Chapter 14 Receptors for gangliosides and related glycosphingolipids on central and peripheral nervous system cell membranes

L Svennerholrn. A K Asbury. R.A Reisfeld. K Saiidhoff. K Suzuki. G l’ettarnanti and G Toffano (Edn 1 Progrerv in Bruin Rulearch. Val. I01 0 1YY4 Elrrvier Science BV. All nghts reserved.

I85

CHAPTER 14

Receptors for gangliosides and related glycosphingolipids on central and peripheral nervous system cell membranes Ronald L. Schnaar, James A. Mahoney, Patti Swank-Hill Michael Tiemeyer and Leila K. Needham Departments of Pharmacology and Neuromence. The Johns Hopkins Unlverrih, School of Medicine, Balnmore, MD 21205. USA

Introduction Advances in glycosphingolipid purification, structural determination and functional analysis have led to a better understanding of the multiple biological roles of this diverse class of molecules. As reviewed elsewhere in this volume, gangliosides and related glycosphingolipids are involved in cell-cell recognition and cellular regulation events in the nervous system and elsewhere (also see Igarashi et al., 1989; Cuello, 1990; Hakomori, 1990; Schnaar, 1991). The molecular mechanisms by which glycosphingolipids control cell behavior are likely to converge on two general motifs (Hakomori, 1990; Schnaar, 1991): Specific binding of cell surface glycosphingolipids to complementary receptors on apposing membranes (trans recognition) and modulation of protein activities in the same membrane (cis regulation). An example of trans recognition is the binding of endothelial cell surface protein E-selectin to complementary fucosylated gangliosides on neutrophils (Brandley et al., 1990). Cis regulation is exemplified by the modulation of epidermal growth factor receptor kinase by ganglioside GM3 and related molecules in the same cell membrane (Hanai et al., 1988). Research in our laboratory focuses on detection and characterization of novel protein receptors for gangliosides and related glycosphingolipids in the nervous system. Our strategy is to identify ganglioside (and related) binding activities using synthetic

glycosphingolipid-based radioligands, to characterize the structural and tissue specificity of binding activities, and to purify the responsible protein receptors. Through these efforts, we hope to help elucidate the detailed mechanisms by which gangliosides and related glycosphingolipids modulate cell behaviors in the nervous system. This chapter reviews our progress towards that goal, including identification of a ganglioside receptor in the central nervous system and a receptor for sulfoglucuronyl glycosphingolipids in the peripheral nervous system.

Glycosphingolipid-based Neoglycoconjugates Neoglycoconjugates are semisynthetic derivatives consisting of a non-glycosylated ‘scaffold’ (e.g. a carrier protein) covalently derivatized with defined carbohydrate structures. Neoglycoproteins consisting of bovine serum albumin (BSA) derivatized with monosaccharide glycosides have been valuable tools for probing vertebrate cell surface lectins (Stahl et al., 1980; Connolly et al., 1983; Lee and Lee, 1992). Likewise, neoganglioproteins, consisting of BSA covalently derivatized with gangliosides or ganglioside oligosaccharides, have been useful in probing for ganglioside receptors on brain membranes. These conjugates have the advantage of presenting the ganglioside oligosaccharide structures in polyvalent arrays which may be critical to detecting high-affinity receptors (Lee et al., 1984; Lee, 1989). At the same

I86

time, the lipid portion is sequestered in covalent association with the carrier protein so that non-specific adsorption and insertion into membranes is minimized. Neoganglioproteins are readily radiolabeled to high specific activity by radioiodination of the carrier. We have used three synthetic strategies to generate the desired conjugates. The first two approaches retain as much of the intact ganglioside structure as possible by removing the original ganglioside fatty acid amide (by strong alkaline hydrolysis; Neuenhofer et al., 1985) and replacing it, via an

amide bond, with a spacer arm terminated in an activated linker moiety (Fig 1). In each case, the activated ganglioside intermediate is purified to homogeneity prior to covalent linkage to the carrier protein. Our initial synthetic scheme (Tiemeyer et al., 1989) used a sulfosuccinimidyl ester as the activating group for attachment to lysine primary amines on a BSA canier (Scheme 1, Fig. 1). While this generated the desired ligand, the active ester was susceptible to hydrolysis during purification of the activated ganglioside intermediate. To solve this problem, we now use a maleimidyl activating group, and link to free

