The EMBO Journal vol.6 no.10 pp.2875-2883, 1987

Characterization of binding properties of the myelin-associatedglycoprotein to extracellular matrix constituents

Thomas Fahrig, Carlos Landal, Penka Pesheva,Klaus Kuhn2 and Melitta Schachner

Department of Neurobiology, University of Heidelberg, Im NeuenheimerFeld 364, 6900 Heidelberg, and 2Max-Planck-Institut fur Biochemie, 8033Martinsried uber Munchen, FRG

'Present address: Universidad Nacional de Cordoba, Facultad de CienciasQuimicas, 5016 Cordoba, Argentina

Communicated by M.Schachner

The myelin-associated glycoprotein (MAG) can be obtainedfrom adult mouse brain from detergent-lysates of a crudemembrane fraction as a 96-100 kd form (detergent solubil-ized MAG), and from 100 000 g supernatants of homogenatesas a 90-96 kd fonn (soluble MAG). The soluble form dis-tributes into the Triton X-114-poor aqueous phase, whiledetergent-solubilized MAG predominantly enters the TritonX-114-rich phase. Both molecular forms bind to heparin inhypo- and isotonic buffers. Soluble MAG binds to several col-lagens (type G, I, II, I, IV, V, VI, IX) with a kd of5.7 x 10- M for collagen type IX and 2.0 x 10-7 for col-lagen type IV. Binding of 125I-labeled MAG to collagen Gcan be completely inhibited by unlabeled MAG and collagenG, but not by heat-denatured collagen. MAG does not bindto itself, laminin, fibronectin, or the neural cell adhesionmolecules Li and N-CAM. Binding of MAG to collagen Gis most effectively blocked by a high molecular weight dex-tran sulfate, heparan sulfate and heparin, with chondroitinsulfate and a low molecular weight dextran sulfate being lesspotent blockers. These fmdings are in agreement withprevious observations on the localization of MAG in basallamina and interstitial collagens of the sciatic nerve in situ.Key words: collagen/heparin/mouse/myelin-associated glyco-protein/nervous system

IntroductionThe myelin-associated glycoprotein (MAG) is a constituent ofcentral and peripheral nervous system myelin (for review seeQuarles, 1983/1984). As a glycoprotein of approximately 100 kdapparent mol. wt with a carbohydrate content of about 30% itis a minor component of myelin, comprising less than 1 % ofthe total protein. Because of its particular localization in peri-axonal membranes it has been implicated in neuron-myelinatingcell interactions (Martini and Schachner, 1986; Stemberger etal., 1979; Trapp and Quarles, 1982, 1984). On the basis of re-cent evidence that MAG belongs to a family of glycoproteinsthat includes the cell adhesion molecules LI1, N-CAM and J1(Kruse et al., 1984, 1985) we have suggested that MAG is alsoinvolved in cell surface recognition. The common feature amongmembers of this family is a carbohydrate epitope which isrecognized by the monoclonal antibodies L2 and HNK-1, thatis also present on an unusual acidic glycolipid (Chou et al., 1985)which contains a 3'-sulfated glucuronic acid as a major antigenicdeterminant (Chou et al., 1986; Noronha et al., 1986). To verify

our hypothesis that all members of the L2/HNK-1 family ofglycoproteins are indeed adhesion molecules, we have isolatedmono- and polyclonal antibodies to MAG and used these in anin vitro assay system to perturb adhesion among neural cells(Poltorak et al., 1987). We could show that Fab fragments ofantibodies to MAG inhibited adhesion of oligodendrocytes toneurons and oligodendrocytes to oligodendrocytes, but notoligodendrocytes to astrocytes. Furthermore, MAG containingliposomes were shown to adhere to neurons indicating that MAGis itself the binding ligand.

In view of the particular functional importance ofMAG in cellrecognition we have studied the expression and localization ofMAG in the developing nervous system in the hope of makinginferences about the biological role ofMAG in the myelinationprocess. In the developing mouse sciatic nerve MAG was firstdetectable in the initial stages of myelination, when the loops ofthe myelinating Schwann cell had turned approximately one anda half times around its axon, just at the time when the cell adhe-sion molecule LI disappears and N-CAM is reduced at the axon-Schwann cell interface (Martini and Schachner, 1986). MAG wasalways expressed in the uncompacted loops of Schwann cell pro-cesses during myelination and in the adult myelin, but ceasedto be detectable in compacted myelin (Martini and Schachner,1986) as had been observed by Trapp and Quarles (1982), butdisputed by Webster et al. (1983). Interestingly, MAG was alsofound in the extracellular matrix consisting of interstitial collagensand basement membrane of Schwann cells (Martini andSchachner, 1986). MAG appeared in the extracellular matrixonly at later stages of myelination and well after the mor-phologically recognized deposition of extracellular matrix struc-tures (Martini and Schachner, 1986).Based on these observations it seemed pertinent to investigate

whether MAG would bind to extracellular matrix constituents,such as collagen or glycosaminoglycans. Such interactions couldplay an important role in the presentation of an adhesion moleculeto regenerating axons which tend to choose the basement mem-brane side of Schwann cells for regeneration (see, e.g. Aguayo,1985; Ide et al., 1983; Schwab and Thoenen, 1985). Here wereport that MAG binds to several types of collagens and heparin,but not to other extracellular matrix components present in theperipheral nerves, such as laminin or fibronectin.

