Ganglioside glycosyltransferase assay using ion-exchange chromatography

ANALYTICAL

BIOCHEMISTRY

152, 154- 159 ( 1986)

Ganglioside Glycosyltransferase Assay Using Ion-Exchange Chromatography’ KEVIN Departments

M. WALTONANDRONALD

L. SCHNAAR'

of Pharmacology and Neuroscience, The Johns Hopkins School of Medicine. Baltimore, Maryland 21205

University

Received June 26, 1985 A rapid method for determination of ganglioside glycosyltransferaseactivity has been developed employing ion-exchange chromatography. Using l3-day chick brain as a source of uridine diphospho-N-acetyl-D-galactosamine: ganglioside GM3 N-acetylgalactosaminyltransferase (ganglioside GM2 synthetase), we were able to accurately measure transfer of N-[‘H]acetylgalactosamine (GalNAc) from UDP-[3H]GalNAc to GM3 by application of the reaction mixture to small columns of DEAE-Sepharose and elution of radiolabeled GM2 reaction product with IO mM potassium acetate in methanol. This method proved to be as reliable and sensitive as previously published assaysbut requires less time and fewer manipulations. 0 1986 Academic Press, Inc. KEY WORDS: gangliosides; glycosyltransferases; nucleotide sugars.

Gangliosides are an important class of cell surface complex carbohydrates which exist at high concentrations in the brain (I ). They are localized primarily on the outer membrane leaflet of neurons, particularly in areas of cellcell contact (2,3). They have been implicated as regulators of neuronal function and intercellular interactions and may be modulated during neoplastic transformation (4-7). The expression of specific gangliosides varies during embryonic development (8) and upon cellular differentiation in vitro (9,10). The biosynthetic pathways for many gangliosides have been determined ( 11,12). They are synthesized by stepwise addition of saccharides from nucleotide sugars to the growing oligosaccharide chain by the appropriate glycosyltransferases (13,14). The control of the activity of these glycosyltransferases by cells is likely to be pivotal in ganghoside expression and, ultimately, function, A rapid, simple as-

say for nucleotide sugar:ganglioside glycosyltransferases would be of value for the examination of these enzymes. This paper reports a new assay for uridine diphospho-N-acetyl-Dgalactosamine:GMs N-acetylgalactosaminyltransferase which should also be applicable for the assay of some of the other enzymes involved in ganglioside metabolism. Previously published assays have separated ganglioside product from nucleotide sugar precursor and other radiolabeled breakdown products in the reaction mixture by time-consuming paper electrophoresis, by paper chromatography, or by the use of relatively large Sephadex G-25 partition columns requiring evaporation of solvents and/or dialysis before the product radioactivity could be determined ( 15- 17). Recent reports of the use of ion-exchange chromatography (18) to purify gangliosides led us to test an alternate and simpler procedure. In this paper we present an assay method using

’ This work was supported by National Institutes of Health Grants HD 14010 and GM 07626 (for support of K.M.W.). R.L.S. is the recipient of an American Cancer Society Faculty Research Award (FRA-280). * To whom correspondence should be addressed.

3Abbreviations used: UDP-GalNAc, uridine diphosphoN-acetyl-D-galactosamine; GalN, Galactosamine; GalNAc, N-acetylgalactosamine; GalN-I-P, galactosamine l-phosphate; and MES, 4-morpholineethanesulfonic acid. Ganglioside nomenclature used is that of Svennerholm (2 I).

0003-2697186 $3.00 Copyright 0 1986 by Academic Press, Inc. All rights of reproduction in any form reserved.