Fig. 1. Preparation of multivalent glycosphingolipid-based derivatives. Three schemes were used to covalently link glycosphingolipids or their oligosaccharides to BSA (a generalized glycosphingolipid is presented at the top of the figure). Scheme I (Tiemeyer et al., 1989): LysoGTlb, prepared by alkaline deacylation of the sphingosine amine of purified glycosphingolipid (Neuenhofer et al., 1985). is reacted with excess bis(sulfosuccinimidy1) suberate (BS’) to generate an active ester intermediate of the lysoglycosphingolipid. The sulfosuccinimidylactivated intermediate is purified from un-reacted BS3 by Iatrobeads chromatography before addition to BSA. Reaction of the sulfosuccinimidyl esters of the intermediate with primary amines on BSA results in formation of stable amide bonds linking the derivatized glycosphingolipid to the protein. Scheme 2: Lyso-GTlb is reacted with a heterobifunctional crosslinking reagent, succinimidyl 4-(Nmaleimidomethy1)cyclohexane-1-carboxylate (SMCC, Pierce Chem. Co., Rockford, IL,USA), and the resulting maleimide-activated intermediate is chromatographically purified. The activated glycosphingolipid is added to BSA containing excess free sulthydryl groups introduced chemically (at lysine amino groups) with the reagent N-succinimidyl S-acetylthioacetate (Pierce Chemical Co.), followed by hydroxylamine (Duncan et al., 1983). Reaction of the N-maleimidyl group with sulthydryls on BSA results in formation of stable sulfate ether bonds linking the derivatized glycosphingolipid to the protein. Scheme 3: Glycosphingolipid is treated with ceramide glycanase (Zhou et al., 1989) to release the oligosaccharide, which is purified chromatographically and added to BSA in the presence of 10% pyridine borane as mild reducing agent (Cabacungan et al., 1982). Spontaneous reductive amination of lysines on BSA with the reducing terminal glucose of the glycosphingolipid results in a stable secondary amine linkage between the oligosaccharide and the protein.

187

sulfhydryls introduced into the BSA camer (Scheme 2, Fig.1). This has led to routine incorporation of = 13 GTlb molecules/BSA. Both of the above schemes use strong alkaline hydrolysis to generate a lysoganglioside starting material, and are therefore inappropriate for glycosphingolipids retaining important alkali-labile groups, such as 0-acetyl groups or sulfates. For these glycoconjugates we use a third method, isolation of the glycosphingolipid oligosaccharide and coupling directly to BSA via reductive amination. This effort has been greatly enhanced by the availability of the enzyme ceramide glycanase (Zhou et al., 1989) which quantitatively releases oligosaccharides from most glycosphingolipids under very mild conditions (Scheme 3, Fig.1). We have used this enzyme to readily generate oligosaccharides from gangliosides

and sulfoglucuronyl glycosphingolipids (see below). The isolated oligosaccharides spontaneously link to BSA under mild conditions in the presence of an appropriate reducing agent (e.g. pyridine-borane complex; Cabacungan et al., 1982). While the resulting conjugate has lost much of the glycosphingolipid structure (the ceramide is removed and the reducing terminal glucose is linearized), it retains the nonreducing saccharides which may be most important for receptor recognition in a trans configuration (see above). The synthesized neoglycoconjugates are purified by gel filtration chromatography and ion exchange HPLC (Tiemeyer et al., 1989), which resolves them from underivatized saccharides or carrier. They are characterized by several techniques including carbohydrate compositional analysis (Table I), and gel

TABLE I Carbohydrate compositional analysis of various neoganglioproteins Neoganglioprotein

sugar (mol/mol)

Sugar/ BSA (moYmol)

Observed ratio (rnoVmol)

Expected ratio (moUmol)

(GTlb),BSA'

NeuAc GalNAc Gal Glc

12.8 4.2 8.7 4.4

3.0

3

2.0

2

NeuAc GalNAc Gal Glc

38.8 12.2 24.5 14.2

3.0 0.9 1.9 1.1

3

NeuAc GalNAc Gal Glc

33.0 13.0 19.1 13.5

3.0 I .2 1.7 1.2

3

(GTlb),,BSAh

(oGT1b), ,BSA'

1 .o

1.o

1 1

1

2 1

1

2 1

Neoganglioproteins were synthesized using one of three methods (see Fig. 1): a

N-sulfosuccinimidyl activation of lyso-GT1b Maleimidyl activation of lyso-GTlb Direct coupling of released GTlh oligosaccharide via reductive amination

In each case, the chromatographically purified conjugate was subjected to acid hydrolysis and the released saccharides were quantitated using a Dionex carbohydrate analysis system (strong cation exchange HPLC separation, pulsed amperometric detection; Lee, 1990).

