Aggregation properties of semisynthetic GD1a ganglioside (IV3Neu5ACII3Neu5AcGgOse4Cer) containing an acetyl group as acyl moiety

Chemistr,/and PhDia of ELSEVIER

Chemistry and Physics of Lipids 77 (1995) 41-49

LIPID$

Aggregation properties of semisynthetic GDla ganglioside (IV3Neu5AclI3Neu5AcGgOseaCer) containing an acetyl group as acyl moiety Paola Brocca, Laura Cantu', Sandro Sonnino* Study Center for the Functional Biochemistry of Brain Lipids, Department of Medical Chemistry and Biochemistry, The Medical School, University of Milan, 20133 Milano, Italy Received 24 November 1994; revision received 18 April 1995; accepted 18 April 1995

Abstract G D l a ganglioside containing an acetyl group as acyl moiety, GDla(acetyl), was synthesized from natural GDIa. The aggregative properties in aqueous solution of GDla(acetyl) have been studied by static and dynamic laser light-scattering measurements. GDla(acetyl) spontaneously aggregates as small micelles showing a hydrodynamic radius and molecular mass of 33 ./k and 96 kDa, respectively. Vibrio cholerae sialidase showed a very high activity on the micelles of GDla(acetyl), compared to GDIa. This has been explained as a consequence of the high surface curvature of the the small micelles. High resolution proton N M R spectra were recorded from micelles of GDla(acetyl) in deuterated water. The low overall correlation time of the GDla(acetyl) micelles was calculated to be about 2 x 10 -:~ s, a value one order of magnitude lower than that determined for natural GDla. Keywords: Gangliosides; Ganglioside derivatives; Ganglioside aggregates; Light-scattering; N M R

1. Introduction Abbreviations: Ganglioside nomenclature is in accordance with Svennerholm [47] and IUPAC-IUB recommendations [48]. GM1, II3Neu5AcGgOse4Cer, fl-Gal-(1-3)-fl-GalNAc-(l4)-[~t-Neu5Ac-(2-3)]-fl-Gal-(l-4)-fl-Glc-(1-1)-Cer; GDla, IVaNeu5AcII3NeuSAcGgOse4Cer, ~t-Neu5Ac-(2-3)-fl-Gal-(l3)-fl-GalNAc-( 1-4)-[ct-Neu5A-(2-3)]-fl-Gal-(l-4)]-fl-GIc-(1- l )Cer; Cer, ceramide; Neu5Ac, N-acetylneuraminic acid; Sph, sphingosine; GMl(acetyl) and GDla(acetyl), gangliosides which contain an zLcetylgroup as acyl moiety; DPPC, dipalmitoylphosphatidyld~tolit/e. * Corresponding; author, Dipartimento di Chimica e Biochimica Medic~t, Via Saldini 50, 1-20133 Milano, Italy. Fax: + 39 2 2363584; E-mail address, [email protected].

Gangliosides [1], glycosphingolipids characterized by the presence o f sialic acid residue(s), are c o m p o n e n t s o f vertebrate cell plasma m e m b r a n e where they play an i m p o r t a n t role in a variety o f surface events such as recognition o f external ligands, biotransduction o f membrane-mediated information and m o d u l a t i o n o f m e m b r a n e - b o u n d enzyme activities [2-16]. These features p r o b a b l y originate from specific ganglioside-protein interactions occurring at the m e m b r a n e surface [17] and

0009-3084/95/$09.50 © 1995 Elsevier Science Ireland Ltd. All rights reserved

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P. Brocca et al. / Chemistry and Physics of Lipids 77 (1995) 41-49

