Glycosphingolipids of human plasma

ARCHIVES OF BIOCHEMISTRY AND BIOPHYSICS Vol. 238, No. 2, May 1, pp. 388400, 1985

Glycosphingolipids

of Human Plasma’

SAMAR K. KUNDU,’ ISABEL DIEGO, SUSAN OSOVITZ, AND DONALD M. MARCUS3 Departments

of Medicine, Micr&ology, and Immunology, Baylor Medicine, One Baylor Plaza, Houston, Texas 77030

Received October 26, 1984, and in revised form December

College of

12, 1984

A number of glycosphingolipids, including 10 gangliosides, not previously identified in human plasma have been characterized. The plasma contains 2 pugof lipid-bound sialic acid/ml plasma and 54% of the gangliosides are monosialo, 30% disialo, 10% trisialo, and 6% tetrasialo. Individual glycosphingolipids were purified by highperformance liquid chromatography and thin-layer chromatography, and were characterized on the basis of their chromatographic mobility, carbohydrate composition, hydrolysis by glycosidases, methylation analysis, and immunostaining with antiglycosphingolipid antibodies. The monosialogangliosides were identified as Gu3, GW2, sialosyl(2-3)- and sialosyl(2-6)lactoneotetraosylceramides, sialosyllacto-N-nor-hexaosylceramide, and sialosyllacto-N-isooctaosylceramide. The major gangliosides in the polysialo fractions contained a ganglio-N-tetraose backbone and were identified as G GDI~, Gmb, and GQlb. The most abundant neutral glycosphingolipids were glFiosy1, lactosyl, globotriaosyl, globotetraosyl and lactoneotetraosylceramides. The other neutral glycosphingolipids, tentatively identified by immunostaining with monoclonal antibodies, contained Hr, Lea, Leb, and la&o-N-fucopentose III (X hapten) structures. 0 1985 Academic Press, Inc.

Glycosphingolipids (GSLS)~ of plasma describe the isolation and characterization are carried by lipoproteins (l-5). Some of of 10 gangliosides that have not been these GSLs are transferred in vivo to identified previously in human plasma. erythrocytes or lymphocytes where they We have also tentatively identified five function as cell surface antigens (6, 7). neutral GSLs of the lactoneotetraosyl Although many erythrocyte GSLs have family that contain four to six sugar been identified only a small number of residues. plasma GSLs have been characterized (l5, 8-10). In relation to our studies on the MATERIALS AND METHODS mechanism of transfer of GSLs from liIsolation of glycosphingolipids. Samples of plasma poproteins to cells, we have undertaken a more extensive characterization of the were dialyzed against water and lyophilized, and the powder was extracted twice with chloroform:methGSLs in human plasma. In this report we anol:water, 50:50:15 (v/v) at room temperature. The i Supported by Research Grants Q-832 from the Robert A. Welch Foundation and AI 17712 from the National Institutes of Health. * Present address: Abbott Laboratories, Diagnostics Division, Department 93H, North Chicago, Ill. 60064. 3 To whom reprint requests should be addressed. 4 Abbreviations used: GSLs, glycosphingolipids; NeuAc, N-acetylneuraminic acid. 0003-9861/85 $3.00 Copyright All rights

0 1985 by Academic Press, Inc. of reproduction in any form reserved.

388

total lipid extract was fractionated on DEAESephadex A 25 (Pharmacia Fine Chemicals, Piscataway, N.J. (11). The ganglioside fraction (Fig. 1) was then separated into the monosialo-, disialo-, trisialo-, and tetrasialoganglioside species (Fig. 2) by DEAE-silica gel gradient chromatography (12, 13), and individual gangliosides were purified by HPLC (14). The neutral GSLs were isolated by the acetylation procedure of Saito and Hakomori (15),