188

Ganglioside binding activity in the central nervous system

Fig. 2. Neoganglioprotein molecular model. The model represents the relative sizes of covalently bound GTlb molecules (linked as described in Fig.1) and BSA. The approximate size and shape of GTlb were determined using Desktop Molecular Modeler (Oxford University Press, Oxford, UK), while the size of BSA is based on X-ray crystallography studies of the closely related human serum albumin (Carter and He. 1990).

electrophoresis under denaturing conditions. The latter technique reveals that the conjugates migrate less rapidly and more diffusely than the parent BSA. In some cases oligosaccharide-specific reagents (such as labeled cholera B subunit) were used to demonstrate co-localization of glycosphingolipid oligosaccharide and protein on SDS-PAGE. These data demonstrated that the desired covalent conjugate was obtained. Following the nomenclature for neoglycoproteins (Kuhlenschmidt and Lee, 1984), we denote neoganglioproteins as, for instance, (GTlb),,BSA, where the glycosphingolipid designation is in parentheses and the average number of molecules attached per carrier is subscripted. Conjugates are readily radioiodinated for use as radioligand probes. Molecular modeling demonstrates that gangliosides form the major surface structures on the resulting conjugates, even though they constitute a minor portion of the total conjugate mass (Fig. 2). This is because the oligosaccharides (<2 kDa) spread out in space while the BSA carrier protein (>66 kDa) folds into a highly compact structure. Correct models of glycoconjugate-protein interactions must consider this disproportionately large sizehass ratio of complex carbohydrates compared to most proteins (Rademacher et al., 1988).

Our initial efforts to detect ganglioside receptors on brain membranes used (GTlb),BSA conjugates. GTlb was chosen because of its relatively high concentration in brain (Yu and Saito, 1989). If structurally specific receptors exist, their concentration may reflect that of the recognized glycosphingolipid. Furthermore, GTlb can be isolated in sufficient quantities to optimize synthetic techniques, and our laboratory had demonstrated that GT1b immobilized in a surface array supported neuronal cell adhesion

0.0

0

1

2

(C,,,),BSA

3

4

6

e

C o n c e n t r a t i o n (nM)

Fig. 3. Saturation isotherm for '251-(GTlb)4BSA binding to rat brain membranes (Tiemeyer et al., 1989). Brain 'P2' membranes were pretreated with 0.3% sodium deoxycholate to remove endogenous glycosphingolipids. and the residual membrane structures (30 pg original membrane protein per reaction) were collected by filtration and incubated with the indicated concentrations of '251-(GTlb)4BSAin a total volume of I ml of binding buffer (50 mM Hepes pH 7.4, 10 mM calcium chloride, 0.016% Triton X100, I m g / d BSA) for 90 min at 8°C in the presence or absence of 10 pM GTlb. Unbound radioligand was removed by washing the membranes on filters, and bound radioactivity was determined. Specific binding (filled circles) is defined as total binding (squares) minus non-specific binding (triangles, binding in the presence of 10 pM GTlb). Inset: Data from saturation isotherms were transformed by the method of Scatchard (Scatchard, 1949) with the abscissa as bound ligand (pmoUfilter) and the ordinate as bound/free ligand. The apparent single binding site has a K, of 2 nM and a ,B of 20 pmoUmg P2 protein.

189

When Iz5I-(GT1b),BSA was incubated with brain membranes (crude ‘P2’fraction from rat brain), specific binding was not readily apparent. The possibility that ganglioside receptors might be masked by endogenous gangliosides in the preparation led us to test delipidated brain membrane preparations as a source of binding activity. Two methods for delipidation were successful in revealing ganglioside-specific binding activity: (1) mild detergent extraction; and (2) solvent solubilization followed by chromatographic resolution (see below). Mild detergent treatment of the membranes removed gangliosides and other lipids (as well as many membrane proteins) but left detergent-resistant membrane-like structures which were recovered by ultracentrifugation or filtration. When probed with lz5I-(GT1b),BSA, the residual brain membranes demonstrated high affinity and saturable radioligand binding (Fig. 3). Scatchard transformation of the binding data was linear with a K, of 2 nM and a B, of 20 pmoVmg ‘P2’ membrane protein. Inhibition of 1251-(GTlb),BSAbinding to residual rat brain membranes by various lipids (added as 120 1

-9

1

-8

-7

-6

-5

-4

Log lnhlbltor Concrntratlon (M) Fig. 4. Inhibition of ’251-(GTlb),BSAbinding to rat brain membranes by underivatized gangliosides (Tiemeyer et al., 1989). Brain ‘P2’ membranes were pretreated with mild detergent and incubated with 0.5 nM ‘Z51-(GTlb),BSAas described in Fig.3. except that the binding buffer contained underivatized gangliosides at the indicated concentrations. Binding in the presence of added ganglioside is expressed as a percent of the binding measured in the absence of added inhibitor (maximal binding was comparable to that shown in Fig.3). The gangliosides tested were: GD3; W, GM3; 0, GQlb ; A,GTlb ; 0, GDlb; A,GDla ; 0, V.GM2 ; V,GM1 ; GA1.