can, in part, be related to the conformational, dynamic and geometrical properties of gangliosides [18-19]. These properties play a role in the formation of a net of interactions, including hydrogen bonding, with the protein, while contributing to determine the organization of the ganglioside microenvironment and the degree of crypticity of the ganglioside oligosaccharide chain embedded and, eventually segregated, in the phospholipid hydrophilic layer [20-21]. The marked amphiphilic character of gangliosides leads them to form aggregates, or mixed aggregates with other amphiphilic compounds [19] of high molecular mass, in aqueous solution. These aggregates are useful models to study several physico-chemical, enzymatic and biological features of membrane- containing gangliosides [22-24]. From the physico-chemical point of view, the aggregation properties of amphiphilic compounds are strictly related to the structural properties of both the hydrophilic and hydrophobic moieties [19]. As far as the hydrophobic contribution is concerned, it has already been shown that GM1 ganglioside, which contains stearic acid as acyl moiety, shows a critical micellar concentration of about 10 -8 M and aggregates as micelles of 470 kDa and axial ratio over 2, while the substitution of the stearoyl group with the acetyl one, which causes a strong reduction of the hydrophobic volume, drives the modified ganglioside to aggregate in small (100 kDa) and spherical micelles [25] in equilibrium with 10-5 M free monomers. On the other side, short acyl chain-containing gangliosides bind in large amounts to cells in culture [26], then becoming membrane components and resulting in a protective effect against glutamate neurotoxicity [27]. In this paper, we describe the semisynthetic preparation of G D l a ganglioside with a ceramide species containing the acetyl group as acyl moiety, GDla(acetyl), and present some characteristics, both physico-chemical and enzymatic, of the ganglioside aggregates, pure or mixed with phospholipids, in solution. We also propose that short acyl chain-containing gangliosides could be suitable systems to perform N M R spectroscopic studies in water solution on ganglioside sugar chain proper-

ties in an environment enriched in gangliosides, with some similarities to a segregated domain in a membrane. 2. Materials and methods 2.1. Materials

Commercial chemicals were of analytical grade or the highest purity available. Common solvents were redistilled before use, and water for routine use was freshly redistilled in a glass apparatus. Methanol was dehydrated by refluxing over and distilling from metallic magnesium. Silica gel 100 for column chromatography (0.063-0.2 mm, 70230 mesh, ASTM), high performance silica gel precoated thin-layer plates (HPTLC Kieselgel 60, l0 × 10 cm), LiChroprep RP-18 (40-63 /Lm), acetic anhydride, (CD3)250 and D20 were purchased from Merck GmbH (Germany). Sepharose 4B was from Pharmacia Fine Chemicals (Sweden); Chelex-100 000-200 mesh, sodium form) and Biogel P2 (200-400 mesh) x;eere from Biorad (USA). N-acetylneuraminic acid and Vibrio cholerae sialidase (EC 3.2.1.18) were from Sigma Chemical CO. (USA). Dipalmitoylphosphatidylcholine was from BDH (England). Ganglioside G D l a was extracted [28] from calf brain, purified to over 99% and characterized [29]. Its lipid composition was: fatty acids: C16:0, 3.8%; C18:0, 88%; C18:1, 1.7%; C20:0, 6.5%; long chain bases: C18:0, 2.3%; C18:1, 43%; C20:0, 2.7%; C20:1, 52.0%. 2.2. Preparation o f GD la(acetyl)

G D l a (500 mg) was dissolved, and maintained for 15 h under continuous stirring at 100°C, in 50 ml of 1 M solution of tetramethylammoniumhydroxide in butan-l-ol/water (9:1 v/v) [30]. The total reaction mixture was neutralized with acetic acid, dried, dissolved in water and applied to a 2 x 10 cm LiChroprep RP-18 column. After washing with 300 ml of water the ganglioside derivatives were eluted with 300 ml of methanol. The desalted ganglioside reaction mixture was dried, maintained under high vacuum for one night and dissolved in 200 ml of dehydrated methanol. Then, 200/~1 of triethylamine and 100 pl of acetic anhydride were added to the solution which was

P. Brocca et aL / Chemistry and Physics of Lipids 77 (1995) 41-49

maintained at room temperature, under stirring, for 30 min. The reaction mixture was then dried and purified on a 2 x 100 cm silica gel 100 column chromatograph equilibrated and eluted with the solvent system chloroform/methanol/water (60:35:5 v/v). The elution profile was monitored by TLC (Section 2.6). Fractions containing G D l a ( a c e t y l ) (-R.fr = 0.5) were collected and submitted to a farther column chromatography purification. GD, la(acetyl) (100 mg) was dissolved in 2 ml of water, dialyzed, freeze-dried, solubilized again in 2 ml of water and precipitated with 8 ml of acetone. GDla(acetyl), as a white powder, after drying under high vacuum, was stored at - 20°C. Chemical characterization of GDla(acetyl) was carried out by N M R spectroscopy (Section 2.6).