ResultsPurification ofMAG from adult mouse brainMAG was isolated from adult mouse brain by two procedures.In one procedure MAG was purified from non-ionic detergentlysates of crude membrane fractions of mouse brain by immuno-affinity-chromatography using a monoclonal MAG antibody col-umn (Poltorak et al., 1987). Membranes were solubilized in 0.5%Triton X-100 and the detergent lysate applied to the antibodycolumn which was then washed extensively with octylglucosideto replace Triton X-100 (Figure la). This MAG preparation willbe referred to as 'detergent-solubilized MAG'. MAG was alsoisolated from 100 000 g supernatants of brain homogenates

() IRL Press Limited, Oxford, England 2875

a b c d e f

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200'.116'_.970'. d 466'.

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48'.

Fig. 1. a, Autoradiogram of 125I-labeled detergent-solubilized MAG (isolatedby immunoaffinity-chromatography on a monoclonal MAG antibody columnfrom detergent lysates of crude membrane fractions of adult mouse brain);b, autoradiogram of 125I-labeled soluble MAG (obtained by immunoaffinity-purification on a monoclonal MAG antibody column from 30 000 gsupernatants of brain homogenate); c, Western blot analysis of soluble MAGwith polyclonal antibody to MAG isolated by the lithium diiodosalicylatemethod; d, Western blot analysis of soluble MAG with L2 monoclonalantibody; e, autoradiogram of 125I-labeled detergent-solublized MAG;f, adsorbed to collagen G by the solid phase radio-ligand binding assay anddesorbed by 1% SDS. SDS-PAGE was performed on 4-10% (a, b) and10% (e, f) gels. Mol. wt standards are indicated at the left (for a, b) andright for (c-f) margins (in kd).

Ozm0

0.z

0

zw

180

TIME (minutes)

Fig. 2. Kinetics of binding of 125I-labeled detergent-solublized MAG(10 ng/well) to collagen G in hypotonic buffer as assayed by solid phaseradio-ligand binding assay. Two independent experiments were carried out.Values (dots) are from one experiment carried out in duplicate. Closedcircles, binding to collagen; open circles, binding to BSA.

using the monoclonal MAG antibody column. This MAG pre-paration will be referred to as 'soluble MAG', since it does notprecipitate or aggregate in hypotonic or isotonic buffers in theabsence of detergents (Figure lb). Polyclonal antibodies preparedagainst MAG isolated from bovine brain according to the well-established lithium diiodosalicylate-phenol method (Quarles etal., 1983) reacted with soluble (Figure ic) and detergent-solubilized MAG. In 4-10% gradient gels, soluble MAGmigrated as a smear with an apparent mol. wt of 90-96 kd anddetergent-solublized MAG with an apparent mol. wt of96-100 kd (Figure la,b). Soluble and detergent-solubilizedMAG could be shown to carry the L2/HNK-1 carbohydrateepitope as detected by Western blot analysis (for soluble MAG,see Figure Id).2876

To investigate the solubility properties of the two MAGpreparations in aqueous and detergent solutions, a Triton X-1 14phase separation (Bordier, 1981) was carried out. Approximatelytwo-thirds of the detergent-solubilized MAG distributed into theTriton X-1 14-rich phase, whereas approximately one-third wasrecovered from the aqueous phase. The apparent mol. wt ofMAGin the Triton X-1 14 phase was - 96 kd and -90 kd for MAGrecovered from the aqueous phase. Soluble MAG was onlyrecovered from the aqueous, but not from the Triton X-1 14 phase.It also migrated with an apparent mol. wt of -90 kd.

Binding ofMAG to collagensMAG was found to bind to various collagen types as measuredby a solid phase radio-ligand binding assay, in which proteinswere adsorbed as substrate onto plastic and binding of '25I1labeled MAG was quantified. Soluble MAG was used in thisassay under hypotonic and isotonic buffer conditions, anddetergent-solubilized MAG under hypotonic conditions.Maximal, i.e. equilibrium, levels of soluble and detergent-

solubilized MAG were bound in hypotonic buffer after -3 hwith tIA of approximately 60 min (Figure 2). In isotonic buffersoluble MAG also reached saturation levels after about 3 h. Solu-ble MAG was found to bind with a higher efficiency thandetergent-solublized MAG either in hypotonic or isotonic buf-fers. Under hypotonic buffer conditions detergent-solubilizedMAG had a binding efficiency of 6-12% of the input countsto collagen G, whereas soluble MAG showed a binding efficiencyof 15-20%. The ratio of MAG binding to collagen G versusbovine serum albumin was almost 10-fold. In isotonic buffer bin-ding efficiency of soluble MAG to collagen G was less (3- 8 %)than in hypotonic buffer. Background levels were generally twiceas high as under hypotonic binding conditions and specific bin-ding was lower, such that the ratio of specific to background bin-ding was reduced to a factor of three to four. However, withincreasing concentrations of 1251-labeled MAG (up to21 /tg/well) the specific binding ratio could be increased to five-fold in isotonic buffer. The large quantities of 1251-labeled MAGneeded for such binding studies were prohibitive for large testseries, so most studies were performed under hypotonic bufferconditions with key experiments done under both conditions.Detergent-solubilized MAG bound only marginally in isotonicbuffer. Binding efficiency of detergent-solubilized MAG to col-lagen G could be increased up to 25% of the input underhypotonic binding conditions, when 1251-labeled MAG was ad-sorbed on a collagen G column and the eluted peak-fractions takenfor binding studies. The fraction adsorbed onto collagen G bythe solid phase radio-ligand binding assay could be shown bySDS-PAGE to have the same mol. wt (Figure le,f). Bindingof '251-labeled MAG (3.5 Ag/ml) could be completely inhibitedin hypotonic buffer by unlabeled MAG (50-fold excess) and col-lagen G, but not heat-denatured collagen (gelatine) (both at10 g/ml) or the RGD sequence containing hexapeptide(100 Ag/ml). Half maximal binding ofMAG to collagen G wasseen at 1 ,sg collagen G/ml.Both soluble and detergent-solubilized MAG bound to all col-