*,

mixed Triton X- 100 micelles) revealed ganglioside specificity (Fig. 4). The most potent ganglioside inhibitors were all of the ‘lb’ series (GDlb, GTlb and GQlb) with similar IC,, values ( 3100 nM). By comparison, GDla and GD3 were one sixth as potent inhibitors compared to GDlb, although of equal anionic charge. GM1, which varies from GDlb only by the absence of the NeuAca2-8 group was the least potent ganglioside inhibitor with an inhibitory potency <4% that of GDlb. Other anionic or neutral sphingolipids including asialo-GM 1, globotetraosylceramide, sphingomyelin, sphingosine, and psychosine (galactosylsphingosine) were inefficient at blocking Iz5I-(GT1b),BSA binding. Although selected phospholipids also inhibited with IC,, values as low as 0.5 pM, hnetic analyses of the inhibition of Iz5I-(GTlb),BSA binding by gangliosides and phospholipids demonstrated that ganglioside inhibition was competitive and reversible while phospholipid inhibition was non-competitive and irreversible (Tiemeyer et al., 1989). These data demonstrate that the 1251-(GT1 b),BSA binding protein on brain membranes is selective for gangliosides carrying a defined oligosaccharide structure. This conclusion was supported by inhibition with the oligosaccharide released from GTlb (K, = 8 pM). In contrast, the oligosaccharide from GM1 inhibited only ~ 2 0 %at 10 pM (the highest concentration tested). Galactose, glucose, N-acetylgalactosamine, N-acetylglucosamine, Nacetylneuraminic acid, lactose, N-acetyllactosamine, and N-acetylneuraminyllactose were non-inhibitory at 5 mM. The relatively poor inhibitory potency of GTlb-oligosaccharide (8 pM) compared to GTlb (100 nM added as a mixed detergent micelle) supports a role for the ceramide portion of the ganglioside in receptor binding. The ceramide could act to enhance self-association of GTlb into multivalent arrays, to orient the oligosaccharide appropriately, or as part of the binding determinant. Data to address these alternatives was obtained by comparing the relative inhibitory potencies of GTlb and various GTlb neoganglioproteins (Table 11). (GTlb),BSA and (GTlb),BSA were an order of magnitude less potent (per saccharide unit) in binding to this receptor than

190

TABLE I1 Inhibition of 1251-(GTlb),BSAbinding to rat brain membranes by various GTlb conjugates - Effect of valency and molecular form Ganglioside conjugate

GTlb (lipid in Triton micelles)

Valency

Kl conjugate (nM)

per saccharide (nM)

1-2

I00

100-200

81 46 2.6 7.2

92 10 58

(GT1b),BSA (succinimidyl ester method)

K,

81

GT1b-oligosaccharide

1

8OOO

8OOO

(oGT1h)BSA (reductive amination)

11

60

660

~~

Inhibitory potencies of GTlb, the oligosaccharide derived from GTlb (oGTlb), and various GTlb related neoganglioproteins were tested by performing inhibition titrations of '2SI-(GTlb),BSAbinding to rat brain membranes as described in Fig. 4. The assay was conducted in the presence of 0.016% Triton X-100, resulting in approximately equimolar GTlb to mixed micelle concentration ratio. Due to variability in Triton X-100critical micelle concentration and micelle size, a reasonable range of valency for GTlb 'lipid in Triton micelles' is given.

(GT1b),BSA, demonstrating the importance of multivalency (although higher order GTl b conjugates were not better than (GTlb),BSA). Likewise, GTlb oligosaccharide was an order of magnitude less potent as inhibitor (per saccharide unit) than a multivalent conjugate consisting of 1 1 GTl b oligosaccharidesiper BSA, linked via reductive amination (see Fig. 1). However, multivalent oligosaccharide conjugate was far less potent as an inhibitor than multivalent glycosphingolipid. These data suggest that both multivalency and the structural contribution of the ceramide are important to ganglioside receptor recognition. Since gangliosides can cluster in the plane of a cell membrane, these factors may play a role in ganglioside recognition in vivo. To gather information about the potential functions of this ganglioside binding activity, we determined its histological distribution and ontogeny in rat brain (Tiemeyer et al., 1990).Tissue and regional distribution studies revealed marked specificity for CNS tissue and differences among the different brain regions