2.3. Laser light-scattering measurements The presence of GDla(acetyl) aggregates in solution, their hydrodynamic radius, molecular weight and shape, were determined by static and dynamic laser light-scattering analyses. The apparatus and the techniques have already been described in detail elsewhere [19,31]. Measurements were performed at 25°C and 36°C, on 10-3-10 -4 M GDla(acetyl), and 3 x 10 -2 M NaCI solutions. Some experiments on G D l a solutions were performed at 36°C. 2.4. Preparation of vesicles of dipalmitoylphosphatidylcholine (DPPC) and gangliosides GD la(acetyl) and GD la DPPC (dried under high vacuum from chloroform/methanol, 2:1 v/vl) was suspended and vortexed in 50 mM, pH 7, Tris-HC1 buffer at 50°C, at a final concex~ttration of 28 mM. The dispersion was extruded through 0.1 p m pre-filter (Nucleopore, USA) using a nitrogen-operated apparatus (Lipoprep, Canada). This procedure resulted, as previously reported [32-33], in the formation of unilamellar vesicles. The polydispersity and size of these vesicles was determined by laser light-scattering technique',. The polydispersity index ~r, as obtained from a cumulant analysis [31] of the time-dependent part of the correlation function of the scattered intensity, was 5% ( + 1%), and the diameter was 1000 _ 50/~.

43

DPPC vesicles were incubated at 50°C for 36 h with different amounts (2.5 and 5 mol% of total lipid) of G D l a or GDla(acetyl), for ganglioside insertion into the outer membrane layer [34]. The insertion of ganglioside into the vesicles and the final DPPC/ganglioside molar ratio were checked after ultracentrifugation of the dispersed lipids, followed by phospholipid and ganglioside analyses of the pelletted lyposomes.

2.5. Sialidase assay Solutions of micelles of G D l a and GDla(acetyl) or dispersion of mixed vesicles of each ganglioside with dipalmitoylphosphatidylcholine, in 50 mM, pH 7, Tris-HCl buffer (90 pl), were incubated with Vibrio cholerae sialidase (10 pl, different dilutions) at 36°C for 20 min and under continuous shaking. The control incubation mixtures (blanks) were complete incubation mixtures, but lacking the enzyme. The enzymatic reaction was stopped by dipping the reaction tubes in cold water and by adding to the solutions 100 pl of sodium periodate. The enzyme kinetic parameters, the maximum activity Vmaxand the Michaelis's constant Km, i.e. the affinity to the substrate, were calculated by the method of Lineweaver & Burk after fitting the experimental data points (as average of three experiments) to a rectangular hyperbola. 2.6. Analytical procedures Ganglioside-bound sialic acid and free sialic acid were assayed by the resorcinol-HC1 [35-36] and thiobarbituric [37] method, respectively, pure Neu5Ac being used as reference standard. Lipidbound phosphorus was assayed according to Bartlett [38]. TLC of the gangliosides and ganglioside derivatives, of the deacylation and acetylation reaction mixtures and of the chromatography purification fractions, was carried out using the solvent systems chloroform/methanol/0.2% aqueous CaCI2 (50:42:11 v/v). Spots were made visible by treatment with a p-dimethylaminobenzaldehyde spray reagent [39] followed by heating at 120°C for 20 min. 1H-NMR spectra were obtained at 500 MHz on a Bruker AM500 spectrometer equipped with the

