Gangliosides

Gangliosides

I99 Wiegandt (ed.) Glycolipids 0 I985 Elseoier Science Publishers B. K (Biomedical Division) CHAPTER 3 Gangliosides HERBERT WIEGANDT Department of ...

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I99

Wiegandt (ed.) Glycolipids 0 I985 Elseoier Science Publishers B. K (Biomedical Division)

CHAPTER 3

Gangliosides HERBERT WIEGANDT Department of Biochemistry, School of Medicine, Philipps University, Marburg an der Lahn, F.R.G.

I . Introduction Gangliosides are distinguished from other glycosphingolipids in that they contain one additional characteristic carbohydrate constituent, namely sialic acid *. It was indeed this sugar amino-acid that by its colour formation upon heating with p-dimethylaminobenzaldehyde/HCl (Ehrlich’s reagent) or orcinol/H,SO, (Bial’s reagent) led to the detection of these lipids [l]. In 1935, E. Klenk characterized a new type of acidic glycolipid, “substance X”, from the brain of patients suffering from amaurotic familial idiocy [2]. G. Blix, in 1938, recognized that such a glycolipid could also regularly be found in a normal brain [3]. Further work from the laboratory of Klenk in 1942 showed that “substance X” was concentrated in the brain grey matter, and it was suspected of being localized in ganglia1 cells. “Substance X” was therefore termed a “Ganglioside” [4]. Work on the characterization of gangliosides and the elucidation of the chemical structures of many of their components was mostly performed during the sixties in the laboratories of Ernst Klenk, Richard Kuhn and Lars Svennerholm (for reviews, see Refs. 5, 200). Since their discovery, the gangliosides have for several reasons elicited much interest. The concentration of complex gangliosides in membranal elements of the brain is suggestive of a functional role in the central nervous system. Furthermore, certain lipidoses that affect the nervous system, such as Tay-Sachs disease are characterized by a ganglioside accumulation within cells of the brain. An additional indication for an involvement of gangliosides in nervous function, was their interaction with the neuro-toxin of Clostridium tetani. This toxin is specifically fixed by brain tissue, a property believed to relate to its ganglioside content [6,7]. During the elucidation of the chemical nature of the sialic acids, it also became evident that they have numerous specific biological properties for which gangliosides frequently are the glycoconjugate carrier. * Sialic acid is the generic term designating all N-acylneurarninic acids.

200

Gangliosides also occur outside the nervous system. It is recognized now that they are important constituents of the surface membrane of most, perhaps all, cells of animals that belong to the phyla of the Deuferosfomiu. Before embarking on current concepts of the biological behaviour and significance of gangliosides, it will be necessary first to describe their chemical composition and physicochemical properties.

2. Chemistry, physics and methods of preparation and analysis 2.1. GENERAL PRINCIPLES OF CHEMICAL CONSTITUTION

Gangliosides are acidic glycosphingolipids due to their content of one or more sialic acid residues in the carbohydrate moiety. In common with other glycosphingolipids they consist of a long-chain base, a “sphingoid” that is linked to fatty acid by an amide bond thus forming the lipophilic “ceramide” portion. The carbohydrate is linked to the sphingoid’s primary hydroxyl by a glycoside. Acyl. N H

I

CH,. (CH2 )12.CH=CH .CH(OH) * C H * C H ~ . O * G I ~ C O S ~ I

Similar to other lipids, the lipophilic portion of the ceramide shows microheterogens ity with regard to the sphingoid and the fatty acid composition [8,9]. In most instances ganghoside components are distinguished and characterized on the basis of their sialo-oligosaccharide (for review, see Ref. 10). In general, glycosphingolipids can be classified into several series that may reflect their presumed biogenic relations, i.e., the sequential addition of monosaccharides to ceramide, and a growing sugar chain via glucosylceramide or galactosylceramide (Fig. 3.1). Of all glycosphingolipids indicated in Fig. 3.1, sialic acid-substituted components with one or two hexose units have been structurally characterized carrying glucose, galactose or lactose. All higher gangliosides so far identified are derived either from the gunglio-, lacto- or globo-series. The ceramide moiety of gangliosides frequently is similar to the fatty acid and sphingoid composition of other neutral glycosphingolipids that are derived from the same cellular source (for reviews, see Refs. 70, 71). It has, however, also been observed that the ceramide composition of gangliosides differs from that expected on the basis of the biogenic relation of their carbohydrate portion. An example of this is the fatty acid composition of ganglioside G,,J (“ hematoside”) which may be quite different from that of lactosylceramide of the same tissues [72], or else that of the human erythrocyte gangliosides IV3NeuAcnLc4Cer as compared to IV6NeuAc-nLc4Cer [582]. Similarly, with increasing sialic acid content, brain gangliosides show an increasing proportion of eicosa-sphing-4enine (Cz0:,)over sphing-4-enine (C18:l)[73-751. The way in which the relation between the composition of the ceramide to that of

201 INVERTEBRATES

/

/ '

Man-Man-Glc - C e r

Man-Glc- C e r

GIc-Cer

/

GlcNAC-Man-GIC-Cer

GaINAc-Gal-GIc-Cer /Gal-Gal-Gal-Glc-Cer Cer

(lactosyl-Cer )

~GalNAc-Gal-Gal-Glc-Cer

F l

GlcNAc-Gal-Glc-Cer VERTEBRATES

Gal -Cer-Gal-Gal

-Cer (galabiosyl -Cer )

Fig. 3.1. Carbohydrate series as a base for classification of glycosphingolipids.

the carbohydrate portion is regulated is as yet unknown. The presence of sialic acid in ganglioside is an indication that these lipids are typical cell surface constituents (for sialic acid reviews, see Refs. 11, 12). Whereas free sialic acid in aqueous solution is present as its P-anomer, in gangliosides it is linked by an a-ketoside [13-151. With the exception of gangliosides discovered in starfish [16], sialic acid substitution always takes place in branching or terminal positions of the oligosaccharide. Sialic residues in ganglioside may form lactones under acidic conditions. Such lactone formation occurs with particular ease in the terminal sialic acid that is linked to another sialic acid by an a2 + 8-ketoside [16]. It is tempting to speculate that such lactonization-delactonizationof gangliosides may also occur under in vivo conditions. This would result in changes of electric charge of membranes that may be of functional significance. This speculation becomes all the more plausible since it was discovered that, in the brain, some sialic acid residues of gangliosides can be reduced by sodium borohydride as would be expected for lactone linkages [457]. The sialic acid of all gangliosides of human origin is N-acetylneuraminic acid or 9-0-acetyl-N-acetyl-neuraminicacid * [18]. The brain gangliosides of other vertebrates, except for trace amounts of N-glycolylneuraminic acid [19], also contain N-acetylneuraminic acid or N,O-diacetylneuraminic acid [20,615]. Teleost fish brain gangliosides are particularly rich in the latter diacetyl-derivative [Zl]. * There is some immunological indication that N-glycolylneuraminic acid might be a tumor-associated antigen in humans I458.6761.

202 Interestingly, the gangliosides of the bovine neurohypophysis, that in their component distribution are similar to those of the cerebral cortex, contain a high proportion of N-glycolylneuraminic acid [228]. This sialic acid is otherwise more typical for gangliosides met outside the central nervous system. In particular, gangliosides of horse and cat erythrocytes are rich in N-glycolylneuraminic acid. It appears that N-acetyl- and N-glycolylneuraminic acids can replace each other since, structurally, no positional preferences have yet been observed. The component profile of the gangliosides generally is quite typical for their orign, and there are certain trends for the occurrence of gangliosides containing lactose or those of the gungfio-, gfobo- and lucto-oligosaccharide series. But insufficient data have been recovered to obtain a clear picture of the biological significance of a particular ganglioside pattern. As an example of the complexity of their occurrence, one may compare gangliosides of the central nervous system and those of the red blood cells of different animals. Whereas brain gangliosides of vertebrates are almost exclusively of the gungfio-series, those of erythrocytes may vary considerably depending on the species. Some of the latter gangliosides may contain galactose (mouse), others lactose (man, pig, horse, cat, dog), or they may belong to the gunglio-series (rat), the fucro-series (cattle) as well as to the gfobo-series (man) [636,637]. 2.2. CHEMICAL COMPOSITION

Gangliosides of simplest chemical structure, G,,J and G,,,l, derived from glucosylor galactosylceramide (cerebroside) are NeuAca2-6Glcp-Cer and NeuAca2-3GalplCer for the gametes of the sea urchin [23]. The latter ganglioside, however, may not be restricted to this animal, since in one report it was also described as occurring in pig platelets [24]. Ganglioside Ggall,besides G,,,l, was identified as a typical and major component of oligodendroglial myelin of the brain [25,28]. Apart from human and chicken brain myelin, G,,,l, besides G,ril, also was detected as a major ganglioside of mouse erythrocytes [29], chicken-embryonic liver [459] and chicken egg yolk [30]. In the latter material, Gg,,l occurs together with the next higher, the lactose-derived gangliosides, G ,,J, NeuAccu2-3Galpl-4Glc/3-Cer and G lac 2, NeuAca2-8NeuAca23Gal/ll-4Glc/l-Cer [30]. Gangliosides G with one (G J), two (G,,,2) or three (Gl,,3) sialic residues, are among the most abundant extraneural gangliosides in vertebrates. In brain, however, where higher hexosamine-containing gangliosides predominate, G laclis oniy a minor constituent [31-34]. Ganglioside G luul with predominantly N-glycolyl-neuraminic acid was isolated in 1951 from horse erythrocytes by Yamakawa and Suzuki [35]. This was the first ganglioside extracted from extraneural material. In order to distinguish it from the brain “gangliosides”, it was named “ hematoside”. Hematosides G lac indeed are the major ganglioside components in erythrocytes of many animal species [36]: these include man [37], rabbit [42], cattle [39], giant panda

203 [36], horse [35], and the Cunidue, dog [40],jackal, dingo and racoon dog with Glacl, as well as the Fefidue, cat [38,42], lion and the closely related hyaena [36] with G,,,2. The latter ganglioside G,,,2 also occurs as a major component of mammalian retinal gangliosides [234,44]. Except for hematoside from human erythrocytes, with only N-acetylneuraminic acid, the gangliosides G,,J and Gl,,2 from other mammalian red blood cells may also contain N-glycolyl-neuraminic acid. The occurrence of these two sialic acids, NeuAc and NeuGc, shows a typical species and interspecies distribution: horse, cat (except Persian cat), racoon dog and giant panda hematosides contain exclusively N-glycolylneuraminic acid. In the european dog the hematoside is of the N-acetyltype, whilst in some oriental dogs it is N-glycolyl-hematoside [36]. Gangliosides from the invertebrate starfish Asterina pectinifera also contain ceramide-linked lactose, substituted by N-glycolylneuraminic acid residues [45-471. In this instance, however, the sialic acid carries additional carbohydrate- and 0-methyl residues, i.e., Arab1-6Galfil-4[ 80Me]NeuGcol2-3Gal~1-4Glcfil-Cer

Ara~l-6Gal~1-4NeuGca2-3Gal~l-4Glc~l-Cer and Arab1 -6Ga1/31-4[GalPl-8]NeuGca2-3Gal~l-4Glcfi-Cer.

The “classical” gangliosides, i.e., the predominant species in the brain of higher animals (deuterostomia), contain carbohydrate moieties of the gunglio-series: Gangliotriaose, GalNAc/31-4Gal~1-4Glc Gangliotetraose, Gal~1-3GalNAc~l-4Gal~l-4Glc Gangliopentaose, GalNAc~l-4Gal~1-3GalNAc~l-4Gal/31-4Glc

In this oligosaccharide series, substitution with single or multiple, a24inked sialic acids takes place at the galactose in 3-position and/or of the N-acetylgalactosamine residues in 6-position. Sialic acid residues are linked to one-another by an a2-8 ketoside (Fig. 3.2). Additional substitution by fucose may occur in 2-position of terminal galactose of the gangliotetraose (see Table 3.2). Other gangliosides, discovered in the fat body of frogs are derived from monosialogangliotetraosylceramide by substitution of the terminal galactose residue in 4

--

pent tet

*

P

g

l

tri lac c

GolNAc-Gal-GolNAc-GoI- Glc- C e r

I

NeuAc N~uAc

I I NeUAC (b)

2b. 3b 4 b

NeUAC I

3 c . 4c

NeuAc ( a )

(C)

1. 2a

3a

5c

Fig. 3.2. Structure of gangliosides of the gmgh-series.

204 3-position by mono-, di- and trigalactopyranosyl units [53,54] (see Table 3.2). A pentasialogangliotetraosylceramide,isolated from fish brain, is the most highly sialylated ganglioside structurally characterized thus far [ 100,565]. In this species the major tetrasialoganglioside has the structure 1V monosialo-,I1 trissialoganghotetraosylceramide (i.e., “C” class in Fig. 3.2). Different from fish brain, the major tetrasialoganglioside from human and chicken brain is a IV 3bisialo-,I13bissialogangliotetraosylceramide(class “B’in Fig. 3.2) [56-581. The gangliosides of the gunglio-series are listed in Table 3.2. A ganglioside discovered by Watanabe et al. [59] in human erythrocytes has the Since it contains Nunique structure, NeuAccu2-3Gal~l-3GalNAc~l-4Gal~l-Cer. acetylgalactosamine substituted in 3-position by galactose, it shows some similarity to gangliosides of the gunglio-series. The gangliosides of the lacto-series occur more typically in extraneural sites. They contain, as neutral carbohydrate core unit, a lactoneotetraose linked to ceramide, [48,59,60-62,461,462,464,6541. i.e., Gal~l-4GlcNAc/3l-3Gal/3l-4Glc~l-Cer Further gangliosides of the lucto-series are derived from the core unit of lactoneotetraose extended with N-acetyl-lactosamine residues in the 3-position of the terminal galactose, in a linear position, to lactoneohexa-, octa-, etc., osylceramide [63]. Additional LacNAc residues are found branching in 6-position of galactose as, for instance, in lactoneo-IV6kladohexaosylceramide * or lactoneo-IV6kladooctaosylceramide of human red blood cells [64-67,636,637,5951: Gal/3l+ [4GlcNAc/31+ 3Gal/31+ ] , 4 G l c ~ l +Cer (lactoneotetra-,hexa-, octa-, etc., osylceramide)(631 Gal/3lJGlcNAc/31\ Gal B1-4Glc/31-Cer Galal-4GlcNAc/31/

(lactoneo-I16kladohexaosylceramide) [64] *

Gala1 --4 4GlcNAcjIl\ Gala1 + 4GlcNAc/31/

Gal/31-4GlcNAc/31-3Gal/31-4Glc/31-Cer ( lactoneo-IV6kladooctaosylceramide) [69,595,637]

By analogy to the gangliosides of the gunglio-series, those of the lucto-series may in addition to sialic acid, carry fucose residues at branching or terminal positions e.g., as in human kidney ganglioside: IV3NeuAc-,1113Fuc-nLc,Cer(681 or in a branched-chain fuco-ganglioside identified in human erythrocytes: V13NeuAc-,

IV6[Fucal-2Gal/31-4GlcNAc]-nLc,Cer.