GLYCOSPHINGOLIPIDS partitioned into lower and upper phases, and further fractionated by HPLC (16). Analyticalprocedures-Carbohydrateanalysis. Carbohydrate composition was determined by two methods: (i) methanolysis, N-acetylation, trimethylsilylation, and analysis by GLC (1’7); (ii) hydrolysis in 90% acetic acid containing 0.5 N sulfuric acid, reduction with sodium borohydride, acetylation, and analysis by GLC (18). Permethylation studies. Individual glycosphingolipids were permethylated (19, 20), hydrolyzed in 90% acetic acid containing 0.3 N sulfuric acid for 8 h under a nitrogen atmosphere, and then reduced and acetylated according to Bjorndal et aL (21). The partially 0-methylated hexitol and hexosaminitol acetates were analyzed by GLC (18). of gangliosides. Gangliosides Periodate treatment (50-60 fig) were oxidized with sodium metaperiodate and reduced with sodium borohydride according to Ando and Yu (22). The reaction products were desalted on a Cls Sep-Pak cartridge (23), and the ganglioside fractions were subjected to mild methsialic acid anolysis in 0.05 N HCI (24). Terminal groups are converted by this procedure to sevencarbon fragments (NeuAc-7) which were identified by GLC (25). Enzyme degradativn. For hydrolysis with Vibrio cholerae neuraminidase, (Behring Diagnostics, Sommerville, N. J.) gangliosides (25-30 pg) were dissolved in 200 ~1 50 mM acetate buffer, pH 5.0, containing 2 mM CaCI,, and incubated with 10 units of enzyme for 24 h at 37’C (26). Similar hydrolysis conditions were used with Arthrobacter ureaftiens (Boehringer Mannheim Biochemicals, Indianapolis, Ind.), except that the acetate buffer contained 1 mg/ml sodium cholate and 100 mU enzyme. After incubation with enzyme, 1 ml chloroform:methanol 1:l (v/v) was added and the reaction mixture was dried under nitrogen. The dried sample was dissolved in 5 ml of water and purified by passage through a C18 SepPak cartridge. Hydrolysis by jack bean fl-galactosidase and P-N-acetylhexosaminidase, kindly donated by Dr. S. C. Li and Dr. Y. T. Li, was carried out as described previously (18). Antibodies and immunological methods. Purified rabbit IgG antibodies against gangliotriaosylceramide, gangliotetraosylceramide, Iactotriaosylceramide, lactoneotetraosylceramide, globotriaosylceramide, and globotetraosylceramide were prepared and characterized in this laboratory (27-29). Mouse monoclonal antibodies against gangliotriaosylceramide (30), sialosyl(2-6)lactoneotetraosylceramide (31), and blood group H type 2 (32) were kindly provided by Dr. S. Hakomori; monoclonal anti-Lea (33) was a gift from Dr. W. Young; monoclonal antiH hapten (WGHS 29-l) (34) was a gift from Dr. H. Steplewski; and monoclonal anti-Leb was a gift from Chembiomed. Edmonton. Alberta. Canada.

OF HUMAN

PLASMA

389

The antigens for complement fixation assays were prepared by mixing glyeolipid, egg lecithin, and cholesterol in a weight ratio of 1:2:10 as described previously (27, 28). Thin-layer chromatography and quantijkation of glycosphingolipkb. TLC of gangliosides was performed on precoated silica gel 60 plates (E. Merck, FRG) with chloroform:methanol:water, 55:45:10 (v/v), containing 0.02% CaC& * 2H20 or chloroform:methanol: 2.5 N NH,OH, 60:40:9 (v/v). The ganglioside bands were visualized with a resorcinol spray (35). TLC of neutral GSLs was performed with chloroform:methanol:water, 60:35:8 or 60:30:5 (v/v) and GSLs were visualized with an a-naphtholsulfuric acid spray (36). The yield of gangliosides in each preparation was determined by GLC of the lipid-bound sialic acid (24). Immunostaining on thin-layer plates. The immunostaining procedure of Brockhaus et al. (34) was used with some modifications. The GSLs were separated on an aluminum-backed HPTLC plate (E. Merck, FRG) in chloroform:methanol:water, 60:35:8 (v/v) or 55:45:10 (v/v), containing 0.02% CaCl,.2H,O. The plate was overlaid with purified rabbit IgG antibodies or murine monoclonal anti-GSL antibody (protein concentration, lo-50 pg/ml). After overnight incubation at 4”C, the plate was overlaid with l%Ilabeled (37) anti-rabbit or anti-mouse Ig (lo6 cpm/ ml) for 3 h and exposed to X-ray film with an intensifying screen for 2-5 h. RESULTS

Gangliosides. The thin-layer chromatographic pattern of the ganglioside fractions isolated from human plasma is shown in Figure 1 and their relative abundance is presented in Table I. The purified monosialo and polysialo compounds are shown in Figs. 2-4. MG-2. This ganglioside comigrated with brain GM2 (Fig. 2) and was similar to the latter in carbohydrate composition (Table II) and in its resistance to V. cholerae neuraminidase. On treatment with A. ureafaciens neuraminidase, MG-2 yielded a desialylated compound which was identified as gangliotriaosylceramide on the basis of chromatographic mobility and immunostaining with rabbit IgG antibody and mouse monoclonal antibody against gangliotriaosylceramide. The oligosaccharide structure of MG-2 appears identical with that of brain GM2 (Table III). MG-3 and MG-4. These two gangliosides occur in approximately equal concentra-

390

KUNDU

WM

REC

PLASMA

ET AL.