44

P. Brocca et al. / Chemistry and Physics of Lipids 77 (1995) 41 49

ASPECT 3000 computer. Before NMR analysis, gangliosides were passed through a Chelex-100 column, sodium salt resin (3 × 0.5 cm, pH 7), to eliminate traces of divalent cations which lead to signal broadening [40]. After this, samples were carefully dried under vacuum and then dissolved in DMSO-d6, DMSO-d6/D20 (20:1 v/v) or DzO. Proton assignment was made by chemical shift correlation spectroscopy (COSY) and total correlation spectroscopy (TOCSY) experiments. For the assignement of Gal IV, Gal II and GalNAc methylen spin systems, heteronuclear single-quantum coherence (HSQC) spectroscopy and 1D-selective NOE in rotating-frame (1D-ROESY) experiments were used. Selective excitations were realized by means of DANTE pulse trains [41]. 3. Results and discussion 3.1. Synthesis o f GD la(acetyl)

GDla(acetyl) was prepared from natural GDla. The procedure consists in an alkaline hydrolysis, in the presence of tetramethylammoniumhydroxide, which yields a mixture of deacetylated and deacetylated-deacylated ganglioside derivatives [30], followed by a N-acetylation reaction. Several G D l a derivatives were found in the total reaction mixture. Treatment with acetic anhydride of the total reaction mixture yield two compounds, which were chemically characterized to be GDla, resynthetized by acetylation of the deacetylated ganglioside derivatives, and GDla(acetyl), coming from the acetylation of those derivatives which lack the fatty acid moiety. The two compounds were separated by silica gel column chromatography, dissolved in monomeric form in dimethylsulfoxide and submitted to NMR analysis. The proton NMR spectra of so obtained G D l a and GDla(acetyl) are reported in Fig. 1. The spectrum of resynthetized G D l a overlaps that of natural G D l a [29], which is a good indication that the alkaline treatment acts only on the acetamide groups. The resonances of the oligosaccharide chain of GDla(acetyl) are the same as for GDla, while some differences were found for the resonances of protons belonging to the lipid moiety. In particular, the lack of the ~-carbonyl

methylene signals at 2.01 ppm and the appearance of only one methyl group at 0.82 ppm (that of sphingosine) confirms the lack of the stearic acid, and the singlet at 1.73 ppm confirms the presence of the acetamide group at position 2 of the sphingosine. 3.2. Aggregation behavior o f GD la(acetyl) in solution

Static and dynamic laser light-scattering measurements (Table 1) show that GDla(acetyl) is present in solution, at 25°C, as micelles of 96 000 _+ 5% Da and hydrodynamic radius of 33 + 0.5 ]~. The polydispersity index, as obtained from a cumulant analysis [31] of the time-dependent part of the correlation function of the scattered intensity, was 8% ( _+ 1%), a value very similar to that found for other gangliosides [31]. As the monomer molecular mass is 1634 the aggregation number is N = 59. The axial ratio of the hydrophobic core was calculated [19,2~] to be 1.03, that is GDla(acetyl) micelles are spherical, as expected for so low an aggregation number. The surface area a and the packing parameter P of the monomer inserted into the micelle were calculated to be 70.2 A2 and 0.352, respectively. Values of P close to 0.3 are characteristic of spherical micelles. Measurements performed at 36°C, the temperature at which the enzymatic reactions are usually carried out (Section 3.3), showed that the molecular mass and the hydrodynamic radius of GDla(acetyl) micelles are not affected by the temperature increase, this being in agreement with the spherical shape of the micelles. On the other hand, similar experiments performed on GDla, which is present in solution as ellipsoidal micelles [19], showed that the change of temperature from 25°C to 36°C, reduces by only 5% (_+ 1%) the micelle molecular mass. This is in agreement with previous results obtained on GM1 [42] and suggests that in the above temperature range the aggregation properties of gangliosides are quite constant. Micellar solutions of GDla(acetyl) added (2.5 and 5 tool% of total lipids) to vesicle dispersions of dipalmitoylphosphatidylcholine, form mixed vesicles, which could be recovered by ultracentrifugation.

P. Brocca et al, / Chemistry and Physics of Lipids 77 (1995) 41-49 GDia (acetyl) in DMSO

GMi(acetvl) in DMSO

LJ

.....L ~ ~

GDia (acei:yi) in water

in water

n a t u r a l GMI in OMSO

J

I k..

n a t u r a l GDia in water

'

'

'

I

_A_]t___

n a t u r a l GDia in DMSO

ppm

J

K.

