo-assie blue staining, a core protein with a molecularass of about 150 kDa was revealed (Fig. 1A). A core

rotein of a brain proteoglycan in this molecular massange could be derived from brevican (apparent molec-lar mass 145 kDa) (6). It could also be derived fromeurocan, which has a native core protein with anpparent molecular mass of 245 kDa (3, 4), but whichan be proteolytically processed producing N- and

FIG. 1. Pooled fractions of a heparin affinity chromatography ofouse brain proteoglycans (P1, unbound molecules; P2, molecule

luting below 180 mM NaCl; P3, between 180 and 300 mM NaCl; P4,etween 300 and 450 mM NaCl; P5, between 450 and 650 mM NaCl;6, at 650 mM NaCl) were precipitated with TCA and analysed byDS–PAGE. Samples were either not treated (2) or treated (1) withhondroitinase ABC. (A) Coomassie blue staining of a 6% gel. S,road range molecular mass standards. The prominent band at 100Da in all chondroitinase-treated samples represents a protein de-ived from the chondroitinase preparation. The 150 kDa core proteinisible in P5 is indicated by an arrow. (B) Western blot of a 5% geleveloped with anti-neurocan antiserum. (C) Western blot of a 5%el developed with anti-brevican antiserum.

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50 kDa, respectively (3, 4). Western blot analysis re-ealed, that the core protein showing heparin affinityas stained with an anti-neurocan (Fig. 1B, P4 and5), but not an anti-brevican antiserum (Fig. 1C). Withhe anti-neurocan antiserum considerably less immu-oreactivity, and a core protein with a somewhat re-uced molecular mass (Fig. 1B, P1), possibly represent-ng the N-terminal neurocan fragment, could beetected in the unbound proteoglycan fraction. Thesebservations demonstrate that neurocan, as a proteo-lycan, has the ability to bind to heparin, and indicateshat it is the C-terminal fragment of the molecule,hich mediates this interaction.To test this assumption, a C-terminal part of neuro-

an starting with amino acid 925, fragment D925, wasnalysed for its ability to bind to the heparin affinityatrix. Fragment D925 comprises the last 25 amino

cids of the central region of neurocan, which containspotential glycoaminoglycan attachment site, and the

ntire homologous C-terminal domain (Fig. 2A). Frag-ent D925 has been shown to be expressed as a so-

alled part time proteoglycan, which means that only aertain fraction of the core proteins is substituted withhondroitin sulfate chains, whereas another fraction ofhe core proteins is secreted as plain glycoproteins (5).ffinity chromatography reveals, that both, the mole-ules substituted with chondroitin sulfate chains,hich can be recognized by their appearance as a dif-

use immunoreactivity between 70 and 120 kDa, andhe molecules not substituted with chondroitin sulfatehains, which can be seen as sharp bands at 47, andeaker, at 51 kDa, were retained by the affinity matrix

Fig. 2B). The chondroitin sulfate substituted mole-ules started to elute in fraction 18, a fraction compris-ng NaCl concentrations between 340 and 380 mM,hereas the fraction of the core glycoproteins not sub-

tituted with chondroitin sulfate chains showed aigher affinity to heparin, starting to elute in fraction1 (Fig. 2B), a fraction comprising NaCl concentrationsetween 510 and 580 mM. The main fraction of thensubstituted neurocan fragment eluted in fraction 23,omprising NaCl concentrations above 640 mM NaCl.These data show that under physiological salt and

H conditions neurocan substituted with glycosamino-lycan chains is able to bind to heparin. Interactionsith heparin have previously also been observed with

ecombinantly expressed fragments of proteoglycansike fragments of perlecan (25), and of versican (26),ut those fragments were not modified with glycosami-oglycan chains. Thus, so far the ability of neurocan to

nteract with heparin as a proteoglycan appears to benique and is likely to have implications for variousspects of the biology of this molecule. This abilityight also help to understand observations made in

inding studies with tenascin-C and in studies withespect to a directional secretion in polarized cells.