MONO

TRI

DI

TETRA

FIG. 1. TLC of human plasma gangliosides. Solvent: chloroform:methanol:water, 55:45:10 (v/v), containing 0.02% CaCl,. 2HaO. Lane WM, gangliosides of human brain white matter; lane RBC, gangliosides from human erythrocytes; Lane plasma, unfractionated gangliosides of plasma, followed by the mono-, di-, tri-, and tetrasialogangliosides. The square bracket in lane WM indicates a nonganglioside impurity.

tions and together comprise approximately 2% of the monosialoganglioside fraction. MG-3 comigrated with human erythrocyte sialosyl(2-3)lactoneotetraosylceramide (data not shown) and MG-4 comigrated with brain GM1(Fig. 2). Both

WM

FIG. 2. TLC of purified brain white matter; lane N-neotetraosylceramide amide. Solvent as in Fig.

STD

MG-I

MG-2

MG-3

gangliosides contained galactose, glucose, glucosamine, and N-acetylneuraminic acid in approximate molar ratios of 2:l:l:l (Table II). On treatment with V cholerae neuraminidase MG-3 and MG-4 yielded asialo glycolipids whose migration rates

MG-4

MG-5

MG-6

MG-7

MG-6

MG-9

human plasma monosialogangliosides. Lane WM, gangliosides of human STD, standard mixture of gangliosides including GYZ, sialosyl(2-3)lacto(SPG), disialosyl SPG (DPG), and disialosyl(2-3)lacto-N-isooctaosylcer1.

GLYCOSPHINGOLIPIDS

OF HUMAN

391

PLASMA

lactoneotetraosylceramide. The oligosaccharide structures of MG-3 and MG-4 DISTRIBUTION OF INDIVIDUAL GANGLIOSIDE SPECIES appear to be identical with that of sialoIN HUMAN PLASMA’ syl(2-3)lactoneotetraosylceramide, and the compounds differ in their fatty acid comGanglioside species Percentage of total position. MG-5 and MG-6. These two gangliosides Monosialo 54 comprise about 2% of the monosialoganDisialo 30 glioside fraction. MG-5 migrates below Trisialo 10 brain Gul and MG-6 comigrates with the 6 Tetrasialo upper band of brain Go3 (Fig. 2). Carbohydrate analysis indicated that both MG’ Determined by measuring lipid-bound sialic acid. 5 and MG-6 contained galactose, glucose, glucosamine, and N-acetylneuraminic acid in approximate molar ratios of 2:l:l:l relative to lactoneotetraosylceramide iso- similar to those of MG-3 and MG-4 (Table lated from human erythrocytes are 1.0 II). On treatment with V cholerae neurand 0.85, respectively. Both asialo com- aminidase MG-5 and MG-6 produced asipounds were identified as lactoneotetraalo glycolipids whose migration rates were osylceramide by immunstaining with identical to the asialo glycolipids obtained purified rabbit anti-lactoneotetraosylcerfrom MG-3 and MG-4. The asialo comamide IgG antibody. Both gangliosides pounds were identified as lactoneotetrayielded the same methylation products, osylceramide by immunostaining with pu1,3,5-tri-O-acetyl-2,4,6-tri-o-methylgalacrified anti-lactoneotetraosylceramide IgG titol, 1,4,5-tri-O-acetyl-2,3,6-tri-o-methylantibody. On methylation analysis both glucitol, and 1,4,5-tri-O-acetyl-3,6-di-OMG-5 and MG-6 gave the same methylmethyl - 2 - deoxy - (N- methylacetamido) - ation products, 1,5,6-tri-0-acetyl-2,3,4-triglucitol, in an approximate molar ratio 0-methylgalactitol, 1,3,5-tri-O-acetyl-2,4,6of 2:1:1, which was identical with that tri-0-methylgalactitol, 1,4,5-tri-o-acetylobtained with standard sialosyl-(2-3)2,3,6-tri-0-methylglucitol, and 1,4,5-tri-OTABLE

WM

DI

DG-1

I

De-2

De-3

DG-4

FIG. 3. TLC of purified human plasma disialogangliosides. Lane WM, gangliosides of human brain white matter; lane DI, total disialoganglioside fraction; and lanes DG-1 through DG-4 contain purified disialogangliosides. Solvents as in Fig. 1; the square bracket in lane DI indicates a nonganglioside impurity.

WM

TRI

TETRA

TRI-1

TET-1

FIG. 4. TLC of purified trisialo- and tetrasialogangliosides. Lane WM, gangliosides of human brain white matter; lane TRI, unfractionated plasma trisialogangliosides; lane TETRA, total tetrasialoganglioside fraction; lanes TRI-1 and TET-1, purified trisialo and tetrasialogangliosides.