rd cord &&I Stem ~~

0

2

4

6

8

10

12

(pmol/mg protein) ( G T ~ ~ ) ~ Binding BSA Fig. 5. Tissue and regional distribution of 1251-(GTlb)4BSA binding (Tiemeyer et al., 1990). Membranes were prepared by differential centrifugation of crude hornogenates from whole rat brain, peripheral nerve, liver, or the indicated brain regions. All membranes were pretreated with mild detergent under optimal conditions, collected by filtration, incubated with 0.5 nM 1251-(GTlb)4BSA, and specific binding determined as described in Fig. 3. The means of the specific binding activities per mg protein were calculated using multiple protein concentrations within the linear binding range.

191

Fig. 7. Histological localization of 1Z51-(GTlb),BSAbinding to rat spinal cord and peripheral nerve structures (Tiemeyer et al., 1990). Transverse serial sections of frozen rat spinal cord were prepared, overlaid with buffer containing '251-(GTlb)4BSA. and processed for autoradiographic determination of specific binding as described in Fig. 6. The resulting autoradiographic image (center panel) is presented along with an adjacent section treated identically except for a pre-incubation with 10 pM ganglioside GTlb as inhibitor (bottom panel). For comparison, another adjacent section stained with cresyl violet is presented (top panel). Note that the white matter within the spinal cord was heavily labeled, while dorsal and ventral spinal roots and the dorsal root ganglion (at the right of the section) did not bind radioligand. b),BSA binding to Fig. 6. Histological localization of 1251-(GT1 coronal rat brain sections (Tiemeyer et al., 1990). Serial coronal sections were prepared from frozen brain, placed on gelatin-coated slides. lightly fixed, treated with 0.3% sodium deoxycholate to remove endogenous lipids, air dried, and overlaid with buffer containing 5 nM 12'I-(GTlb)4BSA.After 90 min at 8°C. unbound radioligand was removed by washing, the slides dried, and subjected to auroradiography. The autoradiographic image (lower panel) is presented along with a cresyl violet stained adjacent section (upper panel) for comparison. Note the absence of labeling of cerebellar grey matter, while the white matter tracts within the cerebellum (and in the underlying medulla) are heavily labeled. An adjacent section incubated with radioligand in the presence of 10 pM GTlb as inhibitor was unlabelled (data not shown).

(Fig. 5). Rat brain membranes supported >lO-fold higher 12SI-(GTlb)4BSA binding compared to liver membranes, and binding of the ligand to peripheral

nerve membranes was insignificant. Within the CNS, areas rich in white matter supported more binding than those rich in gray matter. These data suggested that this receptor might be associated with myelinated fibers rather than neuronal soma or synaptosomes. Subcellular fractionation confirmed that ganglioside binding activity is found in myelin, with an 8-fold enrichment of '251-(GTlb),BSA binding to myelin (per mg membrane protein) when compared to crude 'P2' membranes. Ontogenic studies further supported the association of this activity with myelin; IZ5I(GTlb),BSA binding appeared first at 15 days postnatal and was half-maximal at = 50 days, paralleling the appearance of myelination. Binding of IZ5I(GT1b)4BSA directly to sections of adult rat brain

192

revealed predominant ganglioside-specific binding to white matter tracts throughout the brain. Even in the gray matter-rich cerebellum, which has relatively little neoganglioprotein binding (cf. FigS), the white matter tracts are clearly labeled (Fig. 6). Notably, spinal cord white matter displayed substantial binding while attached dorsal root ganglia, as well as dorsal and ventral roots were unstained (Fig. 7). Therefore, the ‘1b’-directed binding activity we have characterized is specific for CNS myelin, which is elaborated by oligodendrocytes, and is absent from PNS myelin, which is elaborated by Schwann cells. We refer to this activity as the CNS ‘myelin ganglioside receptor’ (MGR). The observation that myelin is very poor in the ‘Ib’ gangliosides while the axons

14

0 Receptor Binding 0 Protein

1

2

3

4 6 8 7 8 Fraction Number

8

they ensheathe are rich in GDlb and GTlb have led us to hypothesize that the MGR is involved in ‘trans’ recognition between oligodendroglia and axons. Further testing of this hypothesis awaits immunological and molecular tools, the development of which are the focus of current research. A major current goal of our studies is the purification of the MGR. Solubilization and chromatographic resolution of the highly hydrophobic proteins of myelin are technical challenges. Recent studies (Diaz et al., 1991) demonstrate total solubilization of myelin membranes in tetrahydrofuradwater mixtures and bulk resolution of solubilized proteins from extracted lipids by LH-60 size exclusion chromatography. Successful application of this method to the myelin ganglioside receptor is shown in Fig. 8. Binding activity (measured using a receptor-ligand complex precipitation assay) is low in THF/water extracts of myelin membranes (data not shown), presumably due to the presence of inhibitory lipids. However, full activity is revealed in the protein fraction after LH-60 chromatographic resolution from lipids. A comparison of the THF-solubilized activity