lagen types tested (collagen G and types I, II, Ill, IV, V, VI,IX) (for soluble MAG, see Figure 3). Binding of 125I-labeledMAG to these collagens in hypotonic buffer was always abovebackground values obtained when bovine serum albumin servedas substrate. No binding of '25I-labeled MAG above this back-ground level was observed when laminin, fibronectin, deter-gent-solubilized MAG, LI, N-CAM or heat-denatured collagenwere used as substrates.To determine the affinity constants for MAG binding to col-

Binding properties of MAG

0

w

25-

G I 11 III IV V VI IX BSA FN NCAMCOLLAGENS LN MAG Li

Fig. 3. Binding of 1251I-labeled soluble MAG (10 ng/well) to varioussubstrates in hypotonic buffer as measured by the solid phase radio-ligandbinding assay. BSA, bovine serum albumin; LN, laminin; FN, fibronectin.Bars represent mean values obtained from three independent experiments 4

standard deviation. For each experimental point three determinations were

carried out in each experiment. Since absolute values of MAG bindingvaried slightly between the three experiments, values are expressed as

relative % MAG bound, i.e. the highest value in each experiment was setas 100%, the other values calculated in relation and normalized to the upperlimit of the error range for collagen Im.

lagens, Scatchard plot analyses both in hypotonic and isotonicbuffers were performed (Norby et al., 1980; Rosenthal, 1967;Weder et al., 1974). Increasing amounts of 125I-labeled MAGthat had been diluted at a ratio of 1:20 with unlabeled MAG wereused to measure saturation of binding under equilibrium condi-tions. Background values of 1251-labeled soluble MAG bindingto bovine serum albumin-coated wells were subtracted from thebinding values obtained with collagens. In hypotonic buffersaturation of binding to collagen G (100 ytg/ml) was approximated(Figure 4A), but never completely reached even at 21 jigMAG/well (not shown). Scatchard plots were constructed fromvalues obtained by subtraction of non-specific background values.Curve-shaped Scatchard plots were obtained indicating bindingsite heterogeneity or negative cooperative effects. Assuming thecase of two independent binding sites these curves were fittedby a hyperbolic function according to the mathematical theoryof Feldman (1972) with a significance level of 0.0001. Scatchardplots are shown for collagen G (Figure 4A,B), type IV (Figure 4C)and type IX (Figure 4D). Two dissociation constants (kd) of7.4 x 10-8 M and 8.0 x 10-6 M were calculated for collagenG in hypotonic buffer (Figure 4A). For collagen type IV a singlekd of 2.0 x 10-7 M (Figure 4C) and for collagen type IX a kdof 5.7 x 10-8 M (Figure 4D) was measured in hypotonic buf-fer. When binding of 125I-labeled soluble MAG to collagen Gwas measured in isotonic buffer (Figure 4B) construction of thecorresponding Scatchard plot according to Rosenthal (1967) was

not possible, suggesting a larger number of binding sites.However, when evaluation of kd values was attempted from theinitial and final slopes of the curve, a two- to three-fold lowerkd was approximated at 2.7 x 10-8 M and a 10-fold higher kd

at 8.0 x 10-6 M when compared to the two dissociation con-stants in hypotonic buffer. Confirmation that saturation valueswere reached for collagen types IV and IX was obtained byanalysis of the data according to Klotz (1982) in that a turningpoint could be seen. These experiments show that, although thebinding efficiencies are different in isotonic and hypotonic buf-fers, dissociation constants are quite similar, indicating the validityof binding experiments in hypotonic buffer.To verify the binding of MAG to the various collagen types