392

KUNDU TABLE

ET AL. II

CARBOHYDRATE COMPOSITIONOF PURIFIED PLASMA GLYCOSPHINGOLIPIDS Component

sugarsa

Glycosphingolipid

Galactose

Glucose

Monosialogangliosides MG-2 MG-3 MG-4 MG-5 MG-6 MG-7 MG-8 MG-9

0.92 1.85 1.78 2.0 1.92 2.8 - 3.7 3.8

1.0 1.0 1.0 1.0 1.0 1.0 1.0 1.0

0 0.95 0.90 0.8 0.96 1.8 2.6 2.7

0.85 0 0 0 0 0 0 0

0.90 0.85 0.80 0.92 0.96 0.85 0.78 0.85

Disialogangliosides DG-1 DG-2 DG-3 DG-4

1.05 1.0 1.80 1.96

1.0 1.0 1.0 1.0

0 0 0 0

0 0 0.75 0.80

1.90 1.85 1.80 1.75

Trisialoganglioside TRI-1

1.75

1.0

0

0.85

2.75

Tetrasialoganglioside TET-1

1.85

1.0

0

0.80

3.80

Neutral GSLs GSL-3 GSL-4 GSL-5 GSL-6

1.90 2.0 1.92 1.85

1.0 1.0 1.0 1.0

0 0 0.95 0.92

0.80 0 0 0

0 0 0 0

’ Expressed as molar ratios relative acid was analyzed as TMS derivatives.

Glucosamine

to glucose as 1.0, as determined

acetyl-3,6-di-O-methyl-Z-deoxy(N-methylacetamido)glucitol, in approximately equimolar ratios. MC-5 and MG-6 contained a terminal 2,6-linked sialic acid residue, which was confirmed by immunostaining with a monoclonal antibody directed against the NeuAc(cYZ-6)Gal structure (31). The carbohydrate structures of MG-5 and MG-6 appear to be identical with that of sialosyl(Z-6)lactoneotetraosylceramide and they probably contain different fatty acids. MG-7. This ganglioside (Fig. 2) is a minor compound that was not detected in the original mixture (Fig. 1). MG-7 comigrates with human erythrocyte sialosyl(23)lacto-N-nor-hexaosylceramide (data not shown), and was similar to the latter in carbohydrate composition (Table II). On

Galactosamine

by GLC of alditol

Sialic acid

acetates. The sialic

treatment with V. cholerae neuraminidase MG-7 yielded an asialo glycolipid with TLC migration identical to that of lactoN-nor-hexaosylceramide. The asialo glycolipid could be immunostained with antilactoneotetraosylceramide, indicating a lactoneotetraosyl backbone. On methylation analysis, MG-‘7 yielded 1,3,5-tri-Oacetyl-2,4,6-tri-0-methylgalactitol, 1,4,5tri-0-acetyl-2,3,6-tri-0-methylglucitol, and 1,4,5-tri-O-acetyl-3,6-di-0-methyl-2-deoxy -(N-methylacetamido)glucitol in an approximate molar ratio of 3:1:2. The carbohydrate structure of MG-7 was tentatively assigned as sialosyl(2-3)lacto-N-norhexaosylceramide. MG-8 and MG9. These gangliosides (Fig. 2) are also trace compounds in human plasma and were not detected in the orig-

GLYCOSPHINGOLIPIDS

OF HUMAN

TABLE

393

PLASMA

III

STRUCTURE OF HUMAN PLASMA GANGLIOSIDES~ Oligosaccharide

Fraction MG-1

GY3

MG-2

GY2

structure NeuAc(a2-3)Gal(pl-4)Glc-Cer

GalNAc(~l-4)[NeuAc(c2-3)]Gal(@l-4)Glc-Cer

MG-3 Sialosyl(2-3)lacto-N-neotetraosylceramide MG-4

NeuAc(~2-3)Gal(~l-4)GlcNAc(~l-3)Gal(~l-3)Gal(~l-4)Glc-Cer

MG-5 Sialosyl(2-G)lacto-N-neotetraosylceramide MG-6

NeuAc(ol2-6)Gal(~l-4)GlcNAc(~l-3)Gal(~l-4)GlcCer

MG-7 Sialosyl(2-3)lacto-N-norhexaosylceramide NeuAc(cu2-3)[Gal(~l-4)GlcNAc(~l-3)]~Gal(~l-4)Glc-Cer MG-8 Sialosyl(2-3)lacto-N-isooctaosylceramide MG-9

Gal@l-4)GlcNAc@l,

6) Gal(~l-4)GlcNAc(pl-3)Gal(~l-4)Glc-Cer 3) NeuAc(a2-3)Gal(fll-4)GlcNAc(B1/ NeuAc(a2-8)NeuAc(a2-3)Gal(fll-4)Glc-Cer

DG-1 DG-2

Go3

DG-3

Gou,

NeuAc(cY2-3)Gal(~l-3)GalNAc(~l-4)[NeuAc(~2-3)]Gal(~l-4)Glc-Cer

DG-4

GDlb

Gal(~l-3)GalNAc(~l-4)[NeuAc(ol2-8)NeuAc(~2-3)]Gal(~l-4)Glc-Cer

Tri-1

GTlb

Tet-1

GPlb

NeuAc(ol2-3)Gal(~l-3)GalNAc(~1-4)[NeuAc(ol2-8)NeuAc(a2-3)]Gal(~l-4)Glc-Cer NeuAc(cu2-8)NeuAc(Lu2-3)Gal(~l-3)GalNAc(~l-4)[NeuAc(ol2-8)~euAc(ot2-3)]Gal(~l-4)Glc-Cer

DAbbreviations: Gal, D-gdaCtOSe; Glc, glucosamine; NeuAc, N-acetylneuraminic

GalNAc, N-acetyl-D-galactosamine; acid; Cer, ceramide (N-acylsphingosine).