GMi(acetyl)

A_A

"~ '='i"

45

'

3U n a t u r a l GMI in water

'

'

'

I

4

'

'

'

'

'

'

'

l

'

'

'

'

2

'

'

I

ppm

'

'

'

'

'

'

'

I

4

'

'

'

'

'

'

'

l

'

'

'

'

2

Fig. 1. tH-NMR spectra of GDla(acetyl) and GMl(acetyl) in DMSO-d6 and DzO, and of natural GDla and GM1 in DMSO-d6 and D20.

3.3. Vibrio cholerae sialidase action on GD la(acetyl) as homogeneous micelles and mixed vesicles with dipalmitoylphosphatidylcholine It has been found that the Vibrio cholerae sialidase, as well as galactose oxidase and fucosidase [22-24], kinetic properties are related to the ag-

gregation features of the substrate. The enzymes show similar or little differences in the Kr. values for the substrate present as homogeneous miceUes or mixed vesicles with phospholipids, while the Vmax can differ up to four orders of magnitude, as in the case of the galactose oxidase action on

46

P. Brocca et aL / Chemistry and Physics of Lipids 77 (1995) 41-49

Table 1 Micellar parameter of GDla(acetyl) and natural G D l a [31]: the molecular mass m (kDa), the hydrodynamic radius R h (A), the aggregation number N, the axial ratio RJRb, the surface area a0 (A2) occupied by the monomer at the water-lipid interface of the micelle, and the packing parameter P of the monomer inserted into the micelle

GDla(acetyl) Natural G D l a

m

Rh

N

R~/R b

ao

P

96 418

33.0 58.0

59 226

1.03 2.00

70.2 98.1

0.352 0.416

GM1. The higher Vrnax values are found for the vesicular aggregates and are explained in connection to a better accessibility of the enzyme to the ganglioside molecules when dispersed and diluted in the phospholipid layer. The behavior of Vibrio cholerae sialidase (Table 2, Fig. 2) on aggregates of natural G D l a is very similar to that already published [22]. The enzyme shows low activity on the G D l a micelles and the Vmax increases up to ten-times, when the natural G D l a is present in mixed vesicles with phospholipids. Vibrio cholerae sialidase shows a very high Vmax for GDla(acetyl), as compared to GDla. The micelles of GDla(acetyl) are very small and, therefore, the surface has a high curvature. This leads us to think that the monomers of GDla(acetyl), inserted into the micelle, are more accessible to the enzyme with respect to the monomers of natural GDla. This hypothesis is Table 2 Vmax (nmol Neu5Ac/min/mU) and Km (mM) values of Vibrio cholerae sialidase acting on micelles of GDla(acetyl) and natural GDla, and on mixed vesicles of each ganglioside with dipalmitoylphosphatidylcholine (DPPC) Substrate

Vmax

Km

Micelles GD 1a(acetyl) natural G D l a

11.5 0.5

0.6 0.8

Vesicles (molar ratio) DPPC/GD I a(acetyl) (97.5:2.5) DPPC/GD 1a(acetyl) (95:5) DPPC/natural G D l a (97.5:2.5) DPPC/natural GDla (95:5)

10.7 9.4 3.6 5.3

0.1 0.1 0.6 0.9

12~ [ |

GDl a(acetyl)

v-----------"~ Micelles

Vesicles2.5%

~

0 0~/~'~0.~;

1.5

;2:5

;

~5

~ Micelles

Ganglicoide concentration, [mM] Fig. 2. Kinetics of Vibrio cholerae sialidase action on homogeneous micelles and mixed vesicles, with dipalmitoylphosphatidylcholine, of natural GDIa and semisynthetic GDla(acetyl). Mixed vesicles contained 5 and 2.5 mol% of ganglioside. The enzyme activity is expressed as Neu5Ac released from the ganglioside.