by another method, an overlay assay was performed in which1I51-labeled soluble MAG binding to collagens was measuredafter SDS-PAGE, transfer to nitrocellulose filter, incubation with'25I-labeled MAG and autoradiography (Figure 5 bottom, capitalletters). Specific binding could be observed particularly to theal chain of all collagen types as revealed by Coomassie Bluestaining (Figure 5 top, small letters) under hypotonic binding con-ditions, except for collagen type IV to which MAG showed hardlydetectable binding (lane F). Collagen tpes I Oane c) and IV (lanef) tended to stain less well with Coomassie in this study, but couldbe shown to transfer as well as the other collagen types tonitrocellulose filters by staining with Ponceau S. Thus, the weakerbinding ofMAG to collagen tpe IV seen by the solid phase radio-ligand binding assay would correspond to a weaker spot in theoverlay assay. Interestingly, MAG bound to heat-denatured col-lagen G (lane A). No binding of '251-labeled MAG was everobserved when laminin, fibronectin or bovine serum albuminwere used in the overlay assay system (not shown). The overlaybinding experiments thus confirm the results of the solid phaseradio-ligand binding assay, except for binding to heat-denaturedcollagen G (see Discussion).Binding of MAG to heparin12514Labeled soluble MAG was retained on a heparin-agarosecolumn and eluted in an NaCl gradient from 0.015-1.0 M at-0.3 M, when the column was loaded in hypotonic (Figure 6A)and isotonic (Figure 6B) buffers, but with a somewhat broadershoulder in isotonic than in hypotonic buffer. The eluted radio-activity migrated by SDS-PAGE at the position of the 125I1labeled MAG that was applied to the column (inset in Figure6B, for the experiment under isotonic binding conditions).To measure the dissociation constants for MAG binding to

heparin a radioimmunoassay on heparin-agarose beads was per-formed. Increasing amounts of 125I-labeled soluble MAG wereused to determine the dissociation constants by Scatchard plotanalysis. Background values of 125I-labeled beads withoutheparin were subtracted from the values obtained withheparin-agarose beads. The binding efficiency of MAG toheparin was 18% in isotonic and 35% in hypotonic buffer. Inhypotonic buffer the ratio of MAG binding to heparin-agaroseversus agarose was almost 200:1. In isotonic buffer backgroundlevels were higher, so that the ratio of specific binding tobackground was only 8:1. In this assay-system saturation wasnot reached due to the high abundance of heparin on beads relativeto the low amounts of MAG ligand. Calculation of affinity con-stants from the initial linear slope of the binding curve gave kdvalues in the range of 10-7 M both under isotonic and hypotonicbinding conditions. Half-maximal binding of MAG to heparincould be achieved by a 40-fold excess of heparin, but not of chon-droitin-4-sulfate or an epimeric mixture of chondroitin sulfates.Binding ofAMG to collagen G in the presence ofglycosamino-glycansBinding of 251I-labeled soluble MAG to collagen G wasmeasured by the solid phase radio-ligand binding assay in thepresence of various glycosaminoglycans (Figure 7). When com-

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Fig. 4. Binding curves and Scatchard plot analysis (insets) of 1251-labeled soluble MAG (10 ng- 15 jAg) binding to collagen G (A, B), collagen type IV (C)and collagen type IX (D) as measured by the solid phase radio-ligand binding assay in hypotonic (A, C, D) and isotonic (B) buffer. In each case, bindingvalues of MAG to BSA were subtracted from those to collagen. Values were obtained from three (for collagen G under hypotonic conditions) and two(collagen types IV, X and collagen G under isotonic conditions) independent experiments carried out in triplicate a standard deviation.

2878

Binding properties of MAG

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Fig. 5. Overlay assay with 125I-labeled soluble MAGtypes. (A-I) Coomassie blue-stained gel after transfenitrocellulose filter. (a-i) Autoradiogram of nitrocelliincubation of 125I-labeled soluble MAG in hypotonic(a, A) Collagen G denatured by boiling before separa(b, B) collagen G; (c, C) collagen type I; (d, D) collE) collagen type El; (f, F) collagen type IV; (g, G) ((h, H) collagen type VI; (i, I) collagen type IX.

petition efficiencies were measured on a mo]according to Koda and Bernfield (1984), a ccbinding was observed in the order of decreadextran sulfate (mol. wt 500 kd) followed(65 kd), heparin (15 kd), chondroitin4-suldroitin sulfate in a mixture of epimers (40(5 kd), sulfate, phosphate and chloride. Thesthat heparin sulfate is approximately 100-folc

g h chondroitin sulfate (mixture of epimers) in inhibiting MAG bind-ing to collagen G.

DiscussionThe present study has shown that the myelin-associated glycopro-tein MAG binds to the extracellular matrix constituents heparinand all species of collagens tested (types I, II, mI, IV, V, VIand IX). This binding appears to be significant, since it can beobserved not only under hypotonic, but also under isotonic buf-fer conditions. Dissociation constants were in the range of10-7 M. Other extracellular matrix constituents, such as lamininor fibronectin did not show specific binding of MAG. Also, MAGdoes not bind to itself or to the other neural cell adhesion

- molecules LI and N-CAM under the conditions of this study.The observation that both forms of MAG, prepared either fromdetergent-free supernatants of brain homogenates or detergentlysates of crude membrane fractions display this specificity showsthat we have been able to isolate MAG in two physiologicallyactive forms that conserve the extracellular binding functions ofthe molecule. The specific binding of MAG to extracellularmatrix constituents in vitro is in agreement with previous obser-

G H I vations in situ on the association of MAG (Martini and Schachner,1986) with the basal lamina and interstitial collagens types I andIH which form the interface between Schwann cells and themesenchymal environment of the endoneurium. Thus, observa-tions in the intact tissue can now be corroborated by those in vitro.