D-ghCOSe;

inal mixture (Fig. 1). Carbohydrate analysis indicated that both MG-8 and MG-9 contained galactose, glucose, glucosamine, and N-acetylneuraminic acid in approximate molar ratios of 4:1:3:1 (Table II). On treatment with V: cholerae neuraminidase, MG-8 and MG-9 yielded asialo glycolipids which, upon further successive treatments with jack bean P-galactosidase and jack bean P-N-acetylhexosaminidase, yielded lactoneotetraosylceramides, which were identified by immunostaining with antilactoneotetraosylceramide IgG antibody. The TLC migration rates of the products were 1.0 and 0.85 relative to standard lactoneotetraosylceramide. The tentative carbohydrate structure of MG-8 and MG-9 appears to be identical with that of sialosyllacto-N-isooctaosylceramide

GlcNac, N-acetyl-n-

isolated from human erythrocytes (US), and these compounds probably contain different fatty acids. DG-1 and DG-2. These two gangliosides comigrated with the two bands of brain Gns (Fig. 3) and were similar to the latter in carbohydrate composition (Table II). On treatment with V cholerae neuraminidase both DG-1 and DG-2 yielded lactosylceramide. On methylation analysis both gangliosides gave the same methylation products, 1,3,5-tri-O-acetyl-2,4,6-tri-Omethylgalactitol and 1,4,5-tri-o-acetyl2,3,6-tri-0-methylglucitol in approximate molar ratios of l:l. Periodate-borohydride treatment of DG-1 and DG-2 followed by mild methanolysis yielded equimolar amounts of intact NeuAc and NeuAc-7, which indicates a NeuAc(Z-8)NeuAc se-

394

KUNDU

quence. The carbohydrate structures of DG-1 and DG-2 appear to be identical with that of Gna. DG-3. This ganglioside comigrated with brain GDla (Fig. 3) and was similar to the latter in carbohydrate composition (Table II). On treatment with V cholerae neuraminidase, DG-3 yielded a ganglioside that comigrated with brain GM1. On treatment with A. ureafaciens neuraminidase DG-3 lost both sialic acids to give an asialo glycolipid which comigrated with gangliotetraosylceramide. The asialo compound was identified as gangliotetraosylceramide by complement fixation with anti-gangliotetraosylceramide IgG antibody. Periodate-borohydride-methanolysis treatment of DC-3 converted both sialic acid residues to the lower homolog, NeuAc-7, which indicated that the sialic acids were both terminal. On methylation analysis, DG-3 gave 1,3,5-tri-O-acetyl-2,4,6-tri-Omethylgalactitol, 1,3,4,5 - tetra - O-acetyl2,6-di-0-methylgalactitol, 1,4,5-tri-o-acetyl-2,3,6-tri-0-methylglucitol, and 1,3,5tri-0-acetyl-4,6-di-O-methyl-2-deoxy(Nmethylacetamido)galactitol in approximately equimolar ratios. These products were identical with the methylation products obtained from brain Gm,. DG-4. This ganglioside comigrated with brain GDlb (Fig. 3). It was similar to the latter in carbohydrate composition (Table II), and V cholerae neuraminidase treatment yielded a monosialoganglioside that comigrated with brain GM*. On hydrolysis with A. ureafaciens neuraminidase, DG-4 gave an asialo compound which was identified as gangliotetraosylceramide on the basis of TLC migration rate and complement fixation with anti-gangliotetraosylceramide. Periodate-borohydride-methanolysis treatment of DG-4 yielded equimolar amounts of intact NeuAc and NeuAc-‘7. On methylation analysis DG-4 gave 1,5-di-O-acetyl-2,3,4,6-tetra-o-methylgalactitol, 1,4,5-tri-O-acetyl-2,3,6-tri-Omethylglucitol, 1,3,4,5-tetra-0-acetyl-2,6di-0-methylgalactitol, and 1,3,5-tri-o-acetyl - 4,6 - di - 0- methyl - 2- deoxy(N- methylacetamido)galactitol in approximately equimolar ratios. Thus, the carbohydrate

ET AL.