A ganglioside of the lucfo-series with a P-N-acetylgalactosamine was discovered in [59]. human erythrocytes, i.e., IV3[NeuAccu2-3GalNAc~l]-nLc,Cer Gangliosides of the globo-oligosaccharide series have recently been discovered.

* This

oligosaccharide was named lacto-“nor”-hexaose. “Nor” the antipode of “homo”, however, designates a compound differing from the parent term by a minus of one carbon unit. Therefore it is suggested that instead of “nor” to apply the syllable “k1ado”-branch, (twig, greek, ~ X d l 8 0 l )for branched chain oligosaccharide isomeres, i.e., lacto-klado-hexaose.

205 From chicken skeletal muscle [462] or human teratocarcinoma cells [672] gangliosides were isolated that contain one or two sialic acid residues linked to globopentaose, i.e., NeuAccu2-3Gal~l-3GalNAc~l-3Galc~1-4Gal~l-4Glc~l-Cer and NeuAccu2-8NeuAccu2-3Gal~l-3GalNAc~l-3Galal-4Galpl-4Glc~l-Cer. A ganglioside from rat intestinal nonepithelial tissue has an isoglobopentaose as neutral carbohydrate core, i.e., NeuAca2-3Gal/3l-3GalNAc~l-3Gald-3Gal~l-4Glc/3l-Cer ~31. 2.3. NOMENCLATURE OF GANGLIOSIDES

A nomenclature of gangliosides was recommended by the Commission on Biochemical Nomenclature of the International Union of Biochemistry (1977). It follows that for neutral glycosphingolipids, which is based on trivial names for specific oligosaccharides (see also Fig. 3.1). Accordingly, the gangliosides are named sialo-Xosylceramide, where X stands for the root name of the neutral oligosaccharide, to which the N-acetyl- or N-glycolylneuraminosyl residue is attached. The position of the sialic acid group may be indicated by a Roman numeral for the number of the monosaccharide residue to which the sialic acid is linked, and with an Arabic numeral superscript indicating the position within that residue, to which sialic acid is attached. The neutral oligosaccharides are represented by symbols, in which the number of monosaccharide units is indicated by Ose,, preceded by two letters giving the trivial name of the oligosaccharide. To conserve space, Ose can be omitted: Gg, ganglio; Lc, lacto; nLc, lacto-neo, etc. (see also Fig. 3.1). For the sake of brevity in this chapter, short hand notations are used for gangliosides that contain as neutral carbohydrate core glucose (glc), galactose (gal), lactose (lac) or the oligosaccharides of the ganglio-series, i.e., gangliotriaose (tri), gangliotetraose (tet) and gangliopentaose (pent). G stands for ganglioside; the index, e.g., Gle,, is the abbreviation for its neutral sugar moiety. To this is added the number and, if necessary, the nature, e.g., NeuAc or NeuGc of the sialic acid residues, a, b to distinguish between positional sialo-isomers, e.g., GI,, 2a (Fig. 3.2). 2.4. CHEMICAL AND ENZYMATIC ALTERATIONS

A number of chemical as well as enzymatic reactions have been applied to ganglio-

sides. The products obtained were often instrumental in the elucidation of physicochemical or biological properties of these sialo-glycolipids.

2.4. I. Alteration of the ceramide Removal of part of the lipophilic ceramide portion yields " 1yso"-ganglioside analogues with properties similar to other amphipathic lyso-lipids. Ceramide cleavage can be achieved by strong alkaline hydrolysis of the long-chain fatty acid followed by re-N-acetylation [76-781 OH

OH

206 R, sialo-oligosaccharide

The secondary hydroxyl-group of the sphingosine, due to the carbon-to-carbon double bond in allylic position can be oxidized to the keto group. OH

n

R, sialo-oligosaccharide DCDCB, dichloro-dicyano-benzoquinone

In order to protect the sensitive sialic acid residues of the ganglioside from oxidative degradation by the dichloro-dicyano-benzoquinone,the reaction is performed with inverted mixed ganglioside/Triton X-100 micelles in toluene [464]. The product " keto-ganglioside" can be conveniently reduced to the parent ganglioside, e.g., with boro-tritiide. This then allows for radioactive labeling of the ganglioside in the ceramide moiety with retention of the carbon-to-carbon double bond but racemisation at carbon 3 [464]. Treatment of the keto-ganglioside with alkali leads to a liberation of the intact free, reducing oligosaccharide moiety from the glycolipid [465]. A point of any easy selective chemical attack at the ceramide of gangliosides or neutral glycosphingolipids, is the carbon-to-carbon bond of the sphingoid. Oxidative cleavage of ganglioside by ozone [86] or osmium tetroxide/periodate [87] yields aldehydic products that are used for further reactions: OH

R, glycosidic residue

207 (a) Mild alkaline fragmentation liberates the total intact sialo-oligosaccharide moiety of the gangliosides [88,89]. (b) Reduction of the ceramide C=C double bond-oxidation product with borohydride yields "lyso" ganglioside analogues, that still have the region of attachment of carbohydrate to the lipophilic molecular portion intact [77,79]. (c) The intermediate of oxidative C=C-cleavage can alternatively be further oxidized to contain a carboxyl function. A direct oxidation cleavage of the -C=Cdouble bond to carboxylic acids can be performed with peracetylated gangliosidesialic acid methyl esters in organic solvent using permanganate in the presence of dicyclohexyl-18-crown-6ether [606]. 0AC

P

o

l

c H 0 2 C q C H 2 . 0 , R (peracetyl)

.YNH 0 Such a fragment-ganglioside derivative can conveniently be used for coupling to matrices such as amino-alkyl-agarose. Prior to oxidation, the carboxyl group of sialic acid must be protected, e.g., by esterification [347,81]. The carbon-to-carbon double bond of the sphingoid can be reduced by catalytic hydrogenation. Using tritium in the presence of palladium, the gangliosides can be radioactively labeled in the ceramide moiety without apparent hydrogenolytic decomposition [82-851. Ganglioside analogues that contain a sialo-oligosaccharide moiety linked to fatty acid have been synthesized. Such derivatives carrying a fluorescent or electron paramagnetic resonance label were used as probes to investigate the properties of sialo-glycolipids in membrane systems [466-4721. To obtain such sialoglycolipids, a ganglioside-derived sialo-sugar is reduced with cyanoborohydride in the presence of ammonia. For tritium labeling, radioactive NaCNB3H3 can be used. The reductaminated oligosaccharide can then be coupled to a fatty acid of choice by an amide. A sialo-glycolipid is directly obtained when reductamination is performed with a long aliphatic hydrocarbon chain amine [90].

R, sialo-glycosidic residue R' and R", aliphatic hydrocarbon chain

208 Ganglioside analogues with two long aliphatic hydrocarbon chains have also been obtained from reductaminated sialo-sugars by the following reaction sequence [91]: R.CH2*NH2 + (p)02N.O,O.CO.CHN3.(CH2)n R .CH2 .NH * CO .CHNJ .(CH2 )n 'CH3

H2/P t

R.CH2 .NH.CO.CH.(NHr).(CH2)n.C~3

.CH3

-

tR'[email protected] (P)

R .CHz.NH . C O . C H .( CH> ) n .CH3

I

N H . C O * R'

R, sialo-glycosidic residue R, aliphatic hydrocarbon chain 2.4.2. Alteration of the carbohydrate moiety Chemical as well as enzymatic reactions involving the sialo-sugar moiety of gangliosides, frequently aim at an introduction of a chemical or radioactive label. Furthermore, enzymes that specifically degrade gangliosides can be used as valuable tools in the elucidation of their chemical constitution. 2.4.2.1. Oxidation of terminal galactose by galactose oxidase Terminal galactopyranosyl, as well as terminal N-acetylgalactosaminyl, groups are oxidized in 6-position to the aldehyde by galactose oxidase (D-Galactose: oxygen 6-oxidoreductase, EC 1.1.3.9) from Dactylium dendroides, frequently misnamed as Polyporus circinatus [92,204,246,487,608,574].Reduction of the aldehyde with tritium can be used for the introduction of the radiolabel [93,94]. Alternatively, the 6-aldehyde is reacted with "S-labelled methioninehydrazide to yield the corresponding hydrazone [176]. 2.4.2.2. Oxidation of terminal sialic acid residues A label can also be introduced into ganghosides after oxidation of their terminal sialic acid residues with a low concentration of periodate. Model studies showed that, by choosing appropriate conditions, sialic acid of brain ganglioside is converted to its 8-C analogue with only negligible destruction of other monosaccharide residues [96]. At the stage of the aldehyde, the oxidation product may be reacted with dinitrophenylhydrazine to yield a colour-labeled ganghoside derivative [587], or else reduced with borotritiide for the introduction of a radioactive label. Another method of chemical alteration of sialic acid residues is by exchange of the usual N-acetyl or N-glycolyl for a N-trifluoroacetyl residue [422]. Interestingly, the obtained N-trifluoroacetylneuraminyl-ganglioside is very inhibitory to cell membrane sialidase, indeed more so than other sialidase inlubitors, such as siastatin, i.e., 2-acetamido-3,4-dihydroxy-5-carboxypiperidine [423] or 2,3-dehydro-2-deoxy-N-acetylneuraminic acid (4241. Galactose-oxidase treatment as well as sialic acid-periodate oxidation, both have been applied successfully for the labeling of gangliosides in intact cells [97,98,587]. 2.4.2.3. Cleavage of terminal sialic acid by sialidase (see Table 3.1) It was the sequential enzymatic removal of sialic acid residues from the higher brain gangliosides by sialidase (N-acetyl-neuraminate glycohydrolase, EC 3.2.1.1 8) that provided the first clue for an understanding of their structural relationships (for review, see

209

Ref. 99). Sialidases of viral or bacterial origin show specificity with regard to the nature of the sialic acid and its linkage to the aglycon (Table 3.1). Sialidases have successfully been used to obtain information on the linkage position of sialic acid at the neutral core oligosaccharide [lOo]. 4-0-substituted sialic acids are not cleaved by the known sialidases. Sialic acid linked to the 3-position of TABLE 3.1 Specificities of sialidases Linkage type Gal c

t3

SV

VC

CP

FPV

NDV

+

+

+

+

AU

+

+

-

+

-

-

+

-

+

+

NeuAca2 GalNAc/ll+ 4Gala 3

+

4Glc

t

NeuAca2 GalNAcal+ 4Gala1 + GlcPl+ Cer 3

t

NeuAca2 GalNAcal+ 4Gal/31+ 4[1-amino-sorbitol]

t3

(cholate)

NeuAca2 Gala --* 3Ga lNAcBl+ 4Gala + 4Glca + Cer 2

r3 NeuAca2

t

laFuc Gala <

r6

(trace)

+

-

+

+

+

+

-

+

NeuAca2

GalNAcfl< 3

t

Neu Ac a 2 GlcNAc[3 + ] a 6 t NeuAca2 NeuAca < 8

+ -

+

t

NeuAca2 SV, Sendai virus neuraminidase (151; VC, Vibrio cholerae neuraminidase; CP. Clostridiurn perfringens neuraminidase; FPV, chicken fowl plague virus neuraminidase; NDV, New Castle disease virus neuraminidase [2.10-12); AU, Arthrobacfer ureafuciens neuraminidase [13,14]. These data are from References: Kuhn et al., 1961 [104]; Drzeniek and Gauhe. 1970 (1051; Suttajit et al., 1971 [loo]; lshizuka and Wiegandt, 1971 [loo]; Cassady et al., 1965 [107]; Wenger and Wardell, 1973 [101,102]; Rauvala, 1976 [108]; Schauer et al., 1980 [109]; Itoh et al., 1981 [llo]; Drzeniek et al., 1966 [ l l l ] ; Drzeniek, 1967 [112]; Huang and Orlich, 1972 [113]; Uchida et al., 1976 [114]; Sugano et al., 1978 [115]; Suzuki et al., 1980 [473].