supported by the enzyme properties for the GDla(acetyl)-phospholipids mixed vesicle preparations. In this case, the Vmax is similar to that shown for the micelles, that is, insensitive to the surface dilution of the ganglioside molecules, thus, suggesting that an 'optimal' availability to the enzyme is realized for GDla(acetyl) independently from the aggregate features. On the other hand, the decrease of about six-times of the Km value, from micelle to vesicle aggregates, could reflect, with respect to GDla, a different ganglioside-enzyme interaction process which could be due to a reduced capability of GDla(acetyl) to segregate into the phospholipid membrane. 4. Conclusion

The synthetic molecular species of G D l a which contains the acetyl group, GDla(acetyl), was prepared by direct acetylation of the reaction mixture obtained by alkaline treatment of natural GDla. Following this scheme, and with a final and sole silica gel column chromatographic step, a few days of work yield a > 99.8% pure GDla(acetyl) sample.

P. Brocca et al. / Chemistry and Physics of Lipids 77 (1995) 41-49

In a previous paper [25] on the preparation and characterization of GMl(acetyl), the great contribution of the hydrophobic portion to the aggregation properties of the ganglioside species was highlighted. The almost complete removal of the second lipid chain, corresponding to the substitution of an extended fatty acid with an acetyl group, reduces the hydrophobic volume of the molecule from 965/~3 to 566/tt3 [25]. The packing parameter becomes smaller and, consequently, GMl(acetyl) forms small, rather spherical, micelles. GDla(acetyl), which has the hydrophobic moiety identical to that of GMl(acetyl), shows similar behavior. It is present in solution as small micelles, which, :showing an axial ratio of 1.03, can be considered as spheres. In GDla, the sialic acid residue linked to the galactose at the terminal position of the neutral oligosaccharide chain can be removed by the action of Vibrio cholerae sialidase [22]. The kinetic properties of this enzyme for G D l a are related to the ganglioside aggregation features and seem to be strictly dependent on the accessibility to the G D l a monomers inserted into the aggregate. In a micelle the oligosaccharide chains are not easily available for the interaction with the enzyme, being the hydrophilic layer very dense. Quite different is the ,;ituation in a system of mixed vesicles, where the ganglioside monomers are somehow dispersed into the phospholipid matrix, with the oligosaccharide chains protruding over the phospholipid headgroups. The accessibility to GDla(acetyl) seems independent from the ganglioside aggregation form. The strong curvature of the small spherical micelles of GDla(ztcetyl) and the high amount of water, which probably separates the oligosaccharide chains, cancels the disadvantages for the enzyme accessibility, which can be overcome by diluting, to some extent, the ganglioside molecules in the vesicle aggregate. Micelles of gangliosides which contain a short acyl chain could, therefore, be a good experimental model to determine some physico-chemical properties and local requirements of the membrane for an optimal ganglioside-protein interaction process. Finally, we would like to consider a further characteristic of ~he micelles of gangliosides with

47

Table 3 IH-NMR chemical shifts (c~) of GDla(acetyl) mieelles in D20 at 310 K. Chemical shifts, in ppm, are referenced to tetramethylsilane indirectely by setting Sph H4 at 5.45 ppm. Sph GIc

H1 HI' H2 H3 H3 H4 H5 H6 H6' H7 H8 H9 H9' COCH 3

Gal II

GalNAc Gal IV

4.11 4.50 4.55 4.83 3.82 4.00 3.38 3.41 4.09 4.12 3.69 4.18 3.84 5.45 3.67 4.15 5.77 3.61 3.77 2.04 3.83 3.85 a 4.01 3.85 a

2.00

4.20 3.76 3.81a 3.81a

2.04

Neu5Ac Neu5Ac A B

4.64 3.57 4.11

1.94ax 2.70eq 3.98 3.83 3.70 3.83 3.76 a 3.53 3.76 a 3.63 3.80 3.68 3.93 2.06 b

1.83ax 2.77eq 3.72 3.87 3.65 3.62 3.92 3.68 3.93 2.07 b

a'bThe values are interchangeable.

a short acyl chain. In Tables 3 and 4, we report the proton assignment, realized at 310 K, for GDla(acetyl) and GMl(acetyl) in deuterated water. Fig. 1 shows the corresponding proton spectra, with evidence of the gain in resolution obtained for the small spherical micelles of Table 4 tH-NMR chemical shifts (c~) of GMl(acetyl) micelles in D20 at 310 K. Chemical shifts, in ppm, are referenced to tetramethylsilane indirectely by setting Sph H4 at 5.45 ppm.