structure of DG-4 was the same as that Of Gmb. Tri-1. This ganglioside occurs as a major compound in the trisialoganglioside fraction (Fig. 4). It comigrated with brain GTn, and was similar to the latter in carbohydrate composition (Table II). Upon treatment with V chokrae neuraminidase, Tri-1 gave a ganglioside which comigrated with brain GM1. Tri-1 lost all sialic acid residues upon treatment with A. ureo$aciens neuraminidase. The asialo compound was identified as gangliotetraosylceramide on the basis of its TLC migration rate and complement fixation with anti-gangliotetraosylceramide IgG antibody. On methylation analysis, Tri-1 gave products that were identical with those of DG-4 (GDlb). On periodate-borohydride-methanolysis treatment Tri-1 gave NeuAc and NeuAc-7 in approximate molar ratios of 1:2. This confirms the carbohydrate structure of Tri-1 as GTlb. Tet-1. This ganglioside comigrated with brain Gglb (Fig. 4). Carbohydrate compo-

CMH-

CDH-

CTH-

STD

RBC

LOW

UP

STD

FIG. 5. TLC of human plasma neutral glycolipids. Solvent: chloroform:methanol:water, 60:35:8 (v/v). Lane STD, standard purified neutral glycolipids; lane RBC, neutral GSLs of human erythrocytes; lanes LOW and UP, neutral glycolipids from Folch lower and upper phases, respectively.

GLYCOSPHINGOLIPIDS

OF HUMAN

sition (Table II) and the appearance of a ganglioside which comigrated with brain GM1 after T/: cholerae neuraminidase treatment suggested its identity with GQlb. Upon treatment with A. ureafaciem neuraminidase, Tet-1 lost all four sialic acid residues. The asialo derivative was identified as gangliotetraosylceramide on the basis of TLC migration rate and complement fixation with anti-gangliotetraosylceramide. Periodate-borohydride-methanolysis treatment of Tet-1 yielded equimolar amounts of NeuAc and NeuAc-7. Upon methylation analysis, Tet-1 gave products that were identical with that of GD-4 (GDlb) and Tri-1 (GTlb). Thus, the carbohydrate structure of Tet-1 is identical with that of brain GQn,. Neutral glycosphingolipids. The thinlayer chromatographic pattern of Folch upper and lower phases of neutral GSLs isolated from human plasma is shown in Fig. 5. GSL-3. These two bands comigrated with the two bands of human erythrocyte globotriaosylceramide (Fig. 6). Carbohydrate composition, methylation analysis, and immunostaining with rabbit IgG anti-

395

PLASMA

globotriaosylceramide antibody indicated that the carbohydrate sequence of GSL-3 was identical with that of human erythrocyte globotriaosylceramide (Table IV). GSL.-4. This GSL comigrated with human erythrocyte globotetraosylceramide (Fig. 6) and was similar to the latter in carbohydrate composition. Methylation analysis and immunostaining with antiglobotetraosylceramide IgG antibody confirmed the carbohydrate structure of this GSL as globotetraosylceramide. GSL-5 and GSL-6. GSL-5 comigrated with human erythrocyte lactoneotetraosylceramide, and GSL-6 migrated more slowly (Fig. 6). Carbohydrate analysis indicated that both GSLs contained galactose, glucose, and glucosamine in approximate molar ratios of 2:1:1, and both GSL5 and GSL-6 were immunostained with anti-lactoneotetraosylceramide IgG antibody. On methylation analysis both GSLs gave the same methylation products, which were identical with the methylation products obtained from lactoneotetraosylceramide. Identification of HI glycosphingolipid. This GSL was not isolated in pure form.

CMH -

CDH -

CTH As GM2 Glob PG Forss AS GMl -

STD

LOW GSL-1

GSL-2

GSL-3

GSL-4

GSL-5

GSL-6

UP

RBC

STD

FIG. 6. TLC of purified individual neutral glycolipids from Folch lower phase. Solvent: chloroform:methanol:water, 60:30:5 (v/v). Abbreviations STD and LOW as in Fig. 5; GSL-1 through GSL-6 are individual purified glycolipids.

396

KUNDU

ET AL.

TABLE

IV

STRUCTURE OF HUMAN PLASMA NEUTRAL GLYCOSPHINGOLIPIDSa Fraction

Oligosaccharide

structure

GSL-1

Glc-Cer

GSLQ

Gal@l-4)Glc-Cer

GSL-3

GbOse&er

Gal(al-4)Gal(fil-4)Glc-Cer

GSL-4

GbOse,Cer

GalNAc(~l-3)Gal(ol-4)Gal(~l-4)Glc-Cer

GSL-5 GSL-6

LcnOse,Cer

Gal(pl-4)GlcNAc(fil-3)Gal(@1-4)Glc-Cer

GSL-7

Hi

GSL-8

X hapten

Gal(~1-4)[Fuc(otl-3)]GlcNAc(~l-3)Gal(~l-4)Glc-Cer

GSL-9

Le”

Gal(~l-3)[Fuc(oll-4)]GlcNAc(~l-3)Gal(~1-4)Glc-Cer

Fuc(ol-2)Gal(/31-4)GlcNAc(~1-3)Gal(~l-4)Glc-Cer

Fuc(oll-2)Gal(~l-3)[Fuc(cul-4)]GlcNAc(~l-3)Gal(~l-4)Glc-Cer

GSL-10 Leb

a Abbreviations: Gal, D-galactose; Glc, D-glucose; GalNAc, N-acetyl-D-Galactosamine; D-glucosamine; NeuAc, acetyl neuraminic acid; Cer, ceramide (N-acylsphingosine).