210 internal galactose that is substituted in 4-position by N-acetylgalactosamine, as in gangliotetraose or gangliopentaose, is characteristically resistant to many sialidases. This has much facilitated structural investigation of gangliosides [293]. The reason for such resistance is as yet not well understood. Ganglioside G,,,l, resistant against Cl. perfringens sialidase, is cleaved, however, by this enzyme in the presence of cholate [101,102]. Surprisingly, monosialogangliotetraose, the free sialo-sugar of this ganglioside resists attack by this sialidase in the presence or absence of cholate. In contrast, N-acetylneuraminic acid is cleaved after reductamination of the monosialogangliotetraose to the parent amino-alditol. I t could therefore be speculated that the sialidase resistance of gangliosides Gtr,l, G,,la, G,,,la, Gpen,lu. G,,,2a and IV2F~~-,113NeuAc-Gg,Cer could be due to “steric hindrance”, caused by an arrest of the sialic acid residue to neighbouring N-acetamido group of N-acetylgalactosamine. This arrest may be released in a ganglioside-cholate mixed micelle. In such a lipid arrangement, cholate-carboxyl may be placed in a sterically favoured position to unlock the sialic acid block. Introduction of a neighbouring amino-group may have a similar effect. Sialidases have been used to probe the localization of ganglioside sialic acid in biological membranes. The results of such studies, however, are rather equivocal (for review, see Ref. 103). A recent analysis of N-18 mouse neuroblastoma cells shows that 728 of ganglioside G,,J and 85% of ganglioside Gte,2aare cleaved by Vibrio cholerae sialidase (5831. Interestingly enough, 50-80% of the sialidase-labile sialic acid residues of the neuronal perikaryon membrane were protected from added sialidase, whereas those of the synaptosomes were found to be accessible to the enzyme [474]. 2.4.2.4. N-Deacetylation-reacetylution Hydrazinolysis of gangliosides under anhydrous conditions leads to a preferential cleavage of N-acetyl residues from sialic acid and N-acetylhexosamine [546]. Specific N-re-acetylation of the product with radioactive acetic acid anhydride allows for a high radio-labelling of the parent ganglioside compound [546]. 2.5. P R E P A R A T l O N A N D A N A L Y S I S

2.5.1. Prepararion

Gangliosides, even though often freely soluble in aqueous media, are not extracted from biological membranes by water, or aqueous 1M KCI and 1 m M ethylenediaminetetraacetic acid [374]. They are, however, solubilized by detergents, e.g., by deoxycholate, and in part by the addition of Triton X-100. For their separation from proteins, it is best to use organic solvents. Gangliosides are extracted from biological material with aqueous chloroformmethanol or tetrahydrofuran [116]. Isolation of the sialo-glycolipid is then achieved by partition against an aqueous phase (“Folch partition”) [117-120,486,519], followed by Sep-Pak@purification [675]. Alternatively, gangliosides can be separated directly from total lipid extracts by sequential elution from anion exchange matrices [121-124,126,1271. The gangliosides

211 can thereby be separated into groups according to the number of sialic acid residues. Single species are then obtained by chromatography on silica [128] or silica-Kieselgur [192]. Based on these techniques, Iwamori and Nagai have devised a useful method for the “mapping” of gangliosides [125]. The application of a liquid partition technique for the separation of gangliosides has also been reported [476]. Gangliosides, can successfully be separated into components by high pressure liquid chromatography. This is performed either with the perbenzoylated derivatives [619,628] or in the underivatized state [620,621]. 2.52. Structural identification and analysis (see Table 3.2) A preliminary identification of gangliosides is achieved by thin layer chromatography [130,474,486,519,629,656]. When an anti-serum is available, specific detection on thin layer chromatograms can be performed by radioimmunoassay [592-5941. For quantitation of gangliosides, thin layer chromatography may be combined with colorimetric determination of single components [28,614]. Microestimations using ‘‘C-acetylation of glycosphingolipids [131]. or of sphingoid obtained after hydrolysis [32], have also been described. The structural characterization of gangliosides comprizes: (a) identification of the nature and number of sialic acid residues and their position along the neutral oligosaccharide core; (b) the neutral oligosaccharide moiety: and (c) the sphmgoid and the fatty acid composition of the ceramide. In recent years, the elucidation of the chemical constitution of gangliosides was greatly advanced by the introduction of newly developed gas chromatographic techniques, particularly in combination with mass spectrometry (for review, see Refs. 133, 134). An initial information of the sialic acid content of a ganglioside as mono-, di-, etc., sialocompound may be derived from its elution profile during anion exchange chromatography [125]. A further step in the characterization is by attempting to cleave sialic acid with Vibrio cholerae sialidase [loo]. Partial hydrolysis of oligosialogangliosides with this enzyme will thereby reveal intermediates in the degree of sialic acid substitution [293]. Dialysis or anion-exchange chromatography separates the liberated sialic acid from the neutral glycosphingolipid for further identification [85]. In the case of the sialic acid proving resistant to enzymatic cleavage, either sialic acid is present as 4-0-Me-substituted derivative or in a “sterically hlndered” configuration (see Section 2.4.2.3). Early structural studies of the total sialooligosaccharide portion of gangliosides were mostly performed by partial hydrolysis followed by identification of the fragment products (for review, see Ref. 99), by Smith periodate degradations [136] or by permethylation [ 1371. The long-chain base sphingoid composition was analyzed by gas-liquid chromatography of the trimethylsilyl derivatives after hydrolysis of the ganglioside under special conditions [ 1491. More recently, mass spectrometry of glycosphingolipids was employed that yielded information on the constituent sugars as well as the ceramide constitution [ 138- 140,4631.

h) CI

h)

TABLE 3.2 Structure Gangliosides of the gonglio-series G a l p l + 4Glcfi1+ Cer 3

Designation according to

Short notations

IUPAC-IUB Recommendations

Wiegandt Svennerholm

I1 NeuAc-Lac-Cer

Refs.

M3

t

2aNeuAc

'

I1 NeuAc2-Lac-Cer

G a l p l + 4Glcfil- Cer 3

G D3

t

2aNeuAc8 + 2aNeuAc Gal/?l+ 4Glc/31+ Cer 3

I1 NeuAc3-Lac-Cer

T3

II 'NeuAc-GgOse,-Cer

MZ

2

t

2aNeuAcS + 2aNeuAc 8

T

2aNeuAc G a l N A c b l 4 4Gal/?1+ 4Glcp1 + Cer 3

t

2 aNeuAc GalNAcbl + 4 G a l a l + 4GIcbI + Cer 3

113NeuAc,-GgOse,-Cer

G D2

I1 'NeuAc,-GgOse,-Cer

Gl-2

+I

2aNeuAc8 + 2aNeuAc GalNAc@l+ 4Gal/31+ 4Glcp1+ Cer 3

t

2aNeuAc8 + 2aNeuAc 8

t

2aNeuAc Gal/.?l+ 3GalNAcpl+ 4Galp1+ 4Glcp1+ Cer 3 t

II NeuAc-GgOse,-Cer

1

4 4

2 d

m’ N

* N

4

0

LL

L

N

c

N

213

TABLE 3.2 (continued) Structure Gal/3l+ 3GalNAc/31+ 4Gal/31- 4Glc/31+ Cer 3

Designation according to IUPAC-IUB Recommendations

Short notation

Refs.

11'NeuAc2-GgOse4-Cer

Gte~2b

GDlh

5

IV3NeuAc-,I13NeuAc-GgOse4-Cer G1,,2a

G Dla

6

Wiegandt Svennerholm

t

2uNeuAc8 + 2aNeuAc G a l S l - 3GalNAc/?l- 4Gal/31+ 4Glc/31+ Cer 2 3 t f laFuc 2aNeuAc8 + 2aNeuAc Gal,% 3

3GalNAcPl- 4Gal/31 3

+

t

f

2aNeuAc

-

4Glc/31+ Cer

IV2 Fuc-,I1 NeuAc,GgOse,-Cer

2aNeuAc

2aNeuAc

1

6 Gala1 + 3GalNAcP1- 4Gal/31- 4Glcj3 + Cer 3

26

IV NeuAc-,IIl6NeuAc-Gg0se,-Cer

t

2aNeuAc Gal/3l+ 3GalNAc/31- 4Gal/31- 4Glc/31+ Cer 3 f 2aNeuAc8 + 2aNeuAc 8

11 NeuAc,-GgOse,-Cer

Gtet3c

G n

6

t

2aNeuAc G a i S l + 3GalNAcBl- 4GalPl- 4Glc/31+ Cer 3 3 f f 2aNeuAc 2 aNeuAc8 + 2 aNeuAc

IV3NeuAc-,I13NeuAc,-GgOse4-Cer GI,, 3b

GTlb

7

Gala1 3

IV3NeuAc2-.I13NeuAc-GgOse4-CerG,,,3a

G,,,

8

t

-

3GalNAc/31

,-..

1-0.

7

-

4Gajl/31+ 4GlcPl- Cer

..

.,..

t

.1

X T .

.

2aNeuAc8 + 2aNeuAc

1

6 Gala1 + 3GalNAcPl+ 4GalS1 + 4GlcP1- Cer 3

IV NeuAc-,III6NeuAc,-GgOse4-Cer

26

IV NeuAc2-.1116NeuAc-GgOse.,-Cer

26

t

2aNeuAc 2aNeuAc 1 6 Gala1 + 3GalNAcPl+ 4Galfi1+ 4Glca1 + Cer 3

t

2aNeuAc + 2aNeuAc G a l P l + 3GalNAcal+ 4GalS1- 4Glc/31- Cer 3 3

t

IV3NeuAc,-,I13NeuAc2-GgOse4-Cer GI,,4b

GQlh

t

2aNeuAc8 + 2aNeuAc 2aNeuAc8 + 2aNeuAc

Gala1 3

t

-

2aNeuAc8 + 2aNeuAc

1

6 3GalNAcSl- 4Gal/31+ 4Glc/3l

+

Cer

IV NeuAc,-,111 NeuAc,-GgOse,-Cer

2aNeuAc8+ 2aNeuAc Galfil+ 3GalNAcSl- 4GalB1- 4GlcS1- Cer 3 3 f t 2aNeuAc 2aNeuAc8 + 2aNeuAc 8

GalSl 3

t

-

IV3NeuAc-.I13NeuAc3-GgOse4-cer G,,&

GQ,,

6

IV3NeuAc,-I13NeuAc,-GgOse4-Cer Glel5c

G,,,

6

t

2aNeuAc 3GalNAc/31+ 4Gal/31+ 4Glc/31+ Cer 3

t

2aNeuAc8 + 2aNeuAc 2aNeuAc8 + 2aNeuAc 8

t

2aNeuAc GalNAc/31- 4GalS1+ 3GalNAcSl- 4Gal/31+ 4GlcSl- Cer 3

t

2aNeuAc

I1 3NeuAc-GgOse,-Cer

9

TABLE 3.2 (continued) Structure GalNAcPl -t 4Gals1- 3GalNAcPI -. 4Gal/31-. 4Glc/?1- Cer 3

Designation according to I UPAC-IU B Recommendations

Short notations

IV NeuAc-GgOse,-Cer

G,,,lw

23

IV3NeuAc-,II'NeuAc-GgOse5-Cer

GFn, 2a

10

Refs.

W iegand t

f

2aNeuAc GalNAcSl- 4GalPl- 3GalNAcPl- 4GalPI + 4GlcP1+ Cer 3 3

t

-

GalPl 3GalNAcBl 3 f 2aNeuAc

-

-

2aNeuAc

t

2aNeuAc 20

4GlcPl- Cer

Gangliosides of the loeto-series

4GlcNAcpl+ 3GalPl + 4GlcP1- Cer

IV NeuAc-nLcOse4-Cer

11-13

2aNeuAc Galpl 4GlcNAc/31+ 3GalB1- 4GlcPl- Cer 6

IV6 NeuAc-nLcOse4-Cer

3. 20

IV NeuAc-.Ill Fuc-nLcOse,-Cer

16.20

IV NeuAc,-nLcOse4-Cer

29.32

I V NeuAc3-nLcOse4-Cer

29

Galpl 3

r

r

2 aNeuAc G a l P l - 4GlcNAcpl- 3GalB1- 4GlcB1- Cer 3 3

t

2aNeuAc

r

laFuc

Gal@l- 4GlcNAcPl- 3GalP1- 4GlcPI + Cer 3

t

2aNeuAcS + 2aNeuAc Gala1 -. 4GlcNAcfil- 3GalP1- 4GlcB1+ Cer 3

t

2aNeuAcS -.2aNeuAcS + 2aNeuAc

m

8 t

t

N Wl

6

U

b

U

t

5

t

5 t

PL

t

9

PL

9

9

t

5

t

9

5

4

c

3 9t

c

g 3 t

9

c

t + 3

xt

L

s

eb 3

t

5

0

8 t

t

t

s

a

0

G

2

5

0

t

s 5 c

9t

3

xt

t

217

2 00 TABLE 3.2 (continued) Structure

Gangliiiaes of tbe gbbo-series Gal/Il+ 3GalNAcbl+ 3Galal+ 4Galpl 3 f 2uNeuAc

+

4Glcp1 + Cer

Galpl- 3GalNAcSl- 3Galal- 4Galp1+ 4Glcb1- Cer 3 f 2aNeuAc8 + 2aNeuAc

Designation according to IUPAC-IUB Recommendations

Refs.

V NeuAc-GbOse,-Cer

28. 31

V NeuAc,-GbOse5-Cer

28

V 3NeuAc-.V6NeuAc-GbOse5-Cer

32

V 3NeuAc-iGbOse,-Cer

27

2aNeuAc

1

6 Gal/3l+ 3GalNAc/31+ 3Galal+ 4Gal/31+ 4Glc/31+ Cer 3 f 2aNeuAc Galfil+ 3GalNAcPl+ 3Galal- 3Gal/31+ 4Glcfi1+ Cer 3 f 2aNeuAc

Key to literature references: 1, Kobata and Ginsburg, 1972 14521; 2, Ohashi and Yamakawa, 1981 1453); 3, Wiegandt, 1973 14541; 4, Ghidoni et al., 1976 [455]; 5, Sonnino et al., 1978 14561; 6, Ishizuka and Wiegandt, 1972 14571; 7, Kuhn and Wiegandt, 1963 14581; 8, Ando and Yu,1977 14591; 9, lwamori and Nagai, 1978a 12851; 10, Svennerholm et al., 1973 16731; 11, Wiegandt and S c h u k , 1969 1611; 12. Uemura et al., 1978 16091; 13, Li et al.. 1973 1621; 14, Rauvala, 1976 [68]; 15, Wiegandt, 1974 [63]; 16, Keranen, 1976 16091; 17, Watanabe et al., 1978 1691; 18, Watanabe et al., 1974 1641; 19, Suzuki et al., 1975 1491; 20, Watanabe et a]., 1979 11981; 21, Ohashi, 1979b 14711; 22, Hirabayashi et al., 1979 12833; 23, Itoh et al., 1981 11101; 24, Watanabe et al., 1979 11981; 25, Iwamori and Nagai, 1980 14511; 26, Ohashi, 1981 1541; 27, Breimer et al., 1982 14621; 28, Hogan and Chien, 1981 [462]; 29, Murakami-Murofushi et al., 1983 16541; 30, Homes and Hakomori, 1982 15911; 31, Kannagi et al., 1983 16361; 32, Kundu et al., 1983 16371; 33, Watanabe and Hakomori, 1979 1591; 34, Kannagi et al., 1983 [595].

