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Biochimica et Biophysica Acta, 588 (1979) 35--43

© Elsevier/North-Holland Biomedical Press

BBA 29083

ISOLATION AND IDENTIFICATION OF A FUCOSE-CONTAINING GANGLIOSIDE F R O M BOVINE T H Y R O I D GLAND

BRUCE A. MACHER a,,, TADEUSZ PACUSZKA b , * * , BRIAN R. MULLIN c, CHARLES C. SWEELEY a, ROSCOE O. B R A D Y b and PETER H. FISHMAN h,*** a Department of Biochemistry, Michigan State University, East Lansing, MI 48824; b Developmental and Metabolic Neurology Branch, National Institute of Neurological and Communicative Disorders and Stroke, The National Institutes of Health, Bethesda, MD 20014, and c Department of Pathology, Cleveland Metropolitan General Hospital, Cleveland, OH 44109 (U.S.A.) (Received March 21st, 1979)

Key words: Ganglioside; Fucose; Carbohydrate analysis; (Bovine thyroid gland)

Summary Bovine thyroid glands are known to contain a complex array of gangliosides. One of the predominant gangliosides was isolated and analyzed by gas-liquid chromatography and mass spectrometry. The carbohydrate composition was fucose, N~acetylneuraminic acid, galactose, N-acetylgalactosamine, and glucose in molar ratios of 1:1:2:1:1. The structure of the ganglioside was identified as: Fuc(1-2)Gal(1-3)GalNAc(1-4)Gal(1-4)Glc(1-1)ceramide (3i2) AcNeu

Introduction Gangliosides are constituents of plasma cell membranes and are involved in certain recognition phenomena. The role of ganglioside GM1 as the receptor for * Present address: Cancer Research Institute. University of California, San Francisco, CA 94143, U.S.A. ** Present address: Zaklad Biochemfi, I n s t y t u t Hematologii, Warszawa, Poland. *** To w h o m correspondence should be addressed. Abbreviations: Fuc, fucose; Me3Si, t r i m e t h y l s i l y l ; GMI , G a l ~ l - 3 G a l N A c ~ l - 4 G a l [ 3 - 2 a A c N e u ] ~ l - 4 G l c ~ l l c e r a m i d e ; GM2 , G a l N A C ~ l - 4 G a l [ 3 - 2 ~ A c N e u ] ~ l - 4 G l c f l l - l - c e r a m i d e ; GM3 , AcNeu~2-3Gal~l-4Glc~l° l c e r a m i d e ; G D l a , AcNeu~2o3Gal~l-3GalNAc~l-4Gal[3-2aAcNeu]~l-4Glc{~l-lceramide;GD3 , AcNeu~28AcNeu~2-3 G a l ~ l - 4 G l c ~ l - l c e r a m i d e .

36 cholera toxin is a prime example ([1] and references therein). Although they are found in highest amounts in brain, there has been considerable interest in gangliosides from extraneural tissues [1,2]. Bovine thyroid gland contains a complex mixture of gangliosides [3--6] * and fractionation by DEAE-cellulose and silica gel column chromatography indicated that at least 30 different gangliosides were present [6]. Preliminary analyses of several of these thyroid gangliosides showed that bovine thyroid gland contains GM3 and GD3 , gangliosides corresponding to the major ones found in brain **, and one that contains fucose. In this communication, we report the structural identification of this fucose~ontaining ganglioside. Experimental procedures

Materials. Bovine thyroid ganglioside fraction 7 was isolated as described previously [6]. Briefly, the total ganglioside mixture was fractionated first by DEAE-cellulose column chromatography; gangliosides enriched in fraction 7 were eluted with 0.022 M NH4Ac, pooled and dialyzed against H20. Fraction 7 was purified further by chromatography on two silica gel H columns; the first column was eluted with chloroform/methanol/H20 (60:35:8, v/v)and the second with chloroform/methanol/2.5 M NH4OH (60:35:8, v/v). 8.2 pmol were obtained from 1.5 kg of thyroid which contained 195 pmol of lipid-bound sialic acid. Gangliosides used as standards for thin-layer chromatography were prepared as described previously [ 8]. Globoside from human kidney as well as GM1 and GM3 were used as reference standards for compositional and structural analyses. Neuramin lactose and phenyl-N-acetyl-~-D-glucosaminide were obtained from Sigma and mucin was kindly provided by Dr. S.S.J. Sung. Gasliquid chromatography column packings were from Supelco (Bellefonte, PA). Carbohydrate compositional analysis. Methanolysis of the gangliosides and preparation of the Me3Si derivatives of the methylglycosides were carried out as described by Laine et al. [9]; less than 10 mg Ag2CO3 were used to neutralize the HC1 remaining after methanolysis. Recovery of amino sugars was 50-60% of that found for neutral sugars. A Hewlett-Packard 5840A gas-liquid chromatograph equipped with glass columns was used for the analysis. For Me3Si methylglycoside analysis, the column packing was 3% SE-30 and the chromatograph was programmed from 140 to 240°C at 3 deg.C/min. Characterization of O-Me3Si derivatives of sialic acid was performed according to Yu and Ledeen [10]. AcNeu from neuramin lactose and N-glycolylneuraminic acid from mucin were reference standards and phenyl-N-acetyl~-D-glucosaminide served as an internal standard. The column (3% SP-2100) was run isothermally at 210°C. * Gangliosides [ 4 ] a n d , in particular, t h y r o i d gangliosides [ 6 , 7 ] h a v e b e e n f o u n d t o i n t e r a c t w i t h t h y r o tzopin. F r a c t i o n 7, h o w e v e r , w a s a p o o r i n h i b i t o r o f t h y r o t r o p i n binding to t h y r o i d m e m b r a n e s c o m p a r e d to o t h e r t h y r o i d gangliosides [ 6 ] . ** T h e s e i n c l u d e d G M 3 and G D 3 c o n t a i n i n g A c N e u and N - g l y c o l y l n e u r a m i n i c acid and gangliosides corr e s p o n d i n g to GM1 and G D 1 a. Similar results w e r e r e p o r t e d b y van D e s s e l et al. [5] ; t h e s e a u t h o r s , h o w e v e r , did n o t describe t h e f u c o s e - c o n t a i n i n g ganglioside r e p o r t e d h e r e . In a d d i t i o n , t h e r e w e r e t w o f r a c t i o n s c o n t a i n i n g N - a c e t y l g l u c o s a m i n e , w h i c h m a y b e sialylated derivatives o f p a r a g l o b o s i d e ( G a i ~ l 4 G l c N A c ~ l - 3 G a i ~ l - 4 G l c f l l - l c e r a m i d e ) . T h e s e s t u d i e s will b e r e p o r t e d e l s e w h e r e .