I54

GANGLIOSIDE

GLYCOSYLTRANSFERASE

small columns of DEAE-Sepharose which allows rapid separation and quantitation of the radiolabeled ganglioside product. In addition, breakdown of the nucleotide sugar substrate to free sugar during the incubation can be readily determined. MATERIALS

AND

METHODS

Materials

DEAE-Sepharose CL-6B was obtained from Pharmacia; uridine diphosphate N-acetyl-D[3H]galactosamine (NET-465, 8.7 Ci/mmol) and [3H]acetic anhydride (NET-O 18,50 mCi/ mmol) from New England Nuclear; uridine diphospho-N-acetylgalactosamine (UDPG~~NAc)~, galactosamine (GalN), and galactosamine 1-phosphate (GalN- 1-P) from Sigma Chemical Company; the buffer 4-morpholineethanesulfonic acid (MES) and Triton X100 from Calbiochem; the detergent Cutscum from Fischer Scientific; silica gel 60 TLC plates from E. Merck, Darmstadt; and embryonated chick eggs from Truslow Farms, Chestertown, Maryland. Ganglioside GM3 was purified from bovine spleen and GM2 from an extract of Tay-Sachs brain tissue kindly supplied by Dr. L. Hoffman and Dr. L. Schneck, Kingsbrook Jewish Medical Center, Brooklyn, New York. The gangliosides were purified by solvent partitioning, ion-exchange chromatography, and silicic acid chromatography as described previously (18). [3H]GalNAc- I-P and [3H]GalNAc were prepared by N-acetylation of the appropriate hexosamine using [3H]acetic anhydride (19). Other materials and chemicals were of the highest available grade from standard commercial sources. Methods Brain microsomes. UDP-GalNAc:GM3 Nacetylgalactosaminyltransferase was prepared in crude form as described previously (15). Briefly, whole brains were removed from 13day embryonic chicks, frozen overnight, and homogenized in 0.25 M sucrose, 0.085% pmercaptoethanol (homogenization buffer), and microsomes were collected by differential

ASSAY

155

centrifugation. Washed microsomal pellets were suspended in homogenization buffer at a concentration of lo-20 mg protein/ml and stored at -20°C. Protein determinations were by the method of Lowry et al, (20). UDP-GalNAc:GM3 N-acetylgalactosaminyltransferaseassay. The reaction was performed

as previously described with minor modifications (17). An aliquot (25 ~1) of purified ganglioside GM3 (2 nmol/Ml) dissolved in chloroform:methanol (2: 1) was mixed in a test tube with 166 ~1 of a detergent solution consisting of 2.7 mg/ml Cutscum and 1.3 mg/ml Triton X- 100 in chloroform:methanol (2: 1). The solvents were evaporated under a stream of nitrogen, and 55 ~1 of assay mixture consisting of 50 mM Tris buffer (pH 7.3) 5 mM M&l*, 0.36 mM UDP-[‘H]GalNAc (sp act, 9 mCi/mmol), and 0.2 mg brain microsomal protein (or as indicated) was added. The mixture was sonicated, mixed, and incubated for 30 min (or as indicated) at 37°C. The reaction was terminated by addition of 1.1 ml of chloroform:methanol (2: 1). Separation and quantitation of reaction products. DEAE-Sepharose CLdB was con-

verted to its acetate form using the manufacturer’s instructions and washed sequentially with water, methanol, and chloroform:methanol:water (4:8:3). Resin was routinely stored in the latter solvent. Small ion-exchange columns were prepared by pipetting a suspension containing 0.3 ml of resin into a Pasteur pipet plugged with a 3-mm glass bead. The column was washed with approximately 2 vol Solvent A [chloroform:methanol:water (120:60:9)]. Reaction mixtures (see above) were applied to columns, and then eluted with 0.5 ml of Solvent A and 2 ml of methanol. The salt-free eluates were routinely discarded. However, on occasion (see below), they were combined and processed for determination of radiolabeled free sugar. After the methanol wash, ganglioside product was eluted with 4 ml of 10 mrvr potassium acetate in methanol and the eluates were collected directly into glass 20-ml scintillation vials. Liquid scintillation fluor ( 12 ml,

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Beckman Readi-Solv EP) was added and radioactivity determined using a liquid scintillation spectrometer (LKB 12 17 Rackbeta). As indicated, subsequent 4-ml fractions containing higher concentrations of potassium acetate in methanol (20 mM, 30 mM, 50 BIM, and 1M) were collected and radioactivity was quantitated. Counting efficiency was determined on selected samples by addition of internal standard. Under these conditions counting efficiency was 22% and was not affected by the concentration of potassium acetate. For occasional determination of the radiolabel in the salt-free (run-through) fraction, efficient counting required evaporation of the chloroform-containing solvent prior to addition of water (1 ml) and scintillation fluor (12 ml). The current method was compared with that of Kemp and Stoolmiller (17) in which the reaction was terminated by addition of 1.1 ml of chloroform:methanol (2: 1) and the entire mixture applied to a column of Sephadex G-25 (0.8 X 4.5 cm) preequilabrated with chloroform:methanol:water ( 120:60:9). The column was washed with 3 ml of chloroform: methanol (2:l) and the eluates were pooled, evaporated, and resuspended in a small volume of water. After dialysis for 6 h against running water, scintillation fluor ( 12 ml) was added and radioactivity measured.