219

For t h s end, glycosphngolipids are permethylated with methyl iodide in presence of dimethylsulfinyl carbanion [640] and hydrolyzed [641] or methanolyzed [642]. After reduction and peracetylation, the partially methylated alditol acetates can be analyzed by gas chromatography-chemical ionization mass spectrometry. This “mass chromatography” yields information on the ratio of the constituent monosaccharides and their linkage positions [148]. Direct probe mass spectrometry of permethylated glycosphingolipids produces ions that are characteristic for the sequence of monosaccharide residues, as well as for the ceramide portion [141-147,463,643-6451. More recent developments of mass spectrometry using negative ion fast atom bombardment, allow the structural elucidation of underivatized gangliosides. With this method, molecular weight, monosaccharide constituent sequence and the molecular species of the ceramide portion can directly be analyzed [648,649]. More specifically, for sequence analysis of the monosaccharides, exoglycosidases can be employed that will also distinguish the anomeric linkages. The anomeric configuration of glycosides can also be assayed by chromium trioxide procedure [646] or with no destruction of the glycosphngolipid sample by proton nuclear magnetic resonance spectroscopy [645,647]. Chemical structures of the gangliosides that have been established until 1983, are summarized in Table 3.2. 2.6. PH YSICOCHEMICAL CHARACTERIZATION (SEE TABLE 3.3)

2.6.1. General Resulting from the molecular combination of hydrophilic carbohydrate that, under physiological conditions, carries a negative electric charge, with a lipophilic moiety of comparable size (the ceramide) the gangliosides are highly polar lipids (for dipole moments, see Ref. 174 and Table 3.3). In addition, the unique properties of both moieties confer complex physicochemical characteristics on these lipids that may not be unrelated to their function in biological membranes. Gangliosides appear as highly rigid structures that allow for tightly packed arrangements, i.e., reducing the mobility of neighbouring hydrocarbon chains [485,581]. On the other hand, gangliosides have molecular properties that apparently promote the contrary effect, i.e., a loosening of lipid phase molecular configurations. It is speculated that the adaptation of such properties to environmental requirements occurs in vivo, by modification of the ceramide constituents (e.g., increase in hydroxyl content) as well as through changes in the carbohydrate portion (e.g., varying the degree of sialylation). The molecular arrangement of the ceramide has some special features that appear to contribute to the physicochemical chariicteristics of gangliosides. Due to the rigidity of the planar amide group, the hydrocarbon tails adopt a preferentially parallel conformation [155]. Ths, in addition to the presence of the trans carbon-tocarbon double bond in sphing-4-enine, promotes condensation of the ceramides into a closely packed arrangement. A further condensation of molecular packing is effected by groups in the

220 ceramide that can partake in hydrogen bonding, e.g., the secondary hydroxyl group of the sphngoid, and the hydroxyl of 2-hydroxy-fatty acids, if present. Hydrogen bonds in the region between the lipophilic and hydrophilic moieties of the gangliosides may contribute to a general hydrogen belt located at the level of the estercarbonyl groups of membrane phospholipids [156]. There is evidence that ganglioside-ceramide in a phospholipid mixture intercalates with the lipid portion of the latter [ 1571. Head group interactions also appear possible, e.g., between ganglioside and phospholipid resulting from a partial matching of opposing electrical dipole vectors [478]. 2.6.2. Gangliosides in solution All gangliosides are soluble in more polar organic solvents, such as alcohols, tetrahydrofuran, dimethylformamide, dimethylsulfoxide or in mixtures of chloroform methanol, with addition of a small amount of water. With the exception of the smaller components Ggall,GIaJ and GJ, the higher gangliosides are lipids that are also freely soluble in purely aqueous media. Their extreme amphipathic nature is influenced very sensitively by the environment provided by the solvent and additional complexing molecules. Thus, in the presence of mono- or divalent cations or protein, gangliosides may have different solubility properties, e.g., in a partitioning system of solvents of different polarity [152,153,476]. In organic solvents gangliosides form monomeric solutions [160,161]. In aqueous systems, however, gangliosides exist as micelles (Table 3.3). The micellar size of gangliosides thereby, in a subtle way, depends on the ratio of C2,,- to C,,-sphingoid, as well as on the sialooligosaccharide [ 1621. The aggregation properties of gangliosides are also influenced by their electric charge, i.e., environmental p H [490] or the presence of metal counter ions [163]. Corti et al. in 1980 [164], using laser lightscattering methods, observed some change in the micellar size of gangliosides at concentrations between and M. Reported values for brain ganglioside micelles range from approximately 200000 to 500000 M , [165] (Table 3.3). The micellar shape is globular and in the form of an oblate ellipsoid. Yohe and Rosenberg [166] suggested that the inner part of the ganglioside micelle may be partly permeable and house penetrating molecules, as shown, e.g., for the triiodide ion. Various physicochemical methods have independently revealed that gangliosides undergo a structural transition in aqueous solution at a concentration range of 10-4-10-5 M. This was interpreted as the critical micellar concentration, below which the gangliosides were assumed to be in a monomeric dispersion [161,162,166-1681. Further evidence for such a transition was obtained by the observation that gangliosides G,,1 and G,J are markedly hydrolyzed by Clostridium perfringens neuraminidase below concentrations that correspond to these previously reported “critical micellar concentrations” [108,169,170].Other data obtained from direct studies in the ultracentrifuge and by gel permeation chromatography, however, make it appear likely that the critical micellar concentration, e.g., for ganglioside G,J (I13NeuAc-Gg4Cer), is in a much lower concentration range, i.e., in the

221

order of lo-’ M [171-173,4901 (Table 3.3). I f t h s indeed were the case, it may be speculated that the unexpectedly low critical micellar concentration results from strong intermolecular non-covalent bonding within the region of the sialooligosaccharide moieties. Such interactions may perhaps become possible because the strong hydrophobic forces in the lipid region orient the sugar portions, and in this way promote micellar aggregate formation. 2.6.3. Gangliosides in membranes and at interfaces Much about the properties which gangliosides show in natural membranes, can be learned from their behavior in artificial model systems (e.g., at the air-water interface (see Table 3.3, for review, see Ref. 479)) or in phospholipid liposomes. According to Czarniecki & Thornton [158,159], gangliosides embedded with their ceramide in a lipid phase have their carbohydrate moiety stabilized by solvation as a network of intermolecular hydrogen bonds “anchored” in solution by sialic acid residues. Thereby the sialic acid neighbouring N-acetylgalactosamine, as, e.g., in gangliosides G,,,l or G,J, shows structural effects that are different from sialic acids positioned elsewhere as revealed by I3C-NMR spectroscopy [611]. This sialic acid residue may adopt a preferred position of its ring being perpendicular to that of the neighbouring N-acetylgalactosamine [157]. It is interesting to note that, according to Maggio et al. [281], the contribution of the sialic acid residues in gangliosides G,J, Glrilor G,J to the molecular dipole moment is opposite to that of a second or t h r d sialosyl group in di- or trisialogangliosides. From electron paramagnetic resonance studies it was suggested that gangliosides are randomly distributed and have their sugars protruding from phospholipid bilayers moving homogenously and comparably unrestricted about 2.5 nm above the interphase [483,665]. Ganglioside sugars, however, under such conditions were reported to be less mobile as compared to the carbohydrate of a glycoprotein such as glycophorin [484]. The surface properties of gangliosides could be studied with films formed with these lipids at the air-water interface. Depending on the charge of the molecules, brain gangliosides show different surface requirements in such films [ 1741 (see Table 3.3). The electric charges of ganglioside molecules due to repulsion and a concomitant larger area requirement of the head group, also induce an increase in fluidity of lipid films or dispersions [ 1741. Fluorospectroscopic studies revealed that indeed the hydrophobic region is influenced by the presence of sialic acid residues linked to the core oligosaccharide, resulting in an increased mobility of lipophilic probing molecules [165]. The lytic properties of gangliosides may also be reflected in their ability to induce membrane fusion in erythrocytes [177] or disruption of the Sendai virus envelope [179]. A role for ganglioside Gte12ain the fusion process of myoblasts to myotubes was also postulated [570]. Ganglioside molecules in artificial membranes tend to cooperate via hydrogen-bonding with clustering, particularly in the presence of physiological concentrations of Mg2+ or Ca2+ [179,483,665]. Lectins may disrupt such clusters of ganglioside molecules [482]. An aggregation of gangliosides around membrane glycoproteins was suggested

TABLE 3.3 Physicochemical properties of gangliosides

Ganghoside

G lac1 GI,2 Gtn1 GI,J

S,.

Stoke's radius (A)

7.8 [2] 6.6 [2] 7.6 121

60[2]

9.7[2] 10.3 [ S ]

60[2]

10191

63[11]

60[2]

M,

CMC

( X lo3)

(MI

322*24[2] 281 *23 [2] 281 i-21 [2] 300k 15 141 337k22 [2] 257 (309)[S]

2 ~10-~[2] 1 X lo-' (21 5 X10-'[2] 7.5 x 10-5 [31 2 X10-8[2] 7 X [9] (sedimentation) 8.5X10-9[9] (gel film.)

Dipole moment

( c . (mD)) Charged

532*50[ll] 244 171 34O+N) [4]

450 [8]

8.5x10-5 [3] 2.8x10-5 [lo] (50 Ac (4.6)) a 8.2X 10-5 [lo]

Surface potential (Wm2/moIecuIe)

Uncharged

Surface area

(A2)

Charged

70111

280[1]

0.95 [l]

1.15 [l]

120[1]

340111

l.lO[l]

1.26111

67 [l] 105 [6] 70[1]

40[1]

270[1]

0.85 [l]

1.02 [l]

75 [l]

Uncharged

60 [l]

GI,, 2 GI,, 2a

9.3 [5] 6.2 [2]

Gl,l2b

5.6 [2] 4.5 [2]

Gm3b

57 [2] 59[11] 57 [2] 55 [2]

257 [5] 181k15 [2] 470540 300 f 30 [4] 160514[2] 113*12[2] 250f25 [4]

1 x10-5[12] 2 X10-6 [2]

1 X10-6 [2] 1 X10-5[2] [3] 1 X

240 [I]

310 [l]

1.59 [l]

1.19 (11

95 [61 103 [l]

71 [l]

500[1]

320[1]

2.44[1]

1.26 [l]

105 [l]

90[1]

* 50Ac (4.6) = Na-acetate buffer, 50 rnM, pH 4.6. Key to references: [I] Maggio, B., Curnar, F.A. and Caputto, R. (1978) Biochern. J. 171, 559-565. [2] Ulrich-Bott, B. and Wiegandt, H. (1984) J. Lipid Res. 25, 1233-1245. [3] Yohe, H.C. and Rosenberg, A. (1972) Chem. Phys. Lipids 9, 279-294. [4] Yohe, H.C., Roark, D.E. and Rosenberg, A. (1976) J. Biol. Chern. 251, 7083-7087. [5] Gammack, D.B. (1963) Biochern. J. 88, 373-383. [6] Halser, H.and Dawson, R.M.C. (1967) Eur. J. Biochern. 1, 61-69. [7] Yedgar. S., Barenholz, Y. and Cooper, V.G.(1974) Biochern. Biophys. Acta 363, 98. [8] Tornasi, M., Roda, L.G., Ausiello, C., DAguolo, G., Venerando, B., Ghidoni, R., Sonnino, S. and Tettamanti, G. (1980) Eur. J. Biochem. 111, [9] [lo] [ll] [12]

315-324. Formisano, S., Johnson, M.L.,Lee, G., Aloj, S.M. and Edelhoch, H. (1979) Biochern. 18, 1119-1124. Rauvala, H. (1979) Eur. J. Biochem. 97, 555-564. Corti, M., DeGiorgio, V., Ghidoni, R., Sonnino, S. and Tettamanti, G. (1980) Chem. Phys. Lipids 26, 225-238. Howard, K.E.and Burton, R.M. (1964) Biochim. Biophys. Acta 84, 435-840.

from studies employing electron paramagnetic resonance spectroscopy [ 1791. In biological membranes, a portion of the ganglioside, strongly bound to integral protein, appears to be present. This is deduced from the fact that some integral proteins, e.g., spectrin-actin-depleted rat erythrocyte cytoskeletal elements, in the presence of neutral detergents tenaciously retain sphingolipids, including gangliosides [491]. In a different approach to studying the behaviour of sialoglycolipids in natural membranes, the lateral diffusion of a fluorescent ganglioside analogue was examined by using the method of fluorescence recovery after photobleaching [469]. With an apparent diffusion constant in the order of DDirr5 X cm2/s, and a fractional fluorescence recovery of R 80-loo%, the fluorescent ganglioside analogue shows an unrestricted lateral diffusion not much slower than that of a typical freely diffusing lipid probe [468,677]. 2.6.4. Cation binding to ganglioside Because of their acidic nature and their concentration in neuronal membranes that are probably active in ion fluxes, the gangliosides were, from early on, suspected of playing some role in special cation binding and transport or release mechanisms [180,5271. Gangliosides bind divalent cations [ 1821. Ca2' thereby complexes to gangliosides twenty-times more effectively than Mg2+. Ca2+ binding to ganglioside depends drastically on the position of the sialic acid residue along the neutral oligosaccharide M, chain. The sialic acid in G,,,1 complexes Ca2' above a concentration of whereas in the terminal position, e.g., in Gtet2ait is above l o p 6M. In contrast, sialic acid linked to sialic acid, as in ganglioside Gl,,2b, binds very little Ca2+ [82,478]. The degree of Ca2' complexing influences the structures of ganglioside aggregates in solution, and possibly also in membranes [613]. This was concluded from changes that occur with increasing Ca" concentration in the Ca2'-binding stoichiometry [183,184], as well as in the solvent and surface-labelling properties [153,613]. Solvent partition experiments have also shown that Ca2+ may complex gangliosides to protein [185]. The Ca2+ binding to gangliosides in turn is influenced by various other cations. Notably, tubocurarine was found to be very effective in the displacement of Ca2' from ganglioside [186]. '3C-Nuclear magnetic resonance spin lattice relaxation data revealed that metal ion binding to ganglioside G,,,l occurs via carboxyl- and the "glycerol" side chain of the sialic acid. Additional ligands are donated by the N-acetylgalactosaminyl-pyranoside and the terminal galactose residue [159,187-1891.