All collagen types tested in this study showed binding to MAGthat was well above background levels, even for collagen IVwhich showed a generally weaker affinity. Binding was notonly observed in a solid phase radio-ligand assay, but also by

ID another technique, the overlay assay system. In both assaysystems a more native conformation of the proteins appeared tobe necessary. Heat-denatured collagen G was not a good bin-ding partner for MAG in the solid phase radio-ligand assay, butbound MAG in the overlay assay, where a certain degree ofrenaturation can occur upon transfer to nitrocellulose filters inphysiological buffer. All collagens, and even heat-denatured col-

3 t lagen G appeared to assume a conformational form appropriatefor binding predominantly to the cxl chain in the overlay assay.The observation that MAG binds to all collagens tested so far sug-gests that a domain common to these collagens forms the basisof such interaction. It is noteworthy in this respect that the adhe-sion molecule vitronectin binds to all collagens tested, i.e. typesI through VI, suggesting a specific binding to triple-helical col-

;on various collagen lagens (Gebb et al., 1986). Interestingly, the RGD sequence con-vr of proteins to taining hexapeptide (Pierschbacher and Ruoslahti, 1984) did notulose filter after inhibit binding of MAG to collagen, suggesting that this struc-binding buffer. ture which is common to several extracellular matrix constituents

agen type II; (e, and has been found in MAG (Arquint et al., 1987) may not becollagen type V; operational under our assay conditions.

Another constituent of the extracellular matrix was found tobind to MAG, the glycosaminoglycan heparin. Heparin or heparansulfate have previously been reported to be ligands for a variety

lar basis and plotted of biologically important molecules, such as protease inhibitors)mpetition for MAG (Lindahl et al., 1986), the extracellular matrix constituentsLsing potencies with laminin (Yamada et al., 1980), fibronectin (Ruoslahti andby heparin sulfate Engvall, 1980; Woodley et al., 1983) and various collagens

[fate (40 kd), chon- (Koda et al., 1985) and growth factors (Gospodarowicz et al.,kd), dextran sulfate 1984; Klagsbrun and Shing, 1985; Wagner and D'Amore, 1986;,e experiments show Risau and Ekblom, 1986). Recently, the neural cell adhesionI more efficient than molecule N-CAM was also shown to contain a heparin (heparan

2879

[Ml

z

LJ

z

FRACTION NUMBER

20FRACTION NUMBER

z

~-

Lo

z

15

Fig. 6. Elution profile of 125I-labeled soluble MAG after binding to a heparin-agarose column in hypotonic (A) and isotonic (B) buffers and eluted with a

0.015-1.0 M NaCl gradient. Inset in B shows autoradiogram of 1251-labeled MAG applied to the column (left lane) and eluted from the column (peakfraction, right lane).

sulfate)-binding domain which appears to be involved in neuralcell-cell and cell-substratum adhesion (Cole et al., 1985, 1986;Cole and Glaser, 1986). Although binding constants were notdetermined, the binding of heparin or heparan sulfate to thesemolecules was proposed to induce a conformational change inthe protein resulting in a higher affinity between the binding part-ners. Glycosaminoglycan-protein interactions may thus modulatethe biological activities of adhesion molecules. On the other hand,heparin may serve as an extracellular binding site for 'soluble'MAG thus immobilizing it and, in turn, mediating attachment ofneighbouring Schwann cells.Our finding that the binding of MAG to collagen G can be

inhibited by glycosaminoglycans is interesting with regard to the

observation that heparin inhibits binding of retinal cells toadherons or N-CAM (Cole et al., 1985; Cole and Glaser, 1986;Schubert and LaCorbiere, 1985; Schubert et al., 1983), althougha direct correlation is difficult because the binding partner on

retinal cells has not been identified. Also, binding of heparansulfate proteoglycans from mouse mammary epithelial cells tocollagens was specifically inhibited by heparin (Koda and Bern-field, 1984; Koda et al., 1985). High mol. wt dextran sulfateis the most efficient inhibitor of all glycosaminoglycans as hasbeen observed also by Koda and colleagues (1984, 1985). It in-hibits more potently than low mol. wt dextran sulfate, suggestingthat sulfate groups in high polymeric form are important. Chon-droitin sulfate was found to be at least an order of magnitude

2880

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30

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Binding properties of MAG

-7 -5LOG MOLARITY OF ANION

Fig. 7. Binding of 125I-labeled soluble MAG to collagen G in the presenceof anions as assayed in the solid phase radio-ligand binding assay inhypotonic buffer. (1) Dextran sulfate, mol. wt 500 000; (2) heparan sulfate,mol. wt 65 000; (3) heparin, mol. wt 15 000; (4) chondroitin4-sulfate, mol.wt 40 000; (5) chondroitin sulfate (mixture of epimers), mol. wt 40 000;(6) dextran sulfate, mol. wt 5000; (7) sulfate; (8) H2POZ/HP42-;(9) chloride. The graph was plotted according to Koda and Bemfield (1984).Values were obtained from four independent experiments carried out in'duplicate. For calculation of relative % MAG bound, see legend, Figure 3.