Its presence was demonstrated in upperphase fractions UP-3, UP-4, and UP-5, and a smaller amount in UP-6 (Figs. 7 and SC) by immunostaining with monoclonal antibody BE2 directed against type 2 H, Fuc(al-Z)Gal(@l-4)GlcNAc(@l3)Gal@l-4)Glc.

GlcNAc,

N-acetyl-

Identification of X glycosphingolipid. This GSL migrated slower than HI GSL and was present in upper-phase fractions UP-3, UP-4, UP-5, and UP-6, and a smaller amount in UP-7 (Figs. 7 and 8B), as demonstrated by immunostaining with a mouse monoclonal antibody WGHS 29-l

CMH-

CDH-

CTHAs

As

STD

UP

UP-1

UP-Z

UP-3

UP-4

Up-5

“p-6

Up-7

up-8

LOW

RBC

FIG. 7. TLC of neutral glycolipid fractions from Folch upper phase; solvent and standard abbreviations as in Fig. 5; lanes UP-l through UP-8 contain partially purified neutral GSL fractions.

GLYCOSPHINGOLIPIDS

STO

LOW

UP

UP

OF HUMAN

UP-3

UP-5

397

PLASMA

UP-6

UP-7

UP-3

UP-5

UP-6

UP-7

FIG. 8. Immunostaining of Folch upper-phase neutral glycolipids. Solvent and abbreviations as in Fig. 5. (A) Detection with oc-naphthol reagent; (B) immunostaining by anti-X monoclonal antibody; (C) immunostaining by anti-H, monoclonal antibody.

directed against lacto-N-fucopentaose III, Gal(/31-4)[Fuc(cJ-3)]GlcNAc(pl-3)Gal(@l4)Glc. IdentiJkation of Le” glycosphingolipid. This GSL migrated in the same area as the X GSL and was present in upperphase fractions UP-3 to UP-7 (Fig. 7). Its presence was demonstrated by immunostaining with a mouse monoclonal antibody CF4-C4 directed against Lea, Gal@l3)[Fuc(c~1-4)]GlcNAc(~l-3)Gal(~l-4)Glc (data not shown). IdentiJicatim of Leb glycosphingolipid. This GSL was a major component of the upper phase and migrated more slowly than the Le” and X GSLs (Fig. 7). It was enriched in fractions UP-6 and UP-7. A monoclonal anti-Leb antibody strongly stained fraction UP-6 (data not shown), and fractions UP-5 and UP-7. Fatty acids. The fatty acids of the most abundant gangliosides and neutral GSLs are presented in Tables V and VI, respectively. The fatty acids of the gangliosides are very heterogeneous and contain more GS and Gs, and less CZOand CZ2, compounds than bovine brain gangliosides (Table V), but the fatty acids of plasma GMs, GD3, and GDla gangliosides are similar to those of the erythrocyte gangliosides. The plasma neutral GSL fatty acids were also more heterogeneous than the erythrocyte GSLs (Table VI) and contained much less CzZ and Cz4 compounds. For example, the upper CDH band of erythrocytes contains approximately 90% of C& and CZ4 (38), in contrast to 40% in the plasma compound, and plasma globoside

contains 26% Cad, compared to approximately 80% for erythrocytes (38). DISCUSSION

Although GSLs constitute a small proportion of plasma lipids, these compounds are of biological interest because of their immunological properties. These properties include acquisition of cell surface glycolipid antigens by erythrocytes and mononuclear leucocytes, and the possible immunomodulatory role of gangliosides [reviewed in (39)]. The level of plasma lipid-bound sialic acid found in this study, 200 pg/dl, is in the same range reported by other investigators (40-43), but differs markedly from the value of 14-18 mg/dl reported by one laboratory (44). The method used by this group to isolate gangliosides, including precipitation of glycoproteins by phosphotungstic acid from the upper phase of a partitioned lipid extract, is questionable. Although a number of gangliosides more complex than GM3 have been detected and tentatively identified on the basis of chromatographic mobility in earlier studies (42, 43), only GMa has been fully characterized. We have extended these studies by a combination of chemical and immunological techniques and have characterized nine additional gangliosides. It is interesting that approximately 50% of plasma gangliosides contain more than one sialic acid residue, in contrast to erythrocytes which contain only 10% polysialogangliosides. In addition, all of

1.7 0.6 3.2 6.1

7.0

band band

a Expressed ‘Expressed

CMH Upper CMW Lower CDH Upper CDH Lower CTH Upper CTH Lower Globoside Paragloboside

‘Expressed

1.7 0.4 17.7 0.6

G6:1

G81

24.6 39.4b 6.0b 9.8* 9.6 6.8 12.3 4.4 5.7 8.3 0.6 17.4

27.7

Cls,O

acids.