3. Distribution of gangliosides 3.1. ANIMAL SPECIES

Gangliosides occur, as far as available data show, only in animals of the Deuterostomia. whereas they have not been detected in Protostomia.

225

More detailed data that allow for a comparison of the gangliosides of different animal species are available for the brain [75,190,488,489]and, to a lesser extent, for the sphngolipids of erythrocytes [36] and the thymus [190]. Mammals, birds, amphibians and teleost fish, all have similar brain gangliosides. These gangliosides are of the ganglio-series with, predominantly, gangliotetraose as neutral core oligosaccharide, though with a varying degree of sialylation. There are, however, phylogenetic differences in the concentrations of brain gangliosides: the lower vertebrates (reptiles amphibia, fish) having 110-500 pg; and the higher vertebrates (birds, mammals) with 500-1000 pg lipid-bound sialic acid per gram brain fresh weight [489]. Among the species, variabilities in the concentrations of brain gangliosides are again found, e.g., fish with 160-390 pg lipid-bound NeuAc/g tissue fresh weight [492]. In contrast to the brain, the erythrocytes show drastic species-dependent qualitative, as well as quantitative, differences in their ganglioside components. Thus, typical major gangliosides of the red blood cells of various animals are: mice, G,J and Gtil; rats, Gtet2a;man, dog and pig, GIJ; cats, Gl,,2; and cattle, NeuAcnLc,Cer. For the gangliosides of the thymus, pronounced species dependence of the component distribution, although different from that of erythrocytes, were reported. It appears, however, that thymus gangliosides of all the species investigated are derived mostly from the lacto-series [190,192,495]. For the brain gangliosides, an evolutionary trend to lower animals seems to be reflected in a shortening of the average neutral oligosaccharide chain length. Whereas gangliotetraose is the core sugar predominant in mammals, birds, teleost fish and ganoids (sturgeon), lactose and gangliotriaose, respectively, are the neutral core saccharides of the brain gangliosides of elasmobranchs, ray and cartilaginous fish [192,193]. The gangliosides from the starfish, Asterina pectinifera, contain ceramide-linked lactose substituted by sialic acid residues [194], whilst the sea urchin has gangliosides derived from only glucose [45-471. Another evolutionary parallelism concerns the ceramide composition of brain gangliosides. In their progressive evolution, the brain gangliosides of vertebrates show an increase in the degree of fatty acid saturation and the relative content of C,,-sphngoid [192,194]. In addition to the systematic position of the species, environmental factors, such as temperature of the dwelling of the animal, affect the composition of brain gangliosides. Thus, changes in the degree of sialylation and in fatty acid composition of brain gangliosides appear to be involved in temperature-adaptive mechanisms (for review, see Ref. 493). Compared to warm-blooded animals, the brain gangliosides of poikilotherms are more highly sialylated, promoting a membrane fluidisation, which was interpreted as an adaption to cold environmental conditions [193,195,196,494] (see Section 4.3.3).

226 3.2. TISSUE DISTRIBUTION OF GANGLIOSIDES

3.2.1. General distribution Gangliosides are present in most, if not all, mammalian tissues [197]. The highest concentration of total gangliosides is found in brain grey matter [200]. Whereas the gangliosides of the central nervous system are mostly derived from the gunglio-series, those of peripheral nerves (approx. 0.11 pmol Neu per g wet weight [1971) and of extraneural tissues (approx. 0.1-0.35 pmol Neu per g wet weight [200]) contain a high proportion of gangliosides of the lacto- [88,461,464,496] or globo-series [462,463]. Ganghosides of the gunglio-series have, however, also been detected at extraneural sites. If, indeed, the presence of ganglioside G,,J confers sensitivity of a cell towards cholera toxin (see Section 6.4) then a ganglioside of the gunglio-series must be present rather ubiquitously. 3.2.2. Central nervous system A detailed analysis of the brain ganglioside component distribution of various animal species was given by Ando et al. in 1978 [28], and by Hilbig and Rahmann in 1980 [489] (for review, see Ref. 209). Expressed as per gram tissue fresh weight, mammalian grey matter has roughly three-times as much ganglioside as white matter [ 129,201,2021. The predominant gangliosides of brain grey matter of mammals are Glell,2a, 2b, 3b and 4b, whereas the major component of brain wlute matter in primates and chicken is G,,,1 with variable amounts of G,,,l [28,203,630]. Amphibia and fish myelin contain no ganghoside GJ [630]. The gangliosides, G,,J and Gtell,are characteristic constituents of differentiated mature oligodendroglial myelin [27]. The component pattern of oligodendroglial perikarya is more complex and contains, in addition to Ggalas major ganglioside, Gla,l, G,,,2 and G,J [205,206], and also Glell, 2a, 2b and 3b [27]. The latter four gangliosides are typical components of neurones and neuronal processes. These, as well as astrocytes, probably do not contain the ganghoside Ggall.Higher sialylated ganghoside components, G,,,4b and G,,,4c as well as hexa- and heptasialogangliosides are typical of early embryonic chicken brain [461]. 3.2.2.1, Cells ofthe nervous system in culture Cells derived from the central nervous system have been cultured and used as a model system of minimal complexity in the study of the presence of gangliosides and regulation of their biosynthesis (for reviews, see Refs. 220, 221, 566). There are, of course, certain constraints on conclusions drawn from the observation of gangliosides in cultured cells with regard to the tissue of origin. Thus, established cells of neuronal or glial origin, that indeed retain many of the electrophysiological and enzymatic characteristics associated with neurones or glial cells, show in general a glycosphingolipid profile not very typical of fresh brain tissue-derived cells, e.g., in their having a neutral tetrahexosyl-ceramide and a lack of tugher oligosialogangliosides (no G,,,3 and 4, only traces of G,,,2b) [221-2231. In addition, such cells do not synthesize the higher gangliosides that are typical for neurones of various types in the central nervous system even in the

227 presence of differentiation inducers and neurite outgrowth [224]. Astroblasts in primary culture (96% Gla,l, 4% G,,,2) or other glial tumor cells show only G,,,1 and 2 [207,208]. Coculturing of established neuronal and glial cells, however, induces synthesis at low levels of the gangliosides G,,,2b and G,,,3 that persist after reisolation of clones [221]. Neuroblastoma cells grown in vivo also produce ganghoside G,,,3. Primary cultures of isolated neurones or glial cells possess ganglioside patterns very close to the tissue cell fractions [567,568]. The homogeneity of cell types in culture allowed a reliable comparison of gangliosides of neurones and of glial cells. Thereby, the gangliosides G,,,2b, 3 and 4 were found to be higher in primary neurones than in ghal cells, even though the latter also contained appreciable amounts of gangliosides G,,,2b, 3 and 4 [221]. As with tumor cells, coculture of primary cells, astroglia and neurones change the expressed ganglioside pattern [2211. 3.2.3. Peripheral nerves Gangliosides have been identified in peripheral nerve tissue, including the neuroand adenohypophysis, the adrenal medulla and the visual tract. The gangliosides of the peripheral nervous system are rather different from those of the central nervous systems. They consist of ganghosides, mostly Gla,l, G,,,2 and NeuAc-nLc,Cer. Whereas, e.g., G,,J and G,,,2 are the major components of trigeminal nerve, ganglioside NeuAc-nLc,Cer was found to be localized mainly in the peripheral nerve myelin [670]. Even though more work on peripheral nerve gangliosides has been reported, they still appear insufficiently characterized: human femoral nerve [1971, rabbit sciatic nerve [226], rat sciatic nerve [227]. The neurohypophysis of cattle contains a high concentration of gangliosides, similar to those of the central cortex, except for a high proportion of N-glycolyl- in addition to N-acetylneuraminic acid [228]. In contrast, the adenohypophysis shows a decisively different ganglioside profile with a high proportion of gangliosides G,,JNeuGc and G,,lNeuAc. Similar to the adenohypophysis, with a component pattern resembling more that of extraneural sources, the ganghosides of the adrenal medulla consist of up to more than 90% of G,,J [229] *. Notably, in this case, in the membrane of an intracellular structure the adrenal chromaffin granules were shown to contain ganglioside [230]. The gangliosides of the visual system, including lens, retina, optic nerve fibres and the tectum opticum, have been studied in detail by several groups. The optic pathway offers certain advantages for investigations of this part of the central nervous system, such as structural simplicity and its light excitability [231]. The structural elements of the optic pathway provide more evidence that the higher gangliosides G,,,l, 2a, 2b and 3 are characteristic for ganglion cells. Lens and iris have some 90% of their ganglioside content as G,,,1 [232,233], whereas mammalian retina consists mostly of G,,,2 ( 3 5 4 6 % of total ganglioside) *

Characterization of ganglioside G,,,3b from bovine adrenal medulla has also been reported [671].

228 (234,2351. All other gangliosides of the retina are the same as those found in the brain. Gangliosides of retina show, however, species specificity. Thus, chcken retina - retinal ganglion cells as well as their presynaptic terminals [499] - contains, in addition to G,,1, predominantly Gl,,2a [231,236]. In frog photoreceptors two gangliosides were detected, tentatively identified as G,,,2a and 3 [237]. From subcellular fractionation experiments there is an indication that gangliosides G,,1 and 2 of the mammalian retina are predominantly localized in the rod outer segments of the photoreceptor, whereas the hexosamine-containing gangliosides may be located in other retinal elements [235,238-2411. It is probable that the hexosamine-containing gangliosides in this location are characteristic for the retinal ganglion cells. In support of this, in mammalian optic nerve, i.e., the axons of retinal ganglion cells, no ganglioside G1,,2 was detected [239]. Instead, the four major brain gangliosides Gl,,l, 2a, 2b and 3 constitute more than 90% of the glycolipid. 3.3. CELLULAR LOCALISATION

As with other glycosphingolipids, it is generally assumed that the gangliosides occur on the outer side of the cellular plasma membrane [250,252-256,6051. In the case of neurones, this includes elements of the nerve endings [257]. This location of gangliosides in conjunction with their complex physicochemical character, in particular with regard to calcium ion binding, has stimulated hypotheses for a functional role of gangliosides in synaptic transmission and memory formation [498]. It is not yet clearly established whether or not gangliosides are evenly or unevenly distributed on the outer neuronal cell surface (for review, see Ref. 10). Whereas, the neuronal cell bodies have a ganglioside content that is lower than that of whole brain [211,212], fractions containing nerve endings show enriched levels of sialoglycolipids [213-217,5691. This, however, might be explained by surface area considerations. Synaptic junctions from adult humans are particularly rich in the higher gangliosides G,,,2b, 3b and 4b [218]. On the other hand, Morgan et al. [219] have isolated synaptic junctions almost devoid of gangliosides. Engel et al. [569], on the contrary, have observed an enrichment of cholera toxin peroxidase labeling of ganglioside G,,J in the synaptic cleft region. Perikaryal as compared to synaptosomal gangliosides show a differential accessibility to bacterial sialidase [474] (see Section 2.4.2.3). Low levels of ganglioside * have also been localized intracellularly in membranal structures of the Golgi apparatus and the endoplasmic reticulum, and also possibly in lysosomes [72,242-2451. It is still an open question, as to whether or not the intracellular ganglioside merely belongs to a transient pool of membrane constituent recycling or is in the process of metabolism. This could also be the case for the soluble ganglioside in the cytosol [72,245,247,652]. However, the ganglioside that was detected in other intracellular structures, the chromaffin granules [230] or in the sacroplasmic reticulum [249], may well serve some special biological function.

*

Matyas, Morr.6 and Keenan (1982). however, report that their experiments indicate that less than 65% of the total ganglioside of rat liver is in the plasma membrane 16331.

229

4. Metabolism 4.1. BlOS YNTHESIS

The biosynthesis of gangliosides proceeds by stepwise addition of monosaccharide units onto the growing carbohydrate chain of the glycosphingolipid, and biodegradation by removal of single monosaccharide residues (for reviews, see Refs. 258-263). Gangliosides are comparatively long lived lipids, those of retinal cells all having similar turnover rates, with half lives of 34-38 days [499]. The transer of nucleotide-activated monosaccharides is primarily controlled by glycotransferases. Some evidence indicates that they may be multienzyme complexes as was postulated by S. Roseman in 1970 [263] and R. Caputto in 1971 [264,301]. According to this view, two pools would exist: one, a very small transient pool containing gangliosides in the process of biosynthesis, e.g., on multienzyme systems; and, the other, a ganglioside end-product pool predominantly at the plasma membrane [226,583]. The latter gangliosides cannot be returned to the transient pool, e.g., for repair synthesis. The biosynthesis of gangliosides on specific multienzyme systems could also help to explain why there is a connection between the constitution of the sugar moiety, e.g., the degree of sialylation of a ganglioside and that of the ceramide, e.g., content of C,,-sphingoid. The subcellular site of biosynthesis of gangliosides may largely be located in the Golgi membranes [242,267,268] (for reviews, see Refs. 269, 270). After intracellular synthesis, the gangliosides reach their destination at the cell surface within some 30 min, perhaps participating in a transport mechanism common to other plasma membrane constituents [584]. In nerve cells the primary site of ganglioside biosynthesis appears to be in the neuronal perikarya [271]. It is speculated that, from the cell soma, the gangliosides may then be translocated to the nerve endings, where further sialylation possibly takes place [272] *. The key steps in the biosynthetic pathway to higher sialylated gangliosides of the ganglio-series (pathway I) is the sequential transfer of sialic acid to lactosylceramide [273]: followed by addition of N-acetylgalactosamine to ganglioside G

- -

Pathway I Lac-~er

CMP-NeuAC Lac-Cer UDP-GalNAC GalNAc-4'LaC-Cer 3' step 1

t

NeUAC

Step

2

(Giocl)

1'

NeuAc (Gtr, 1 )

Pathway II

-

Lac-Cer

UDP-GolNAcGalNAC-4'Lac-Cer UDP-GalGal-3GalNAc-4'Lac-Cer

CMP-NeuAc

*

NeUAC-3Gal-3GalNAc-4' L a c - c e r ( G tet 1 w )

Dreifus et al., (1981) report the presence of ecto-glycosyltransferase (NeuAc, Gal, Fuc) on chick embryonic neurone primary cells [579].