less potent an inhibitor than heparin and heparan sulfate. Thestructural characteristics of the inhibitory glycosaminoglycanssuggest a particular binding site on the glycosaminoglycans.Heparan sulfate, and to a lesser degree also heparin, containhighly sulfated regions. These are potentially present also in dex-tran sulfate. The glycosaminoglycans which are less effectivecompetitors of the binding, such as chondroitin sulfate, lack thesehighly sulfated regions and show a more uniform distributionalong the polymer rather than in 'blocks' (for review, see Lin-dahl et al., 1986; Lindahl and Hook, 1978). It appears also thatthe degree of sulfation is not responsible for inhibitory poten-tial. For instance, dextran sulfate (5 kd), which has 3.3 sulfateresidues/disaccharide (Windholz et al., 1976), is less potent thansimilarly sized heparan, which bears 2-3 sulfate residues/disac-charide. Sulfate concentration is also not the decisive feature ininhibition, since the less inhibitory glycosaminoglycans, such aschondroitin sulfate, have a 10- to 50-fold higher sulfate concen-tration than the more inhibitory glycosaminoglycans. The obser-vation that block-sulfated polysaccharides can inhibit the bindingof MAG to collagen would point to the possibility that the bin-ding domains on collagens for glycosaminoglycans and MAGare situated in close proximity, such that the occupation of theheparin-binding domain would sterically hinder the binding ofMAG to collagen. It is important to note here that neither MAGnor the collagens contain detectable amounts of contaminatingglycosaminoglycans which could theoretically form a linker be-tween collagen and MAG, since both contain heparin-bindingsites. The ternary complex between MAG, collagen and heparinmay well exist in vivo, but is not the basis of MAG binding tocollagen in our experiments.The present study has shown that the neural cell adhesion

molecule MAG is a multifunctional protein that contains bothcollagen- and heparin-binding domains. Since MAG is also in-volved in cell adhesion (Poltorak et al., 1987), a distinction be-tween cell- and substrate-adhesion molecules seems thereforeartificial. We would further like to propose that MAG may existin a dual state, the membrane-associated form which links the

cell surface to neighbouring cells or substrates and a soluble formthat associates with the basal lamina and collagens. This solubleform may be generated from the membrane-bound form by pro-teases (Sato et al, 1982; Yanagisawa et al., 1984), but it re-mains to be seen whether this proteolytic cleavage subserves aphysiological role. As an integral membrane protein, MAG mayplay a role in axon-myelinating glial cell recognition or surfaceapposition of turning loops of myelin-forming cells. As an extra-cellular matrix constituent MAG could bridge cell surfaces, e.g.of Schwann cells, with the extracellular matrix. It is pertinentin this respect that axons that regrow into denervated parts ofperipheral nerves seek the Schwann cell-fibroblast interface withbasement membrane and interstitial collagens as sites for growth.Whether MAG does indeed play a functional role as extracellularmatrix constituent in regeneration of the peripheral nervoussystem remains, however, to be seen.

Materials and methodsPunfication ofMAGMAG was purified from brains of adult mice from several inbred strains by twodifferent procedures. In one procedure, MAG was prepared from detergent lysatesof a crude membrane fraction and in the other, MAG was obtained from detergent-free supematants of brain homogenates clarified by centrifugation. For purifica-tion ofMAG from detergent lysates (detergent-solubilized MAG) a crude mem-brane fraction was obtained from brain homogenates by centrifugation for 1 hat 30 000 g and 4°C (Rathjen and Schachner, 1984). The pellet was solubilizedin 20 mM Tris, 0.15 M NaCl, 1 mM EDTA, 1 mM EGTA, pH 7.3 (solubiliza-tion buffer) containing 0.5% Triton X-100 for 1 h at 4°C. After a 100 000 gcentrifugation step the supernatant was applied to Sepharose 4B columns coupl-ed with an IgG fraction of monoclonal MAG antibody (Poltorak et al., 1987)purified over a Protein A-Sepharose column (Pharmacia). The MAG antibodycolumn was washed in sequence with solubilization buffer contaning 0.5% TritonX-100, solubilization buffer containing 0.5% Triton X-100 and 0.3 M NaCl,solubilization buffer without detergent and solubilization buffer cnaining 8 mg/min-octyl-O-D-glucopyranoside (Sigma) in 10 column volumes each. Antigens wereeluted with two column volumes of 0.1 M diethylamine, 1 mM EDTA, 1 mMEGTA, pH 11.5, and 10 mg/nil octylglucoside. The eluates were dialyzed at 4°Cagainst phosphate buffered saline (PBS), pH 7.3, containing 10 mg/mil octyl-glucoside, concentrated in Amicon chambers by pressure dialysis and stored at-70°C. Antigen concentrations ranged from 50 to 150 ,g/ml.For preparation ofMAG in detergent-free form (soluble MAG), supernatants