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The neurocan tenascin-C interaction site was mappedy affinity chromatography of various tenascin-C frag-ents on a matrix with a covalently coupled C-terminaleurocan domain to a tenascin-C fragment composed ofhe 4th and 5th FN III domain (19). In those experimentsnly this fragment was retained by the affinity columnnd could be released in significant amounts by EDTAFig. 3A, lanes 7 and 8) (19). In the presence of heparinhe amount of bound peptide was increased, while thealcium dependence of the interaction was maintainedFig. 3B). This observation indicates that heparin is ableo enhance the interaction of neurocan and tenascin-C.hese three molecules could actually form ternary com-lexes perhaps similar to complexes which can be createdy aggrecan, link protein, and hyaluronan (27).The differential mRNA and protein distributions of

eurocan in certain parts of the brain (12, 15) indicateshat neurocan is directionally secreted, and, for exam-le, in the cerebellum transported from the granuleells to the molecular layer where the axons of theranule cells are located. A directionally transport ofecretory molecules in polarized cells like neurons andpithelial cells is likely to depend on interactions withembrane molecules, which carry, or recognize target-

ng signals. In epithelial cells, mainly in Mardy Darbinanine Kidney (MDCK) cells, a wide variety of molec-lar targeting signals, especially those who are appli-able for secretory molecules have been elucidated. Inhe basolateral compartments of filter grown MDCK-ell monolayers, for example, an enrichment of hepa-an sulfate proteoglycans over chondroitin sulfate pro-eoglycans was observed (28). This observation is in

FIG. 2. (A) The protein modules (-mod.) and potential glycosylatioglycan chains (GAG-att.) of neurocan and fragment D925. p, peptiedium of 293 HEK cells secreting the C-terminal neurocan fragmen

roteins were transferred to nitrocellulose and developed with the m

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ine with the findings that heparan sulfate chainsould direct proteins to the basolateral side (29),hereas chondroitin sulfate chains had no apparentffect (30). Proteins containing N-linked oligosaccha-

sites for N-asparagin-linked oligosaccharides (N-glyc.) or glycosami-hom, homology. (B) Heparin affinity chromatography of conditioned925. Single fractions were analysed by SDS–PAGE on a 10% gel. Theoclonal anti-neurocan antibody 1D1.

FIG. 3. (A) Tenascin-C fragment TNfn4,5 was incubated withhe affinity matrix containing the C-terminal neurocan domain inBSTCM. The resin was washed in a column with 5 volumes ofBSTCM (fractions 2–6), 3 volumes TBSTCM with 25 mM EDTA

small arrow, fractions 7–9) and 3 volumes TBSTCM containing 25M EDTA and 1 M NaCl (big arrow, fractions 10–12). Aliquots of all

ractions were precipitated with TCA and analysed by SDS–PAGEnd Coomassie blue staining. (B) Tenascin-C fragment TNfn4,5 wasncubated with the same matrix in the presence of 400 mg heparin.he resin was washed and the fractions collected and analysed as in. Note the higher TNfn4,5 content of the samples eluting afterpplication of buffer containing EDTA (fraction 7 1 8) in the pres-nce of heparin.

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ains (32), have been found to be preferentially trans-orted to the apical surface.According to these observations neurocan and brevi-

an, both containing N-linked oligosaccharide attach-ent motifs and O-glycosylation domains, should both

e preferentially secreted to the apical compartment.owever, MDCK II cells transfected with neurocan

ecreted this molecule predominantly (73 6 10%) intohe basolateral compartment (Fig. 4A, Table 1). Inontrast, when MDCK II cells were transfected with aDNA coding for the secreted variant of brevican (Fig.B, Table 1), brevican was mainly, although much lessronounced (58 6 6%), detected in the apical compart-ent. When MDCK II cells were transfected with the

DNA coding for the heparin binding C-terminal neu-ocan fragment D925, for this fragment a similar dis-ribution as for the entire neurocan molecule could bebserved, with a secretion of 71 6 7% of the moleculesnto the basolateral compartment (Fig. 4C, Table 1).