230 Omitting the sialic acid transfer (Pathway I) other glycolipids of the ganglio-series could be formed by Pathway 11. Such compounds include: the neutral glycosphingolipids, gangliotriaosylceramide from guinea pig erythrocytes [274] (also detected as a characteristic constituent of neuroblastoma NB41A cells [20]. KIMSV tumour cells in Balb/c mice [275] and mouse lymphoma (LS 178c 127) cells [282]) or gangliotetraosylceramide found in immune-cells of rat and mice [276-2811. There also exist extraneural gangliosides originating by biosynthesis via Pathway I I, such as: G,,,lw (IV3NeuAc-GgOse,Cer) present in human erythrocytes [198], rat hepatoma cells [283], and mouse lymphoma L 5178; Gpentlw(IV3NeuAc-GgOse,Cer) [284] or the gangliosides discovered by Ohashi [460] in the frog brain (e.g., IV3NeuAc-. 1116NeuAc-GgOse,Cer). Little is known of the in vivo signals received by the cell that may regulate biosynthesis of gangliosides. Examples of such regulation are the enhancement effect of cocultivation of neuronal and glial cells on the biosynthesis of higher gangliosides. and similarly the increased amounts of gangliosides present when cells are grown as a solid tumor as compared to single cells in culture. The major route for biosynthesis of the higher sialylated classical brain ganglioside proceeds via pathway I a, b, and c: G a l - G l c - C e r 4 Gal-Glc-Cer ----o keUAc

I

GcllNAc-Gal-Glc-Cer bUAC

I

I

Gal-Glc-Cer t+UAc NeUAC

I

-

I I

GVI-Glc-Cer YeuAC YeuAc NeUAC

GalNAC-G?l-GlC-Cer ?WAC yeuAc NWAC

GalNAc-Gal-Glc-Cer (JeuAc NeuAc

Gal-GalNAc - Gal-Glc-cer keunc

Gal-GalNAc- Gal-Glc-Cer t+Ac NeUAC

Gal-GalNAc- Gpl-Glc-Cer YWAC yUAC NeuAC

Gal-GalNAc- Gal-Glc-Cer keUAC keuAc

G:I-GalNAc-G?l-Glc-Cer NeUAC YeuAc NeuAc

Gpl-GalNAc- GaCGlc-Cer NeuAc @UAC YeuAc NeuAc

I

I

I

4.2. BIODEGRADATION AND STORAGE DISEASES

In the past, particular attention was paid to the enzymes and enzyme activators participating in ganglioside biodegradation because of their involvement in certain hereditary diseases (for reviews, see Refs. 305, 306, 565). Apparently, all gangliosidoses are caused by deficiencies in the activity of degrading hydrolases. The reason for this may be missing or faulty enzyme or activator proteins that bind the ganglioside and present it to the enzymes. Table 3.4 is a classification of gangliosidoses according to the basic defects. “G ,,-gangliosidosis” with an accumulation of ganglioside Gt,,l is believed to result from a structural gene mutation causing the biosynthesis of a P-galactosidase

231 with a much decreased activity. This was concluded because near normal levels of P-galactosidase protein could be demonstrated in this disease [307]. Classical Tay-Sachs disease B and 0 variants are caused by a reduction or absence of ganglioside hexosaminidase. An explanation for the interrelationship of the gangliosidosis variants can be derived from the structural constitution of these enzymes [309,310]. Hexosaminidase A consists of subunits a2 and /I2, each containing two peptide chains linked by a disulfide. Hexosaminidase B has a P2P2 configuration with only one type of peptide. Hexosaminidase S is another enzyme found only in trace amounts. It consists of a2a2subunits only. This explains why hexosaminidase A never occurs without concomitant expression of hexosaminidase B [311]. It is concluded that in variant B of Tay Sachs disease, the a-chain, and in variant 0 the P-chain, of the hexosaminidase are inactive or missing. The degradation of gangliosides by specific hydrolases is greatly enhanced by low molecular weight ( M , , 22 000-25 000 [503]) glycoprotein activators, first isolated from human liver [312,313,504]. The activators are more or less specific for the gangliosides to be hydrolysed. Thus, one activator has been isolated that enhances the hydrolysis of ganglioside G,,J to G,ril by human P-galactosidase [313], and another for degradation of ganglioside G,J to G,,J by P-hexosaminidase [314]. It appears that the specificity requirements of the latter activator glycoprotein are also met when a sulfate group replaces the sialic acid residue, as in the sulfatide I13S0,-GgOse3Cer. This compound, present in rat kidney [472], shows increased hydrolysis by sulfatase in the presence of G,,,l-specific P-hexosaminidase activator (Li and Ishizuka, personal communication). A possible mechanism for the action of glycosphingolipid hydrolase activator protein has been suggested [316,317]. According to this view glycolipid monomers are complexed by the activator protein [503]. For enzymatic hydrolysis, the glycoTABLE 3.4 Ganglioside storage diseases Gangliosidosis

Defect

Storage product

G ,,-gangliosidosis

8-Galactosidase

II'NeuAc-Gg,Cer

Tay-Sachs disease (Variant B)

Hexosaminidase A, S (defect a-chain)

I13NeuAc-Gg,Cer a

Tay-Sachs disease (Variant 0)

Hexosaminidase A, B (defect 8-chain)

11'NeuAc-Gg,Cer Gg'Cer, Gb,Cer

Tay-Sachs disease (Variant AB) New type AB-variant (502)

Activator protein for hexosaminidase /3-Hexosaminidase

I1 'NeuAc-Gg3Cer

Mucolipidosis I1 (I-cell disease)

Acid neuraminidase

113 NeuAc-Gg,Cer

In Tay-Sachs brain, increased amounts of the following gangliosides were also detected: &,l, GF,,lw and Gpen,2a[110,308].

a

232

lipid as well as the activator simultaneously bind to the respective enzyme [316]. This may help to explain why the activator also determines the specificity of a hydrolase towards a glycolipid [317]. Thus, hexosaminidase B is more active towards GgOse,Cer than hexosaminidase A. However, only hexosaminidase A activity against substrate ganglioside GJ, and not that of B-enzyme, is enhanced by activator [318]. This could be the reason for the storage of ganglioside Gtrilin the B-variant of Tay-Sachs disease. Whereas in one type of Tay-Sachs disease AB-variant (AB variant = phexosaminidases A and B, both are present), the accumulation of ganglioside Gtrilis caused by a defect in the enzyme P-hexosaminidase A [502], another type of AB variant shows only a deficiency of an activator for this hydrolase [ 3191. Other sphingolipid storage disorders that are caused by a deficiency of catabolic enzymes not quite directly related to the ganglioside metabolism, by way of a derangement of glycolipid turnover, can also produce an increase of gangliosides, as shown in the case of Gaucher’s disease [602]. 4.3. CHANGES I N I N VIVO COMPOSITION

The component distribution of the gangliosides for a given cell is not always constant. It may change considerably as a result of the interplay of the varying activities of metabolizing enzymes. Occurring ganglioside pattern changes are, however, within the respective cell-specific sialooligosaccharide series. The factors that regulate such changes are not known. The ganglioside profile, significantly and sensitively, depends on the developmental state of the cells and their growth condition. From studies on the changes in the ganglioside pattern it is hoped that perhaps yet unknown functional aspects of these membrane constituents might be revealed. Other changes of gangliosides under pathological conditions, e.g., in the central nervous system, may signal a metabolic derangement with the disappearance of certain cell types by destruction, perhaps even aggravated by autoimmune events, as seen, e.g., in multiple sclerosis [538] (see Section 5.2). 4.3.1. Developmental changes Developmental alterations of ganglioside composition were investigated mostly with embryonal, postnatal, adult and senescent brain, but also with other tissues [510] or with cultured cells. As for sialic acid-containing glycoconjugates, it appears possibly to be a general trend that during embryonal until postnatal life the degree of substitution with sialic acid residues decreases (for reviews, see Refs. 320, 509). Gangliosides are already prominent constituents of the central nervous system at foetal stages of the brain development. Thereby, the quantity of gangliosides increases particularly during periods of rapid outgrowth of dendrites, axons and the formation of neuronal interconnections. But the ganglioside pattern also changes, as has been shown in mammals [321-3231, birds [324-326,248,609,507,569,6691, fish [327] and amphibians [329,669].

233 The available data from different laboratories are not always unequivocal, but justify the following generalizations. At “ very early stages” of development of the embryonal nervous system, gangliosides may function in the transition of neuroblasts into functionally mature neurones. Whereas premitotic cells have a high proportion of G ,ac2,this ganglioside is replaced by higher gangliosides, e.g., Gle12a during the formation of synaptic and dendritic membranes. Gangliosides G,,,3 and G,,,4 are formed during synaptogenesis and thereafter. At “early foetal stages”, the brain gangliosides of birds and mammals show a preponderance of comparably more highly sialylated compounds with Gle13a,3b and 4b more common than Gle12a and 1. Similarly, in embryonic extraneural tissues, chick thigh and leg muscles, the prenatal ganglioside G,,,2 and a GlcNAc-containing di-sialo-component are predominant, whereas the less sialylated ganglioside becomes the major sialo-lipid postnatally [510]. G During “later” foetal brain development, the tri- and tetrasialogangliosides, Gle13a,3b and 4b, decrease in relative amount in favour of Gle12aand 1. At hatching or birth, ganglioside G,,2a is the major brain ganglioside over G,,,3b, 2b and 1. A timely increase in the level of ganglioside G,,J appears to reflect myelination during embryogenesis [609]. The high foetal multisialoganglioside component distribution of birds is similar to that of the adult fish brain [329,330,334,609]; this may be suggestive of certain links between phylogenesis and mammalian brain ontogenesis [348]. Indeed, the brain ganglioside composition during ontogenetic development appears to reflect a phylogenetic recapitulation. During phylogeny to higher species an increase in brain ganglioside concentration is accompanied by a decrease in the more highly sialylated molecular components [508]. At present, a general interpretation of the developmental changes of brain gangliosides is difficult. They may represent alterations in morphology, but equally also reflect differentiation and maturation of membrane structures. Ganghosides Gte,3b and 4b perhaps are typical of early completed connections of foetal brain, whereas ganglioside Glel2a might reflect a later stage and newly completed structures. Gangliosides G,,J and G,,J are typical of mature myelination. A change in cerarmde composition with increasing age was shown for individual human brain gangliosides [332]. Until the age of ten, the ratio of C,, to C,,-sphingoid increases rapidly. It then levels off with 60-70% C,,-sphingoid after 30 years. The fatty acids of ganglioside-ceramide also change with age. At birth, 93% of the ganglioside fatty acids is stearic acid, whereas at age 98 only 78% of this C,,-fatty acid is present. At the same time C,,-fatty acids increase from 3 to 9%. 4.3.2. Changes after nerve stimulation Studies have been reported that deal with the effects of physiological stimulation on ganglioside composition. Thus, the rod outer segments of calf retina show a 40% increase in ganglioside content after stimulation with light, with no change in their component distribution [231]. Behavioural stimulation with rats that were forced to swim in a deep water tank, caused an increase of brain ganglioside G,,,3 with

234 concomitant decrease in G,,,2b [333]. When the weak electric tapir fish was stimulated with a tapir fish dummy, an increased ganglioside biosynthesis could also be observed [511]. 4.3.3. Temperature-adaptive changes in the brain Rahmann and co-workers [512] discovered that the polarity, i.e., the degree of sialylation of brain gangliosides of poikilothermic animals is correlated with the climatic temperature of their habitat. Such animals have increased proportions of more highly sialylated gangliosides the lower their environmental temperature [493]. Thermal adaptation by changing temperature appears possible. Thus, during hibernation, animals have more polar brain gangliosides as compared to those under non-hibernating conditions [513]. Rahmann also speculated that, during the neonatal heterothermic development of birds and mammals, their gangliosides are still correlated with t hermoregulation [493,4941. 4.3.4. Changes in disease The destruction of certain cell types in the central nervous system by disease may be reflected in a quantitative as well as qualitative alteration of the content of ganghoside components. A preferential elimination of nerve cells in Creutzfeld-Jacob subacute spongiform encephalopathy results in a drastic decrease of gangliosides. Thereby, those components are preferentially affected that are typical for neurones, i.e., those with a high content of the C,, long-chain base [635]. In multiple sclerosis, the gangliosides of the central nervous system also show abnormalities. Sclerotic plaques have a complete loss of ganglioside Gga!l and a decrease of G,J and GJ as compared to normal white matter. Interestingly, an elevation of the disialo- and higher oligosialogangliosides in plaque was reported [538]. Tumor cells shed constituents of their plasma membrane. Also among these are ganghosides typical for the respective type of malignant cell of origin. This may explain the increased levels of malignant melanocyte-specific gangliosides in the circulation of tumor bearing individuals [657]. 4.3.5. Changes at the cellular level 4.3.5.1. Normal growth conditions The glycosphingolipid composition of cells, including that of the gangliosides, changes according to the cellular growth phase. Reduction in cell growth or cell arrest, e.g., by cell contact, serum deprivation or induction by drugs, may cause alterations in glycosphingolipid profiles, with an increase in chemical quantity [335,336,340,653]. Furthermore, the mode of growth, in monolayer as compared to suspension culture, can influence the ganglioside pattern in a direction similar to that observed in resting versus rapidly dividing cells [334]. In addition, developmental changes may also be paralleled by an increase in ganghoside. Thus, during differentiation of a cloned rat myoblast cell line to myotubes a three-fold elevation in a ganglioside, tentatively identified as G,,, 2a, was