10

Fig. 8. Solvent-solubilization of the myelin ganglioside receptor: resolution from endogenous lipids via LH-60 column chromatography. Myelin prepared from rat CNS (Norton and Poduslo, 1973) was collected by ultracentrifugation and nearly completely solubilized in 80% tetrahydrofuran, 0.1% trifluoracetic acid (Diaz et al., 1991). The resulting extract was subjected to LH-60 size exclusion chromatography in the same solvent to resolve myelin proteins from lipids. Protein was detected in each fraction by scanning UV spectroscopy, while lipids were detected by thin layer chromatography using a general lipid stain (technique as in Fig. 10). Ganglioside receptor activity was determined by incubating aliquots of each fraction with 50 pM 1251-(GTlb)l,BSA,essentially as described in Fig.3, except that the reaction was terminated by addition of an equal volume of 15% polyethylene glycol in 50 mM Hepes (pH 7.4) to precipitate the receptor-ligand complex, which was then collected on glass fiber filters. Background binding (in the presence of 10 pM GTlb) was generally
Fig. 9. Ganglioside binding activity in myelin from various species. Myelin was prepared using CNS tissues from the species indicated. The membranes were extracted and the solubilized proteins resolved from lipids by LH-60 column chromatography (see Fig. 8). Protein-containing, lipid-depleted fractions were combined and aliquots removed for determination of protein (using a dye binding assay) and specific radioligand binding (using 60 pM llSI(GTlb),,BSA) as described in Fig. 8. The mean of the specific binding per pg protein was calculated from data using multiple protein concentrations within the linear binding range.

193

to that in mild detergent-treated membranes demonstrates similar binding kinetics and inhibition by gangliosides (data not shown). Recovery of total myelin proteins free of lipids aided in the comparison of myelin ganglioside receptor activity from various species. Myelin was prepared from various vertebrate species (including human), solubilized in THF/water, the proteins separated from lipids by LH-60 chromatography, and MGR activity measured. Significant ganglioside-specific binding of 12SI-(GTlb),,BSAwas detected in all species tested, although at different levels (Fig. 9). Binding activities from all species were similarly inhibited by GTlb (Ks0values = 100 nM), and GM1 was a markedly less potent inhibitor in all cases (data not shown). The ‘1b’-directed receptor described here is dis-

tinct from the major myelin protein, myelin basic protein (MBP), which has been reported to bind to ganglioside GMl (Yohe et al., 1983). Total brain membranes from normal and ‘shiverer’ mutant mice had identical levels of 1251-(GTlb),,BSA binding (data not shown). This is notable since ‘shiverer’ mice carry a large deletion mutation in the MBP gene (Campagnoni and Macklin, 1988). Current efforts are directed at utilizing chromatographic and molecular biological techniques to purify the MGR.

Sulfoglucuronyl glycosphingolipidbinding activity in the peripheral nervous system A striking observation from our studies on GTlbBSA binding activity is its absence from the peripheral nervous system (PNS), where myelination of

Fig. 10. Adhesion of primary Schwann cells to TLC-resolved SGNL-lipids (Needham and Schnaar, 1990). Total polar lipids from dog sciatic nerve, isolated using the method of Svennerholrn and Fredman (1980). were applied to DEAE-Sepharose in organic solvent and anionic lipid classes eluted stepwise using ammonium acetate in methanol (Magnani et al., 1987). Each of six fractions was subjected to HPTLC in chloroform/methanol/0.25% aqueous KC1 (60:35:8) on replicate plates. As indicated, replicate plates were treated as follows: General Lipid Stain was via copper sulfate/phosphoric acid char (Yao and Rastetter, 1985). Ganglioside Stain was via the resorcinol reagent of Svennerholm (1963) as detailed previously (Dahms and Schnaar, 1983). Cell binding was performed in specially designed TLC adhesion chambers as previously described (Swank-Hill et al., 1987). Rat primary Schwann cells were isolated, cultured, and metabolically radiolabeled with inorganic phosphate (Needham et al., 1987). The labeled cells were collected by mild tqpsinization, placed in contact with the TLC plate for 30 min at 37°C. then non-adherent cells removed by centrifugation. Adherent cells were fixed in place and detected by autoradiography . HNK-I Stain refers to immunochemical detection using a mouse monoclonal antibody which recognizes SGNL lipids, and was performed as described previously (Magnani et al., 1987). Su(fare Ester Stain was via dye binding with Azure A (Iida et al., 1989). Phosphofipids Szuin was via molybdenum blue reagent (Alltech Assoc, Deerfield, IL,USA). Note that Schwann cells bound selectively to the major species of the SGNL lipid, which is a minor component of total PNS lipids (general stain) but are readily detected (along with other minor members of this structural family) by the intensely staining HNK-I monoclonal antibody.