H1 HI' H2 H3 H3 H4 H5 H6 H6' H7 H8 H9 H9' COCH 3

Sph

GIc

Gal lI

GalNAc

Gal IV

4.12 3.82 3.99 4.12

4.49

4.56

4.85

4.58

3.39 3.68

3.38 4.18

4.09 3.83

3.56 3.67

5.45 5.76

3.66 3.60 3.86 4.00

4.17 3.78 3.82 a 3.82 a

4.19 3.77 3.80 a 3.80 a

3.94 3.72 3.79 3.79

1.99

aThe values are interchangeable.

2.04

Neu5Ac

1.95ax 2.70eq 3.85 3.83 3.53 3.63 3.79 3.67 3.91 2.09

48

P. Brocca et al. / Chemistry and Physics of Lipids 77 (1995) 41-49

GDla(acetyl) and GMl(acetyl), with respect to the high molecular mass ellipsoidal micelles of natural G D l a and GM1, when dissolved in aqueous solvent. This is a well known effect in liquid NMR. It is a consequence of the decreased relative importance of low frequency components of spectral density function J(o9, o9 ~ 0), over the high frequency ones. The decrease in the size of the micelles in solution results in a cut-off of the long-living modes of the electromagnetic field, that is, of those fluctuations of the local field which are brought about by the slow motions present in the sample. For micelles in solution, the diffusion processes of the aggregate as a whole constitute a major contribution to such motions. An estimation of the correlation time zc associated to diffusion gives a measure of the change in the dynamical properties when passing from a micelle of a natural ganglioside to that of a shortchain one. Using the Debye-Stokes approximation of spherical particles of radius Rh in viscous solvent, 'one gets an overall re, at 310 K, of about 2.0 × 10 -8 for GDla(acetyl) and GMl(acetyl), and about 1.0 x 10 - 7 for natural G D l a and natural GM1. This need to deplete the local field from longliving modes in order to increase resolution, has to be balanced against the need to use an experimental model which could mimic the physiological situation. The conformational and dynamic properties of gangliosides were first studied by NMR in dimethylsulfoxide [29,43-45], that is in monomeric dispersion, Rh ~ 5~. This is a first step, which allows a quite easy peak assignement and interaction recognition, but nothing can be said about the secondary structure modifications, which are brought about by the interactions with a physiological water-like solvent. As the dissolution of gangliosides in a water-like solvent gives rise to the formation of micelles too big for NMR resolution, Rh ~ 60A, a further step of approximation, i.e. a second more refined experimental model, was proposed in order to study the effect of the aqueous solvent on the ganglioside secondary structure. It consists of a mixed system of small micelles of dodecylphosphocholine in water containing one single ganglioside molecule per

micelle [29,45-46], Rh ,~ 20/k. This has the advantage of surrounding the ganglioside headgroup with a water-like solvent, but still nothing can be said about the headgroup conformation, when neighbouring similar headgroups are present, as in the case of a segregated ganglioside-enriched domain in a true membrane. As a third step of approximation, we propose that the small spherical micelles, which are formed in water solution by semisynthetic gangliosides, like GDla(acetyl) and GMl(acetyl), R h ~ 35A, are a good new membrane model to study the conformational and dynamic properties of the ganglioside oligosaccharide chain in an environment enriched in gangliosides. Moreover, this model gives the possibility to study the interactions, if any, between the oligosaccharide chains. Experiments in this direction are currently in progress in our laboratory.

Acknowledgements We would like to thank Mr. Riccardo Casellato for his expert technical assistance during ganglioside purification and ganglioside chemical modifications. This work was partially supported by grants from the Consiglio Nazionale delle Ricerche (CNR), Rome, Italy (grant 91.00438.PF40, target project 'Aging'; grant 92.02268.PF39, target project 'ACRO').

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