of the 30 000 g centrifugation step of brain homogenates (see above) were re-centrifuged at 100 000 g for 1 h and applied to the antibody column. Columnswere washed with PBS, PBS containing 0.3 M NaCl and eluted with 0.1 M di-ethylamine, 1 mM EDTA, 1 mM EGTA, pH 11.5. Eluates were dialyzed againstPBS, concentrated by pressure dialysis and stored at -70°C. Antigen concen-trations ranged from 100 to 200 ,g/ml.Radiotoination ofMAGImmunoaffinity-purified MAG (100-300 mg) was dialyzed overnight against PBS,pH 8.2, for soluble MAG and against PBS, pH 8.2, containing 10 mg/ml octyl-glucoside for detergent-solubilized MAG. Antigen solutions were concentratedto a final volume of 200 ju by centrifugation in Amicon tubes (Centricon 30).Bolton -Hunter reagent (Bolton and Hunter, 1973; from Amersham) was driedunder a nitrogen stream and the antigen solution applied to the dried ester andincubated for 30 min at room temperature or for 1.5 h on ice with similar results.The reaction was stopped by adding 50 i1 1 M glycine, pH 8.2. The hydrolyzedester-glycine conjugate was separated from the iodinated antigen by extensivedialysis against binding buffer (see binding assays below). The iodinated proteinsolution was diluted by addition of binding buffer to a volume of 1 ml and specificactivity determined by precipitation with trichloroacetic acid (TCA). Specific ac-tivities for soluble and detergent-solubilized MAG ranged between 1 and5 x 106 c.p.m./4g protein.AntibodiesMonoclonal MAG antibody, an IgG from mouse, has been described andcharacterized previously (Poltorak et al., 1987). Monoclonal L2 antibody hasalso been described previously (Kruse et al., 1984). Antibodies were purifiedover a Protein A-Sepharose column. Polyclonal antibodies to MAG isolatedby the lithium diiodosalicylate-phenol method (Quarles et al., 1981) were preparedaccording to the immunization protocol for the cell adhesion molecule LI (Rath-jen and Schachner, 1984) and immunoaffinity purified over a MAG antigen col-umn as described (Martini and Schachner, 1986). Antibody eluates were dialyzedagainst PBS and concentrated to 5-10 mg/ml.

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Analytical proceduresProtein determinations were camied out according to Bradford (1976) and Duhamelet al. (1981). SDS-PAGE was carried out according to Laemmli using 10%and 4- 10% gradient slab gels (Laemmli, 1970). Western blot analysis was car-ried out according to Faissner et al. (1985). Staining of proteins on nitrocellulosefilters was carried out with Ponceau S as follows: directly after protein transfernitrocellulose filters were washed briefly in 3% TCA and stained for 5-10 minwith a solution of 0.2% (w/v) Ponceau S (Sigma) in 3% TCA. Staining wasvisualized by washing with water and disappeared by washing in 20 mM Tris,150 mM NaCl, pH 7.2.Purification of extracellular matrix constituents and of the cell adhesion moleculesLI and N-CAMFibronectin was prepared from human plasma according to method A describedby Miekka et al. (1982). Laminin was isolated from the EHS mouse tumor (Timplet al., 1979). Collagen G was obtained from Seromed (Heidelberg) as a proteinsolution of 3 mg/ml in 12 mM HCI and consists of 90% of collagen type I and10% of collagen type Im. Collagen type I was extracted from rat skin with aceticacid (Bornstein and Piez, 1966). Collagen type II was prepared from chick car-tilage (von der Mark et al., 1982). Collagen type Im was prepared from fetalcalf skin (Timpl et al., 1981). Collagen type IV from mouse and human wasisolated after mild pepsin treatment of the EHS mouse tumor or human placenta,respectively (Timpl et al., 1981), whereby the carboxyterminal NCI domain wasdestroyed. Collagen types V and VI were purified from human placenta (Bentzet al., 1978; Odermatt et al., 1983). Collagen type IX from chick cartilage wasdissolved by pepsin and isolated in the form of the two fragments Ml and M2.The aminotermmnal, globular domain has been digested during the procedure (vonder Mark et al., 1982). The dextran sulfates, chrondroitin sulfate (mixed isomers),heparan sulfate (isolated from bovine kidney) and heparin (sodium salt, GradeI, from porcine intestinal mucosa, code No. H 3125) were obtained from Sigma.Chondroitin-4-sulfate was obtained from Serva (Heidelberg). N-CAM and LIwere purified from adult mouse brain by immnunoaffinity-chromography on mono-clonal antibody columns as described previously (Him et al., 1981; Rathjen andSchachner, 1984).Triton X-114 phase separation of MAGTriton X-114 phase separation of soluble and detergent-solubilized MAG wasperformed according to Bordier (1981). In brief, 100 A1 protein samples (60 tg/ml)in 10 mM Tris, 150 mM NaCl, pH 7.4, containing 1% Triton X-1 14 were in-cubated on a sucrose-cushion (6% sucrose (w/v), 10 mM Tris, 150 mM NaCl,0.06% Triton X-1 14) for 30 min at 37°C. After centrifugation, pellet and super-natant were separated and further extracted individually. The pellets wereresuspended in 200 1l TBS containing Triton X-1 14 at a final concentration of1%. Resuspended pellets were extracted with TBS and supernatants with TBS con-taining 1% Triton X-1 14 by three cycles of incubation for 30 min at 4°C andthen once for 30 min at 37°C. Samples were then centrifuged and pellets or super-natants analyzed for MAG by SDS-PAGE.Solid phase radio-ligand binding radioimmuunoassayIndividual wells of microtest ITM flexible assay plates (Falcon 3911) were in-cubated overnight at 4°C with different proteins at concentrations of 100 lAg/mlin 0.1 M NaHCO3, pH 8.1 (coating buffer). Plates were washed twice withcoating buffer containing 0.1% bovine serum albumin (BSA) and then treatedfor 1 h at room temperature with coating buffer containing 0.2% BSA. Wellswere then washed three times with hypotonic or isotonic buffers (bindingbuffers). The hypotonic binding buffer was 15 mM NaCl, 10 mM KH2PO4/K2HPO4, 0.1% BSA, pH 7.2. The isotonic binding buffer was PBS, pH 7.2,containing 0.1% BSA. These buffers were used for soluble MAG. When detergentsolubilized MAG was used the binding buffers contained 30 mM octylglucoside.Wells were incubated with 1251-labeled MAG diluted (usually 10-100 ng,70 000-100 000 c.p.m.) in 70 p1 binding buffer/well for 3 h at room temperaturefor the standard binding assay. Wells were then washed five times with bindingbuffer and bound radioactivity was counted by cutting out the bottom of the plasticwells and placing them directly into vials for counting.When the plastic substrate was coated with collagen concentrations of 100 Ag/ml