194

axons is performed by Schwann cells rather than oligodendroglia. Demyelinating peripheral neuropathies (Quarles et al., 1986) appear in individuals producing monoclonal antibodies reactive with an unusual class of acidic lipids found predominantly in peripheral nerves, the sulfoglucuronyl neolactosylceramides (SGNL-lipids). These glycosphingolipids have neolactotetraosyl or neolactohexosyl cores substituted with a 3-0-sulfated glucuronic acid moiety on the 3-position of the non-reducing terminal galactose (Chou et a]., 1986; Ariga et al, 1987). To test the possibility that SGNL lipids serve as recognition molecules in PNS myelination, we first tested their ability to support intact Schwann cell

0

200

400

['esI-SGNL-BSA]

800

recognition using direct cell adhesion to TLCresolved glycosphingolipids (Needham and Schnaar, 1990; Needham and Schnaar, 1991). Radiolabeled primary Schwann cells readily bound to small amounts of SGNL lipids from peripheral nerve when resolved on TLC plates, while gangliosides were completely non-supportive of adhesion (Fig. 10). Direct probing and characterization of a potential SGNL receptor on PNS membranes was accomplished (Needham and Schnaar, 1993) by synthesizing a radioiodinated neoglycoconjugate consisting of SGNL oligosaccharides covalently attached, via reductive amination, to BSA (using Scheme 3, Fig. 1). The structure of the resulting 1251-(SGNL),BSA was confirmed by carbohydrate analysis and SDSPAGE electrophoresis, with detection using a mouse monoclonal antibody, HNK-1, specific for the sulfoglucuronyl neolacto structure (Ilyas et al., 1990).

800

(pM)

Fig. 11. Saturation isotherm for '251-SGNL-BSA binding to rat PNS myelin. Rat PNS myelin membranes were isolated from rat sciatic nerve by homogenization and differential centrifugation (Oulton and Mezei, 1976). An aliquot of membrane suspension (containing 0.25 pg membrane protein) was incubated with the indicated concentrations of lZSI-SGNL-BSAin 0.25 ml of binding buffer (50 mh4 imidazole acetate (pH 7.4). 0.032% Triton X-100, 1 mg/ml BSA, 20 mM CaCI,). After 30 min at 4°C membranebound radioligand was separated from unbound ligand by filtration (open circles, total bound). Ligand that was non-specifically bound (open triangles), determined in the absence of tissue, was subtracted from the total bound at each ligand concentration to calculate specific bound (filled circles). Values are plotted versus the concentration of free lZSI-SGNL-BSAcompetent to bind PNS myelin membranes (the percent of radioligand which bound to a saturating amount of membrane protein). The solid line was generated by a non-linear fit of the specific binding data using a single site model and yielded the indicated apparent dissociation constant (K,) and total receptor concentration (B,) values (Needham and Schnaar, 1993).

0.0

0.5

1 .o

1.5

2.0

2.5

PNS Yyelin Membranes (pa protein)

Fig. 12. Binding of I2'I-SGNL-BSA to rat PNS myelin: Effect of calcium ions. Rat PNS myelin membranes at the indicated protein amounts were incubated with 0.5 nM 12sI-SGNL-BSAfor 30 min at 4°C in a total volume of 250 p1 binding buffer in the presence or absence of 20 mM CaCI,, and membrane-bound ligand was separated from free ligand by filtration. Calcium-specific binding (closed circles) was calculated by subtracting non-specific binding (open triangles, binding in the absence of CaCI,) from total binding (open circles, binding in the presence of 20 mM CaCI,). Nonspecific binding was essentially identical when defined as binding in the absence of calcium ions, in the presence of calcium plus 10 pM SGNL lipid as inhibitor (see Table III), or in the absence of tissue (see Fig. 1 I ) (Needham and Schnaar, 1993).