235 observed [570]. During cell aging, as shown in the case of human fibroblasts, higher gangliosides are drastically decreased with concomitant relative increase in G lacl [631]. In Balb/c 3T3 mouse fibroblasts, the synthesis and quantity of ganglioside G,,J and 2a is particularly h g h at early stages of cell contact; in parallel with a decrease in sialidase activity at the touching phase of the cells [338]. In contrast to this, in plasma membranes obtained from transformed oncogenic cells, as compared to their normal parental untransformed cells, an increase in sialidase activity was observed [339]. In C1300 mouse neuroblastoma cells also, sialidase activity increased in confluent cultures with a concomitant reduction of total ganglioside, but no change in ganglioside composition [653]. The cell contact phenomenon, with an increase in ganglioside G ,aclor G,J concentration in 3T3 mouse fibroblasts, can apparently be mimicked by adding antiganglioside G laclor G1,,1 antibody (Fab-fragment) to the cells [440]. An increase in cell proliferation, however, is not always accompanied by a decrease in the biosynthesis of higher, more complex gangliosides. When stimulation of cell division is paralleled by the expression of differentiation properties, such as in lectin-induced changes in lymphocytes, an elevated biosynthesis of gangliosides can be observed [277,341,342,344]. As yet unrelated to such ganglioside changes as described above, it was reported that treatment of human platelets with thrombin caused a two-fold increase in concentration of G,,J within 10 min [344]. 4.3.5.2. Changes related to cell transformation Oncogenic transformation results in the loss of growth regulation mechanism such as the cell-to-cell contact-dependent inhbition of growth. This loss is generally paralleled by an irreversible reduction in the levels of more complex neutral glycosphingolipids and gangliosides. A reversible phenomenon in a similar direction may be seen in vitro with normal, non-transformed cells that are in a state of rapid division as compared to the resting state, and in vivo with foetal as compared to adult tissues [345] (for reviews, see Refs. 260, 347-349,371). The change in ganglioside biosynthesis in cells undergoing transformation could in many instances be correlated to reduced activities of corresponding glycosyltransferases, thus explaining increases in neutral precursor glycosphingolipids or less complex gangliosides [351,517,5201. There are, however, exceptions where, instead of a decrease, an increase in apparently more complex ganglioside (G,ril)was observed after transformation with an oncogenic virus [518,519]. Infectious processes or drugs that inhibit protein biosynthesis may also interfere with the production of biosynthetic enzymes. The result is a change in ganglioside component profile similar to that generally observed after cell transformation [286,288,352,353,419]. 4.3.5.3. Drug-induced ganglioside changes Since little is known of how ganglioside biosynthesis is regulated by cell factors, drugs that have an influence on ganglioside metabolism may possibly help to shed light on the regulatory mechanism involved in glycosphmgolipid metabolism.

236

An inhibition of ganglioside biosynthesis was observed, when opiate receptorpositive rat neuroblastoma cells were treated with P-endorphin, enkephalins and opiates [354,501,625]. Since these agents effect an alteration in the level of cAMP in the cells with an initial decrease in concentration, it was speculated that ganglioside biosynthesis is somehow regulated by this nucleotide, and hence via a CAMP-mediated kinase system. This hypothesis is supported by the finding that agents which raise cAMP levels, such as cholera toxin, prostaglandin E, , phosphodiesterase inhibitors and dibutyryl- or 8-bromo-CAMP derivatives, also stimulate ganglioside biosynthesis [501]. On the other hand an involvement of protein kinase C in the stimulation of ganglioside biosynthesis is suggested by the action of phorbol esters on cells [638,639]. Other reports on the stimulation of ganglioside biosynthesis by dibutyryl-CAMP are, however, controversial [225,337,354,355]. It is also not clear, whether or not in cases where effects of dibutyryl-CAMP were observed, these were due either to the drug itself or its products of hydrolysis, cAMP or butyrate. An increase of cellular ganglioside biosynthesis is observed following the addition of short-chain fatty acids, including propionate, butyrate and pentanoate. The effect of butyrate could be related to an induction of a CMP-sialic acid Aactosylceramide sialyltransferase, while other glycosyltransferases remain unaffected [286-2881. Norepinephrine, which increases the cAMP level in cells, also stimulated ganglioside biosynthesis in C 1300 mouse neuroblastoma cells due to increased activity of a UDP-GalNAc: G GalNAc-transferase [3561. 4.3.5.4. Changes by uptake of exogenous-ganglioside It is still largely unknown, which particular biological properties of a membrane result from the presence of ganghosides. To solve this problem with a different approach, attempts were made to artificially incorporate exogenous gangliosides into cells. Gangliosides are taken up from the incubation medium by cells or isolated cellular membranes [357]. This uptake is saturable and may depend on the presence of calcium [528]. The cell-associated ganglioside is detected at the cell surface by galactose oxidase-labeling or susceptibility to neuraminidase [586]. Ganglioside uptake varies with the cell type and cell growth conditions, e.g., monolayer versus suspension cells [359,360].In addition, a quantitatively different uptake of exogenous ganglioside is observed with cells in M-phase as compared to randomly growing cells [360,514]. Exogenous ganglioside becomes membrane-associated by different modes of binding. Part of the added sialoglycolipid may be released from the cells by mild trypsinization or incubation with serum or serum albumin [514].During longer incubation with ganglioside (a few hours at 37 "C),the serum- and trypsin-stable portion increases, and a true insertion of the glycolipid into the lipid bilayer of the biological membrane is achieved. Sialoglycolipid incorporation into the membrane lipid phase could be shown with fluorescent or electron paramagnetically labeled ganglioside analogues [470,472,5 1 4 315,5641. When ganglioside is added to preformed phospholipid liposomes, the sialoglycolipid is rapidly incorporated into the membrane bilayer [361-363,5601. Such liposome-associated ganglioside is not removed by incubation of the liposomes with

237 serum, serum albumin or chase-ganglioside. There is no indication that ganglioside incorporated into the outer lipid bilayer leaflet undergoes “flip-flop” to the inner leaflet or that it may disrupt the bilayer integrity of the liposome [560]. Incubation of intact cells with exogenous sialoglycolipids results in their slow lysosomal degradation and also in an increased biosynthesis of more complex gangliosides [291,666]. Thus, free ganglioside added to primary cultures of chick embryonal neurons was a substrate for glycosyltransferases of the cells catalyzing the reactions [579]: G,,,1

+ UDP-(14C)Gal

-

G,,,l and

G,,,I+ CMP-( I4C)NeuAc + GIeI2a

Cell accumulation of exogenous ganglioside may be paralleled by various biological alterations. Most generally, after association of ganglioside, cells display an increased adhesiveness to the substrate [603], and a reversible inhibition of proliferation [358,599,616,627]. Under these conditions, a prolongation of the generation time, and of all phases of the cell cycle, was seen with astrocytoma cells [655]. The mechanism of ganglioside-induced growth inhibition is not yet known. There are, however, indications that a modulation of growth factor receptors by ganglioside could be involved [585]. On the other hand, a shielding effect for surface receptors by exogenously added ganglioside may explain its inhibitory effect for the mitogenic response of lymphocytes to concanavalin A (6161 or rosette formation with autologous erythrocytes [651]. Ganglioside association to neuronal cells may cause an outgrowth of neuritic processes that is interpreted to represent a cell differentiation phenomenon [526,578,580,650]. In addition, synapse formation is accelerated at neuromuscular junctions [525]. Involvement of gangliosides in these processes is also indicated by the observations that antiganglioside antibodies can inhibit neuritic outgrowth [596,612]. In primary neurons, added ganglioside effects a considerable extension of their survival time [573]. In vivo, ganglioside is able to stimulate reinnervations after seizure of peripheral nerve [42]. This regenerative property may involve the immune system, since ganglioside antibodies are produced following nerve injury [607]. Exogenous ganglioside taken-up by the cell may act as a functional receptor or mediator for signals from effector molecules, viruses or cells. This was shown towards cholera toxin [521], lymphokines (MIF/MAF) [529] or infection by Sendai virus [522]. An instance where exogenously incorporated ganglioside might function in the cellular reception of signals from other cells was reported for immune cells. Splenocytes, after incubation with ganglioside Gt,,4b, could be induced to transform in a system with autologous lymphocytes [559]. In explaining the biological activities of exogenous ganglioside, it must be

238 considered that its cell or cellular membrane association may also result in alterations in the activity of certain enzymes. Some of these are membrane-bound as in the case of an activation of adenylate cyclase * [524,576] and of (Na+,K+)-ATP-ase [523,575]. In other instances soluble (cytosol Ca*+-dependent nucleotide phosphodiesterase [525,575]) and solubilized ((Mg'+)-ATPase [526]) enzymes, are activated by added ganglioside.

5. Immuno-properties of gangliosides 5.1. GENERAL

Several reviews have in the past comprehensively dealt with the general immunological properties of the glycosphingolipids, including gangliosides (Rapport and Graf (1969) [364], Marcus and Schwarting (1976) [365], Alving (1977) [388], Yogeeswaran (1980) [622] and Marcus and Kundu (1980) [389]. Gangliosides contribute to the immunological expression of cells. Antibodies to ganghosides have therefore been used as markers for the specific immunological distinction of cell types of the immune [531,532] and nervous systems [367,368,533, 6171. They have further been applied to ultrastructural localization and in neurophysiology. Another example of the cell-typical expression of gangliosides is their detection by monoclonal antibodies, in particular as prominent cell surface antigens in certain tumor tissues, such as colon carcinoma [535] and human melanoma [536,664-6681. The immunological expression of a ganglioside may involve complexing proteins. An example for this was shown with ganglioside G,J which binds to protein that can be coextracted from bovine red blood cell membranes. This protein displays Paul-Bunnel antigen specificity, which is strongly enhanced by the presence of the ganglioside [369]. Gangliosides by themselves are poor immunogens. However, with particular immunization procedures specific antisera can be raised that have widely been used for the study and biological characterisation of gangliosides. Ganglioside antibodies can be induced by injection of lipid micelles and methylated bovine serum albumin [370] (for reviews on methodology, see Refs. 366, 371, 388). With this immunization procedure high titre antisera could be raised against gangliosides G,,,1 and G,,, 2b [373]. Gangliosides with terminal sialic acid residues, however, produced only negligible levels of specific antibodies. In another approach effective production of antiganglioside antibodies was induced by immunization with sialoglycolipid coupled, through their carboxyl groups, to a protein carrier [390,534]. With this technique, IgG-antibodies were obtained against ganglioside G ,==2.Antibodies that are directed against the sialooligosaccharide moiety of a ganglioside can also be produced with a sialooligosac* An inhibition of this enzyme by ganglioside was also recently reported [674].

239 charide-protein conjugate prepared from the liberated ganglioside sugar portion [391,392]: R . CH=O + H 2 N CH 2 C H 2 . $J.N H 2 +matrix - - N=C=S b

NaBH,

R .CH 2 . N H,CH 2 . @ .N H 2

R . C H 2 . N H . C H , . @ . N H . C S .NH-+matrix

The specificity of anti-ganglioside G ,aclNeuGcantibodies was shown to be directed mainly against the N-glycolylneuraminic acid residue [3741. The immunogenicity of this sialic acid also appears to be involved in the immunopathology observed in humans, i.e., “serum sickness”. In patients with t h s condition, who have received animal sera, heterophilic antibodies were found directed against gangliosides with terminal N-glycolylneuraminic acid residues [375]. Another example of the occurrence of anti-sialic acid antibodies was the isolation of a monoclonal IgM (agglutinin MKV) from a patient with Waldenstrom’s macroglobulinemia. The macroglobulin was reported to specifically cold-agglutinate erythrocytes by binding to N acetylneuraminic acid containing glycoconjugates including ganglioside G ,acl[376]. Antisera to gangliosides exhibit a number of biological actions in cellular systems that reflect the possible involvement of gangliosides in cell surface membrane-mediated processes of cell regulation. Thus, ganglioside G,,,l-antibodies are able to stimulate mitosis in rat thymocytes [377]. Monovalent Fab-fragments of ganglioside G ,,,lNeuAc- and G,,,l-antibodies decrease cell growth of Balb/3T3 and Nil fibroblasts and effectively inhibit the expression of transformed phenotypes in virus-transformed cells [378,350]. 5.2. INVOLVEMENT IN DISEASE

There is reason to believe that brain gangliosides can induce experimentally pathological reactions in nervous tissue that closely resemble encephalomyelitis, with concomitant extensive degeneration of peripheral nerves [372,471,537]. 5.2.1. Animal models

An autoimmune neurological syndrome similar to experimental allergic encephalomyelitis could be induced in rabbits by immunization with gangliosides G,J and G,,,2a [658]. Induction of experimental allergic neuritis by myelin protein fraction P2 is enhanced when applied together with ganglioside [659]. A multiple sclerosis-like central chronic neurological disease with no peripheral damage could also be observed in rabbits immunized with ganglioside [543,632]. 5.2.2. Anti-ganglioside immune activities in human pathology Sera from patients with a wide variety of disorders - schizophrenia, brain tumours and amyotrophc lateral sclerosis - show the presence of anti-ganglioside activities [663]. Sera from animals that were immunized, with ganglioside derivatives of

240

gangliotetraosylceramidefrequently show cross-reactivity with this neutral GSL. The presence of antibodies against gangliotetraosylceramidewas found in lupus erythematode patients [660] and in autoimmune thyroid disorders [661]. Certain cold agglutinins, in particular Pr, Gd, Sa and Fe antibodies, are directed against sialic acid-containing determinants, whereby the latter antibody reacts with a branched chain ganglioside of the structure NeuAccr2-3Gal~l-4GlcNAc~1-3[Fucal-2Gal~l4GlcNAcP1-6]Gal/31-4GlcNAc/31-3Gal/31-4GlcPl-Cer [5951. Antibodies against gangliosides, however, were also demonstrated in apparently normal healthy subjects [662]. Multiple sclerosis (MS) is one other neurological disorder where immunoreactions against brain gangliosides appear to be involved. Peripheral lymphocytes of MS patients show sensitization against MS-brain extracts when measured by an active erythrocyte-rosette formation test [539,540]. In this test system, gangliosides as well as cerebroside could produce a rise in active erythrocyte rosettes [541]. The most effective gangliosides thereby were tri- and tetrasialo species [542]. Total gangliosides from MS patients were more effective in the stimulation of active erythrocyte rosetting of MS lymphocytes than normal brain gangliosides, a fact that might be related to a higher content of ganglioside G,,,4 in MS brain [538]. In another assay system, i.e., by immune lysis of ganglioside-containing liposomes, it was also found that sera of MS patients contained a humoral serological activity directed against gangliosides such as Gga,l,G,,1 and G,,,1 [544,545].