maximal binding of soluble and detergent-solubilized MAG was seen at inputsbetween 10 and 100 ng/well. Coating efficiencies of collagens were measuredby protein determination using a modification of the Bradford assay (Duhamelet al., 1981) and found to be approximately 8% of the input. Although somevariation was seen in the absolute values measured in three independent tests relativebinding values were the same for all proteins used with the exception of col-lagens type I and 111 which coated with a slightly lower efficiency (approximately30% less than the other collagens).Binding of 1251-labeled MAG to collagen was also carried out in the presence

of unlabeled MAG, collagen G, gelatine (collagen G boiled for 30 min), the RGDsequence containing hexapeptide after two hours of preincubation of the substrate(Pierschbacher and Ruoslahti, 1984) or polyanionic extracellular matrix consti-tuents. These were mixed together with 1251-labeled MAG and applied to the

substrate for the binding assay as described above. MAG and collagens werefound to be free of contaminating glycosaminoglycans by carbohydrate methyla-tion analyses.Affinity-chromatography on heparin-agaroseHeparin-agarose suspension (1 ml, Sigma H 6508) was filled into a glass col-umn and washed extensively with binding buffer. '25I-labeled MAG(4 x i07 c.p.m.) was diluted in 5 ml binding buffer and passed over the col-umn twice. The column was then washed with binding buffer until no more radio-activity could be detected in the run-through. Bound material was eluted witha linear salt gradient (20 ml) ranging from either 0.015 M to 1.0 M NaCl or0.150 M to 1.0 M NaCl in binding buffer.

Radioimmunoassay on heparin-agarose beadsAliquots (20 1l) of a 10% (v/v) suspension of heparin-agarose beads (see above)were diluted in 750 u1 binding buffer and incubated in 2.5 ml Eppendorf tubeswith varying amounts of 125I-labeled MAG (50 A1) for varing time intervals at4°C on an Eppendorf shaker. Aliquots were then layered onto sucrose cushions(0.6 ml, 1.0 M sucrose in binding buffer containing 0.1% BSA) and centrifugedat 4000 g for 5 min at 4°C. Cushion supernatants were discarded, cushionsoverlayered with 1 ml binding buffer and centrifuged for 3 in at 1000 g. Overlayand cushion were then removed, the bead pellet briefly vortexed in 50 A1 3 MNaCl, 10 mM KH2PO4/K2HPO4, pH 7.4, centrifuged for 2 min at 4000 g andsupernatants counted for radioactivity.

Affinity-chromatography on collagen columnCollagen G (20 mg) was coupled to cyanogen bromide-activated Sepharose 4B(5 ml suspension) according to Stoeckl et al. (1976). The suspension (1 ml) wasfilled into a Pasteur pipette. After washing with hypotonic binding buffer con-taining 30 mM octylglucoside 125I-labeled detergent-solubilized MAG(6 x i07 c.p.m.) was applied. The column was washed again with binding buf-fer until no more radioactivity appeared in the washing solution. Bound MAGwas eluted at 0. 12-0.15 M NaCl with a linear gradient (20 ml) from 0.015 to1.0 M NaCl.Overlay assayCollagens were boiled for 5 min in sample buffer, separated by SDS-PAGE(10 jg/lane) and then transferred to nitrocellulose as described for Western blotanalysis (see above). The efficiency of the transfer was tested by staining withPonceau S. The nitrocellulose filter was then blocked with 5% BSA in PBS for1 h at room temperature and incubated with 1251-labeled MAG in binding buf-fer for 3 h at room temperature. The nitrocellulose filter was rinsed four timeswith binding buffer containing 0.1 % Tween 20 until no more radioactivity wasdetectable in the washing solution. The filter was then dried and exposed to anX-ray film (Kodak XAR-5) at -70°C and developed according to the supplier'sinstructions.

AcknowledgementsThe authors are grateful to Andreas Faissner for help with the heparin bindingexperiments. Erkki Ruoslahti for the RGD peptide. Frank Kirchhoff and Jac-queline Trotter for preparation of ascites fluid and monoclonal MAG antibodies,Rainer Probstmeier, Gautam Thor and Remy Sadoul for helpful discussions, Alex-ander von Humboldt-Stiftung (C.L.) and Deutscher Akademischer Austausch-dienst (P.P.) for fellowships and Deutsche Forschungsgemeinschaft (SFB 317)and Fonds der Chemischen Industrie for support.

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Received on March 25, 1987; revised on July 3, 1987

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