195

TABLE Ill

PNS Membranes

CNS Membranes

Inhibition of tz'I-SGNL-BSA binding to PNS Myelin by glycosphingolipids Glycosphingolipid ( n )

SGNL-lipid. bovine ( 5 ) SGNL-lipid, human (7) Desultated" SGNL-lipid. human (2) Methyl ezter of desulfated SGNL-lipid" ( I )

0.99 (0.55-1.2) 0.48 (0.3-0.6) 1.5 (1-2) >25

Sulfatide (4)

8.2 (4-12)

sialylneolactotetraosylcermide ( 1 ) 2.3 sialyl Lewis x glycolipid ( I ) 2.6 sialyl Lewis x glycolipid ( I )

40 >I0 >10

GT I b (4) GDla (2) GDlb ( 2 ) GMl ( 1 )

33 (6-64)

Globotetraosylceramide (1 ) Galactosylceramide (1)

>I00 >I00

58 (15->100) >I00 >I00

Binding studies were performed as described in Fig. 12 using 7.5 pg membrane protein per reaction, except that membranes were preincubated (30 min, 4°C) with various concentrations of the indicated lipid inhibitors. Specific binding at each inhibitor concentration was determined using calcium-free buffer to define nonspecific binding. The mean (and range) concentration of each inhibitor resulting in 50% inhibition of specific '*'I-SGNL-BSA binding was determined graphically from the indicated number ( n ) of independent binding experiments performed in triplicate (Needham and Schnaar, 1993). Prepared as described previously (Ilyas et al., 1990).

When peripheral nerve membranes (predominantly PNS myelin) were probed with lZsI-SGNL-BSA,saturable binding was detected (Fig. 11). Unlike the CNS myelin ganglioside receptor, SGNL receptor was absolutely dependent on calcium ions for binding (Fig. 12), with maximal binding in the presence of 10 mM calcium. Of various lipids tested as inhibitors of 12SI-SGNL-BSAbinding to PNS membranes (Table III), SGNL lipid itself was the most potent (ICs0< 1 pM),while gangliosides were <3% as effective. Of the glycosphingolipids tested (Table 111) only

myelin axolemma

, , myelin synaptosomes

Fig. 13. Tissue and subcellular specificity of '"'I-SGNL-BSA binding. Leji panel: Rat peripheral nerve endoneurium homogenate was fractionated by differential centrifugation and discontinuous sucrose gradient centrifugation to separate PNS myelin from axolemma (Oulton and Mezei, 1976). Right panel: Rat brain homogenate was fractionated similarly to isolate CNS myelin and synaptosomes (Gray and Whittaker, 1962). Specific binding of 0.5 nM '2'I-SGNL-BSA to aliquots of these fractions containing 1 pg membrane protein was performed as described in Fig. 12.

sulfatide was within an order of magnitude as effective as SGNL lipid as an inhibitor. Structural studies using SGNL lipid derivatives were revealing, in that desulfation (to generate IV3GlcA-nLc,Cer) resulted in only a modest (3-fold) decrease in inhibitory potency. In contrast, methyl esterification of the desulfated derivative (at the GlcA 6-position) eliminated inhibitory potency (Table 111). These data indicate that the carboxylic acid on the terminal glucuronic acid is a major determinant for SGNL binding to its PNS membrane receptor, while the 3-0-sulfate group contributes but is not critical to binding. It is interesting to note that non-sulfated forms of the SGNL. lipid have been found broadly distributed phylogenetically (Dennis et al., 1988), and may be more ancient than gangliosides or SGNL lipids. When PNS membranes are separated into axolemma1 (denser membranes) and myelin fractions, the activity fractionates with the myelin (Fig. 13), suggesting a possible role in 'trans' interactions between Schwann cells and axolemma. Furthermore, limited tissue distribution studies suggest that SGNL receptor serves a specific role in the PNS, in that binding

196

activity is essentially absent from CNS membranes, whether tested with crude ‘P2’, purified myelin, or synaptosomal membranes.

Conclusions and perspectives Through use of semi-synthetic glycosphingolipidbased neoglycoconjugates, which combine multivalence, sequestering or removal of the lipid moiety, and facile radioiodination, we have been successful in demonstrating unique binding activities for gangliosides and sulfoglucuronyl glycosphingolipids in the CNS and PNS respectively. Although identification of the functional roles of these glycosphingolipid-specific binding activities await further studies, their differential tissue and membrane distribution suggests that they may be involved in ‘trans’ interactions between myelinating cells and axons.

Acknowledgements The work described was supported in part by National Institutes of Health (NIH) grant HD14010, Multiple Sclerosis Society grant RG-2223-A- 1, and NIH training grants MH18030 (to MT & LKN) and GM07626 (to JAM).

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