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Permethylation and mass spectrometry. Permethylation was performed as described by Hakomori [11]. The intact permethylated ganglioside was studied by direct probe electron impact mass spectrometry with a Varian MAT CH-5 mass spectrometer under the following conditions: ion source temperature, 260°C; sample probe temperature program, from ambient to 350°C; electron energy, 70 eV; accelerating voltage, 3 kV. Partially methylated alditol acetates were prepared from the permethylated ganglioside as follows. A portion of the permethylated ganglioside was hydrolyzed in 1 ml of concentrated HCl/absolute ethanol/H20 (1:1:1, v/v) for 4 h at 100°C. After cooling, the reaction mixture was extracted three times with equal volumes of hexane. The aqueous phase was dried at 45°C under a stream of N2 and the residue was suspended in 1 ml of 1 M HC1. After hydrolysis for 1 h at 100°C and cooling, the hydrolysate was extracted five times with equal volumes of ether and the aqueous phase was dried; the residue was suspended in 2 ml of 1 M NtLOH and treated with 20 mg of NaBH4 at 4°C overnight. This solution was extracted three times with equal volumes of ethyl acetate and then acidified with glacial acetic acid (2--3 drops) to destroy excess NaBH4. Methyl borate was removed by five successive dryings with methanol. The dried residue was acetylated by adding 0.5 ml of a mixture of acetic anyhydride/pyridine (1:1, v/v) and heating at 100°C for 90 min. The sample was dried under a stream of N2 with several additions of toluene to remove excess acetic anhydride. The residue was partitioned with chloroform and H20; the aqueous phase was discarded and the lower phase was extracted with H20 until clear (5--6 times). The lower phase was dried and the partially methylated alditol acetates were analyzed on a 3% OV-225 column installed in a HP-5985A combined gas-liquid mass spectrometer under the following conditions: column linear program from 180 to 250°C at 10 deg.C/min; separator temperature, 275°C; ion source temperature, 250°C; electron impact ionization, 70 eV; electron energy, 300 pA. Other methods. Lipid-bound sialic acid was quantified by a modified resorcinol method [6]. Analysis of gangliosides by thin-layer chromatography and treatment of gangliosides with Vibrio cholerae neuraminidase were done as previously described [6,8]. Fatty acids were determined by gas-liquid chromatography of the corresponding methyl esters [9]; the column was packed with 3% DEGS and run isothermally at 170°C. Results and Discussion

Thin-layer chromatography Bovine thyroid ganglioside fraction 7 appeared homogeneous when separated on thin-layer silica gel (Fig. 1). It migrated between GM1 and GDIa in the first solvent system (Fig. la) and the same as GDIa in the solvent system containing NH4OH (Fig. lb). These differences in migration were useful in its purification. It also appears to be a major thyroid ganglioside; based on scanning densitomerry of the chromatogram, it represented 25.6 _+3.1% (mean _+S.D. of three separate analyses) of the lipid-bound sialic acid. As indicated in Experimental procedures, only 8.2 t~mol were recovered in pure form for further analysis from 195 t~mol of lipid-bound sialic acid. This ganglioside was completely resis-

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Fig. 1. Thin-layer c h r o m a t o g r a m s of bovine t h y r o i d gangliosides. Ganglioside standards (lane 1), bovine thyr o id ganglioside fraction 7 (lane 2), and bovine thyroid gangliosides (lane 3) were separated on silica gel 60-coated glass plates (E. Merck, Darmstadt, F.R.G.) in the following solvent systems: (a) c hl oroform/ m e t h a n o l / 0 . 2 5 % CaC12 (60:35:8, v/v)~ and (b) c h l o r o f o r m / m e t h a n o l / 5 M NH4OH/2% CaC12/H20 (60: 35:4 : 1 : 3, v/v). Gangliosides were visualized with resorcinol reagent [ 6 ].

tant to bacterial neuraminidase under conditions where an equal a m o u n t of GD1a Was converted to GM1 (data not shown).

Carbohydrate structure Based on gas-liquid chromatography of the Me3Si methylglycosides prepared from fraction 7, this ganglioside contained fucose, galactose, glucose, N-acetylgalactosamine and sialic acid in the molar ratios of 0.8:2.0:1.0:0.9:1.0 (Fig. 2). In a separate analysis, the sialic acid was identified as N-acetylneuraminic acid. Combined gas-liquid mass spectrometric analysis of the partially methylated alditol acetates derived from the permethylated ganglioside are summarized in Table I and Figs. 3 and 4. Five peaks were separated by gas-liquid chromatography (see Table I for the retention times) and six compounds could be identified by mass spectrometry. Peak 1 was identified as 1,5~li-O-acetyl-6