0.6 2.8 13.5 1.2 0.3 0.3 0.9 0.8 6.8 0.9

0.7 10.4

16.2 0.1 0.8 14.2 29.3 13.3 22.8 35.0 15.2 3.6

as the percentage of total fatty acids. as total of saturated and unsaturated fatty

32.6 45.6 9.0 12.2

43.lb 55.46 13.6* 60.2b

C16.0

as a percentage

7.9 27.7 12.6 9.0 25.9 32.2 35.6

38.7

of the total fatty

2.1

5.9

band band

Bovine brain gangliosides

GQlb

G Tlb

Gu,-upper Gy,-lower GM?. Goa upper Gn3 lower G ma

0.3 0.2 11.4

0.4

ACIDS

VI

0.3 0.8 15.0

0.40

0.10

Go:1

2.7 0.8 1.4 1.7 3.4 12.3 2.5

6.7 4.6 2.5 2.0 2.5 0.2 2.5 4.3

a

1.30 1.0 5.9 3.7 11.7 9.5 9.1

cm

‘7.8

9.8

3.7 0.4 15.2 0.9 4.0

of carbon atoms.

10.1

0.5

cm

GLYCOLIPIDS~

5.9 0.1 4.3 12.9 8.7 8.9 14.3 0.7 11.6 0.3

GANGLIOSIDES

0.1 0.2 0.8 0.3 5.2 0.1

OF NEUTRAL

TABLE

2.6 6.0 11.7 13.8 3.1 8.9 5.1

1.4 18.0

OF PLASMA

19.9 21.1 2.6 4.3 2.3 3.8 2.9

8.4

19.3

1.2 21.0 1.1

ACIDS

V

acids with the same number

14.2b

FATTY

13.8 1.7 19.2 23.9 3.8 14.9 26.2 3.7 3.1 0.7

FATTY

TABLE

0.70 0.6 7.1 3.1 3.6 4.7 3.8 10.6 21.5

Cl!%1

0.4 1.2 3.0 8.0 2.4 2.8 2.9 2.0 1.2 30.7 2.2 2.7 2.1 2.3 2.1 3.2 1.4

1.1 0.7 3.7 0.8 2.0

0.90

G.%o

1.3 1.4 1.8

4.0 1.9 0.7 1.0

1.7

0.3 8.5 1.3

1.3 0.6

cm

5.5 2.0 1.8

4.6 0.6 7.7 3.8 4.0 3.2

3.0

0.1 1.4 7.1 2.7 12.0 5.3 8.6

cm

5.1 1.5 1.6

0.4

1.2 0.1 2.2 3.2

0.1 0.2 19.3 4.1 12.7 7.3 1.7 9.1 1.3

c,:,

2.4

1.4

C24:OH

0.5 2.2

r

GLYCOSPHINGOLIPIDS

OF HUMAN

the longer monosialo gangliosides are members of the lactoneotetrasyl series, whereas the polysialo gangliosides belong to the ganglio series. The predominant fatty acid species of plasma gangliosides are GM, CIW , Gee, and CM, and GD~ and GDla contain small quantities of hydroxy fatty acids. This pattern is similar to that of erythrocyte gangliosides but differs from brain gangliosides. The presence of neutral glycolipids containing one to four sugars has been reported previously (l-5, 45), but the identification of these compounds was based primarily on sugar composition and chromatographic mobility. We have extended these reports by analyzing methylated derivatives, performing immunological identification, and examining the fatty acid composition in these compounds. Plasma globoside and the upper bands of CDH and CTH contain much less CZ2 and CZ4 fatty acid than the erythrocyte compounds, which suggests that the major portion of the plasma compounds are not derived from the erythrocyte membrane. The blood group H, Lea, and Leb GSLs have been isolated and characterized extensively by Hanfland and collaborators (8-10). We have also tentatively identified another GSL, X hapten, which contains the Type 2 sequence, Galpl-4(Fuccul3)GlcNAc-, by its reactivity with a monoclonal antibody. The site(s) of synthesis of the plasma glycolipids have not been identified, but the long-chain base composition of the Le” and Leb compounds (9) suggests that they may be synthesized in the intestine (46). The identification of the plasma GSLs reported here provides useful information for future studies of the transfer of these compounds among lipoproteins, and from lipoproteins to cells, and for analysis of alterations in plasma glycolipids in patients with cancer. ACKNOWLEDGMENT The authors thank Ms. Charlene Shackelford her outstanding secretarial assistance.

for

PLASMA

399

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