The results obtained with neurocan did, thus, notgree with the currently known targeting signals inpithelial cells. Assuming that neurocan is an axonallyransported molecule, the results did, in addition, notgree with a concept, which evolved from a comparisonf the specific sorting of various molecules to distincturfaces in neurons and epithelial cells, and proposeshat these cells share common sorting mechanisms,ith the apical and axonal surfaces, and the basolat-ral and dendritic surfaces being analogous membraneomains (33). In support of this concept it was re-ently shown, that detergent insoluble cholesterol-richlycolipid-rafts are involved in the sorting of axonalnd apical molecules (34). However, also several mol-cules have been found which are not sorted according

FIG. 4. Western blots of proteins secreted by MDCK cells trans-ected with the neurocan (A), brevican (B), and D925 (C) cDNA andrecipitated from equivalent volumes of the apical (Ap) and basolateralBa) compartment of the transwell cultures. Blots A and C were devel-ped with anti-neurocan monoclonal antibody 1D1, blot B with a poly-lonal anti-brevican antiserum. The protein were separated on 5% (A),% (B), and 10% (C) acrylamide gels. A band visible at 170 kDa in Aresumably represents a proteolytic fragment of neurocan. A bandisible at 100 kDa in A and C is derived from a cross-reactivity with arotein present in the chondroitinase ABC preparation.

453

o this concept, and in the transmembrane and cyto-lasmic part of membrane spanning molecules specificxonal targeting signals have been identified which arepparently not recognized in epithelial cells (35, 36).hus, the observed basolateral secretion in MDCK IIells does not necessarily exclude an axonal transportf neurocan, and might reflect its ability to bind toolecules, like heparan sulfate proteoglycans, which, by

hance, are directed into the basolateral compartment.Interestingly, a differential distribution of mRNA

nd protein has also been shown for the GPI-anchoredell surface heparan sulfate proteoglycan glypican,hich is actually the molecule showing the basolater-lly directional effect of heparan sulfate chains, and forerebroglycan, a homologous heparan sulfate proteo-lycan molecule, and it was observed that the cellodies and dendrites of cells expressing these two mole-ules are only weakly labeled by antibodies, whereas thexons of those neurons are quite heavily stained (37).In summary, we have identified neurocan as a hep-

rin binding brain proteoglycan, and mapped the hep-rin binding ability to the C-terminal domain. Inddition, we observed that the interaction of the-terminal neurocan domain with the neurocan bind-

ng tenascin-C fragment can be enhanced by the addi-ion of heparin, and that, although currently knownargeting signals present in neurocan would have pre-icted an apical secretion, neurocan and the C-termi-al domain of neurocan are, like heparan sulfate pro-eoglycans, mainly basolaterally secreted by MDCKpithelial cells.

CKNOWLEDGMENTS

The authors thank Gerlinde Kulbe and Gunnel Roos for excellentechnical assistance, Dr. Ray Boot-Handford for reading the manu-cript, and Dr. Reinhard Faessler and Dr. Rupert Timpl for support.unding was provided by the German Research Council (DFG, Ra44/4-1 to U.R.), the Swedish Natural Science Research CouncilNFR), by the Alfred Osterlund Stiftelse, and by the Carl Tesdorpfstiftelse.

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The Percentages of Neurocan and Brevican in the UpperApical) and Lower (Basolat.) Compartment of the Transwellhamber, as Determined by Scanning Densitometry after

mmunodetection

Apical Basolat.

eurocan (n 5 6) 27 6 10% 73 6 10%revican (n 5 6) 58 6 6% 43 6 6%eurocan fragm. D925 (n 5 4) 29 6 7% 71 6 7%

Note. The values represent the mean 6 SD.

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Neurocan Is a Heparin Binding Proteoglycan