6. Ligand-binding properties of gangliosides 6.1. GENERAL

It can be expected that whatever the biological significance of gangliosides will be, they will function in conjunction with other molecules - possibly proteins - that are able to more or less tightly interact with them. Due to their highly amphiphilic nature and their capacity to carry an electrical charge, the gangliosides are very “sticky” molecules. In an aqueous environment, gangliosides aggregate either with themselves or with other complexing molecules in the medium. Such interactions may thereby involve only lipophilic forces via the ceramide, or only hydrophilic binding via the sugar moiety. Complexing to the sialooligosaccharide will show the greater degree of specificity and allow third partner binding to the ceramide. Several instances of ganglioside-ligand binding, however, may involve both the lipophilic, as well as the hydrophilic, portion of their molecule. 6.2. GANGLIOSIDE COMPLEXING WITH LlGAND PROTEIN

Albumin is known to have a preferential binding capacity for anionically charged lipids and, as such, strongly binds to gangliosides. This phenomenon was therefore studied as an example of an interaction between ganglioside and protein that may have relevance to other similar, more biological instances [172,363,546,547,610].Gel

241 permeation chromatography, as well as ultracentrifugation studies, revealed the existence of two types of ganglioside-albumin complexes depending on the molar ratio of the ligands. Both complexes are dispersed by 0.1% Triton X-100 an indication of the comparatively nonspecific nature of the hydrophobic interactions. At a high ganglioside ratio, the complex is made up of ganglioside micelles that contain one or more molecules of albumin, whereas, at a low ratio, the ligands bind in a molar one-to-one fashion. The latter complex consists of a ganglioside monomer bound to albumin. It appears that albumin may associate with the ganglioside micelle, then again leaving this lipid aggregate carrying a molecule of ganglioside with it. Serving as a carrier for ganglioside in a one-to-one molar ratio, albumin seems to behave similarly to the purified lipid transfer protein from beef liver [561] or the /3-hexosaminidase activator protein [363,503]. In contrast to the latter two proteins, however, albumin was not shown to be able to extract single ganglioside molecules from liposomal phospholipid membranes. 6.3. INTERACTION WITH LECTINS

Since gangliosides contain complex carbohydrate they are able to bind to lectins of various origins. Wheat germ agglutinin has a specific N-acetylglucosamine binding site. This site obviously is responsible for interaction also with sialic acid and sialic acid-containing glycolipids [379]. Another lectin, limulin (Limufus polyphemus) agglutinates preferentially horse erythrocytes known to contain a high amount of ganglioside G JNeuGc [380]. It was found that limulin specifically binds to the N-glycolylneuraminic acid of this ganglioside [379]. Recently, a perhaps similar lectin was isolated from Carcinoscorpius rotunda cauda, named carcinoscorpin [420]. This lectin specifically binds NeuAca2-6[2-deoxy-2-N-acetamido-arabitol]. The lectin RCA, of Ricinus communis specifically complexes terminal /3-galactopyranoside. It therefore also interacts with gangliosides having this terminal group, as, e.g., in ganglioside G,,,l [381,382]. A case where perhaps specific cellular adhesiveness is mediated by a cell-surface lectin recognizing terminal N-acetyl-galactosamine of ganglioside G,,1 was reported by Marchase for chick neural retinal cells and surfaces of intact optic tecta [548]. 6.4. INTERACTION WITH TOXINS, HORMONES, INTERFERON AND CELL GROWTH AND

DIFFERENTIATION FACTORS

The pioneering work of W.E. van Heyningen opened up one of the most exciting chapters of ganglioside research, namely their interaction with the exotoxins of Clostridium tetani and Vibrio cholerae (for reviews, see Refs. 383-386,616). It was felt that the interaction between gangliosides and bacterial toxins deserved special interest, because this phenomenon indeed showed many of the characteristics of the binding of physiological effectors, such as hormones, to cell-surface receptors. The following toxins were reported to bind to ganglioside (References are initial reports): the Clostridia neurotoxins, tetanus toxin [393-3951; botulinus toxin [396];

242 the exo-enterotoxins, cholera toxin [397]; Escherichia cofi toxin [398]; Staphylococcus a-toxin [399]; the haemolysins of Streptococcus parahemolyticus [400]; and that of the sea wasp [401]. The toxins most thoroughly investigated with regard to ganglioside interaction are those of tetanus and cholera. Tetanus toxin in its extracellular form consists of two peptide chains ( M , approx. 100000 and 40000) linked by a disulfide bond [404] (for review, see Ref. 601). The heavy chain contains the ganglioside binding site [402,403]. There is no strict specificity on the side of the gangliosides. Several of them belonging to the ganglio-series are able to fix tetanus toxin. There is, however, some selectivity of binding, i.e., G,,,2b = G,,,3b >> G,,,1 = G,,,2a >> Glril [405-4081 with a preference for gangliosides that carry two sialic acid residues at the non-terminal galactose of gangliotetraose. In one assay system, employing Sephadex-adsorbed tetanus toxin, however, all gangliosides tested, including G,J, G,ril, G,,,l, 2b and 3b as well as unrelated synthetic sialoglycolipids, bound the toxin in a 1 : 1 molar ratio with high affinity, and to a comparable extent [403]. It can at present not be excluded that this preference for binding to certain ganglioside structures is influenced by matrix molecules used in the assay systems, e.g., a cerebroside preparation [405,407], ganglioside-containing liposomes [408] or native membranes of brain [409,410]. A prerequisite for binding to tetanus toxin appears to be the presence of a lipophilic moiety in the sialoglycoconjugate. Ganglioside-derived free sialooligosaccharide could not be shown to bind to tetanus toxin (Wiegandt, unpublished observations). Whereas at the molecular level the specificity of binding of tetanus toxin to certain isolated ganglioside species is less pronounced, its association with cells shows a high degree of selectivity. Cells of central neuronal origin, preferentially bind tetanus toxin [604]. T h s may be related to an exposure of the central neuron-typical di- and trisialogangliosides G,,,2b and 3b at the surface of these cells. It is speculated that, after its fixation to ganglioside centers, the tetanus toxin may be translocated and sequestered by other membranal structures that provide for its further intraaxonal transport to presynaptic terminals [634]. There is obvious parallelism for the binding of tetanus toxin and thyrotropin (thyroid-stimulating hormone) to membranes of the thyroid gland [413]. Ganglioside binding to thyrotropin shows an efficiency similar to that observed for tetanus toxin: G,,,2b >> G,,,3b > G,,,1 > G,ril = CI,,,~> G,,,2a [412,416,417].It was therefore postulated that gangliosides are involved as receptors for both the hormone and the toxin [412,414,415]. Speculations that only gangliosides act as cell receptors for tetanus toxin and thyrotropin are contradicted by the observation that neuroblastoma C 1300 cells, pretreated with neuraminidase and fl-galactosidase, still are able to fix tetanus toxin by a mechanism that may be unrelated to ganglioside [411]. Both effectors also specifically bind to a glycoprotein component from thyroid gland [418]. Perhaps both ganglioside and a specifically binding membrane glycoprotein may be involved

243 in the mechanism of reception, and the mediation of effector information [412]. Further doubt was cast on the assumption that gangliosides might serve as thyroid-stimulating hormone receptors by the finding that neurarninidase treatment of thyroid cells converting more complex gangliosides to ganglioside G,,,1 did not change binding of the hormone. Furthermore, down regulation of its receptors by thyrotropin has no effect on the distribution of gangliosides [549]. Cholera toxin binds specifically and multivalently to ganglioside G,J. The ganglioside G,,,2b binds some ten-times less strongly to the toxin as compared to G,,,1 [626] (for reviews, see Refs. 425, 385, 426, 486, 550, 616). Of the two cholera toxin promoter subunits, the A-protein, carrying ADP-ribosyltransferase activity, and the pentamer B-protein, only the latter binds to ganglioside [427]. In the binding, only part of the monosialogangliotetraose, the carbohydrate moiety of ganglioside G,,,l, is specifically involved [428-430,6281. Alteration of the 113-monosialogangliotetraose by substitution of the terminal galactose by fucose in 2-position, or reduction of the sialic acid-carboxyl group, as well as removal of galactose or sialic acid lead to a loss of binding capacity for cholera toxin. Integrity of the glucose residues appears not to be necessary for the toxin binding. Detailed studies showed that with cholera toxin only the sugar moiety of ganglioside G,,,1 is involved, whereas the ceramide provides an anchorage for the toxin at the cell membrane [429,431]. At present the mechanism of the ganglioside-dependent transmembrane events induced by the cholera toxin are not yet known in detail. After attachment to a cell-surface membrane the disulfide bond between the two subunits ( M , , 24 000 and 5400) of the A-protein is broken, and the larger peptide, that is hydrophobic, penetrates deeply into the lipid bilayer [551,623,624]. Specific disulfide bond reduction therefore appears necessary for the choleragenic action of the toxin. Since cholera toxin can induce redistribution of membrane constituents that are believed to be connected with the cytoskeletal system, it is also speculated that ganglioside membrane protein interactions could be involved [432-434,552,5531. A possible role of membrane ganglioside as receptor for interferon has also been suggested [435,437,554,555].Similar to tetanus toxin or thyrotropin, the specificity of binding to interferon is not unequivocal and restricted to one ganglioside, but decreases in effectiveness in the following order; G,,1 >> G,,,3b > G,,,l >> Gte12a> G,aJ *. Interferon-ganglioside interaction, however, appears not to be a general property of all types of interferon. Of the two interferon species detected in mouse fibroblasts, only one (type I) binds to ganglioside [438,555,572]. There is some indication that gangliosides may possibly function as receptors for certain cell growth and differentiation factors. One example for this is an L-cellderived factor that can stimulate the clonal growth of granulocyte macrophage ~~

* See, however, Ref. 555: mouse interferon type 1 is neutralized in the following order: G,,,3b.

G1,,2b >> Glr,l: no binding by G,J or neutral glycosphingolipid. Also Ref. 554: Glr,l, G , J and Lac-Cer, all neutralize the antiviral action of interferon.

244 progenitor cells. Tlus factor is fixed by ganglioside in the following order of efficiency: G,,,1 > G,,1 > Gtet2a> G,,,3b [439]. Other findings are also in support of a possible receptor function of gangliosides for growth factors. Culturing cells in media that have been passed over a gangliotetraosylceramide or a ganglioside GJ-affinity column do not support the growth of 3T3-mouse fibroblast cells [440]. In addition, pretreatment of 3T3-cells with monovalent antibodies to gangliotetraosylceramide or to ganglioside G,,,l inhibits growth stimulation by serum. This might be interpreted as a masking of serum growth factor reception sites [440]. Another interesting example of the possible role of gangliosides as receptors for factors that influence cell behavior may concern lymphokine action on macrophages [556]. The macrophage migration inhibition factor (MIF) and macrophage activation factor (MAF) activities of culture supernatants of concanavalin A-stimulated lymphocytes can be abolished with a total brain ganglioside fraction [529]. Further indication of the involvement of ganglioside is the report that macrophages show an enhanced responsiveness to MIF after incubation with ganglioside-containing liposomes [529]. Even though the putative ganglioside receptor has not yet been characterized, it is believed to carry a terminal a-fucose residue, since this sugar is inhibitory for the MIF [557,558]. Sialooligosaccharide structures at the cell surface present receptors for viruses, such as paramyxovirus, influenza virus, encephalomyocarditis virus and Sendai virus [597,598]. A specific function of ganglioside in the cell reception for a virus was described in the case of Sendai virus [522]. Whereas sialidase treatment of cells makes them resistant to infection, incubation with ganglioside carrying a NeuAc-Gal-GalNActerminus restores susceptibility for the virus [522]. Due to their highly amphiphilic nature, gangliosides can act as rather sticky molecules. It is for this property that “receptor” functions may be observed for gangliosides that indeed are of no true biological significance. An example for this perhaps is reflected in the ability of gangliosides to inhibit a fibronectin-mediated cell attachment to collagen- or fibronectin-coated substrates in a nonspecific manner [5 88- 5901. 6.5. INTERACTION WITH NEUROTROPIC AGENTS

Wolley and Gommi [441,442] originally observed that the serotonin sensitivity of a neuraminidase-treated fundus preparation could be restored by adding ganglioside, in particular G,,,2. The involvement of sialic acid conjugates in the serotonin transport system could also be demonstrated in rat brain synaptosomes [445]. The question, however, whether or not gangliosides constitute serotonin tissue receptors has not yet been answered unequivocally (for review, see Ref. 383). Serotonin not only binds to gangliosides but also to other sialoglycoconjugates, e.g., fetuin [443]. Whereas the ion permeability of ganglioside-containing liposomes was not changed [443], release of glucose could be effected with serotonin and other biogenic amines

245

[a]. Tamir et al. [559]could see no binding of gangliosides to serotonin at relevant concentrations. These authors, however, made another interesting observation that may shed more light on the heretofore equivocal subject. In the presence of other lipids, e.g., lecithin and Fe2+, ganglioside, especially G ,ac2, strongly enhances the fixation of serotonin by the serotonin-binding protein [559]. It was speculated that possibly the interaction of ganglioside with serotonin-binding protein may regulate the concentration of the biogenic amine in the synapse. Other drugs that bind to ganglioside are d-tubocurarine [446], chlorpromazine [447] and colchiceine [448]. 7. Concluding remarks It is obvious that gangliosides appear to be involved in an embarrassing multitude of biological phenomena. Still, it is not yet possible to name clearly one universal role played by gangliosides in the life of cells, singly or in a tissue. The same holds true for the neutral members of the glycosphingolipid family, for which also no unified explanation of their biological significance can be offered at present. Even though t h s review attempts to keep the gangliosides “ under surveillance”, it may perhaps not be justified to consider the possible physiological function of the sialic acid-containing species only. However, considering the ubiquity of distribution of these plasma membrane constituents, future research of gangliosides is encouraged by the intriguing possibilities inherent in the complexity of these molecules as an expression of cell differentiation properties.

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