20

40 60 TIME (mid

00

RESULTS

AND

DISCUSSION

Using the rapid ion-exchange chromatography assay described under Materials and Methods, the transfer of GalNAc from UDPGalNAc to ganglioside GM3 to form ganglioside GM2 (see product characterization below) proceeded linearly for at least 90 min as shown in Fig. 1A (other experiments showed linearity for 3 h). Synthesis of GM2 was also proportional to protein concentration in the reaction mixture up to approximately 2 mg microsomal protein/ml (0.1 mg protein/reaction, Fig. 1B). The nonlinearity at higher protein concentrations was not due to depletion of substrates since increasing the concentration of GM3 or UPD-GalNAc threefold did not yield an increase in GM;, production at the higher concentrations (data not shown). Background radiolabel in our assays was very low, as shown in Table 1. Elimination of the acceptor (GMJ) from the reaction mixture resulted in a 92% reduction in radioactivity eluting with 10 IBM potassium acetate (monosialoganglioside fraction). Heat denaturation of the enzyme eliminated product formation, and omitting manganese or detergents from the reaction reduced enzyme activity to 15% of control levels (Table 1). These activity requirements are the same as those reported

0.1 PROTEIN

a2 ADDED

0.3 0.4 Implmaction)

FIG. 1. Enzymatic synthesis of GM2 from UDP-[3H]GalNAc and GMs as a function of time (A) and protein concentration (B). (A) Reaction mixtures with 0.2 mg microsomal protein were incubated for the indicated period of time and processed as described under Materials and Methods. (B) Reaction mixtures containing the indicated amounts of protein were incubated for 30 min and processed as described under Materials and Methods.

GANGLIOSIDE

GLYCOSYLTRANSFERASE

ASSAY

157

previously (17). The detergent mixture of Cutscum:Triton X-100 (2:l) has been used previously in determination of glucosyl, galactosyl, and N-acetylgalatosaminyltransferases ( 16,17). The concentration of detergent described under Materials and Methods was optimized for production of GM2 in our assay, and could be replaced with an equal concenI 2 3 4 5 tration of Triton X- 100 alone without changELUTION VOLUME (ml) ing the activity. When enzyme activities were FIG. 2. Elution of [‘H]GMg from DEAE-Sepharose. compared using the current method and that Complete reaction mixtures (open circles) or control repreviously published by Kemp and Stoolmiller actions lacking GM, (closed circles) were incubated for 30 (17) using the same enzyme source, specific min and processed as described under Materials and Methods. [‘H]GMz was eluted with sequential l-ml aliactivities agreed within 7%. quots of 10 mM potassium acetate in methanol and raThe volume used to elute monosialoganglioside product was optimized as shown in dioactivity determined. Specifically transferred radioactivity (open squares) was determined by subtracting the Fig. 2. Near-maximum elution of ganglioside counts in control reactions from counts eluted from comproduct was accomplished with 3 ml of 10 mM plete reaction mixtures. potassium acetate in methanol; elution was routinely performed with 4 ml of this solution etate4 (see above) was evaporated, resuspended to ensure maximal product recovery. in a small volume of water, dialyzed overnight Identification of the quantitated product was performed using TLC. The eluant col- at 4°C to remove salts, and concentrated. lected upon addition of 10 mM potassium ac- When this sample was chromatographed on silica gel-coated TLC plates, the radiolabel comigrated with authentic GM2 (Fig. 3). No TABLE 1 radiolabel migrated in positions which would correspond to the free sugar, sugar phosphate, REQUIREMENTS FOR UDP-GalNAc:GMs IV-ACETYLGAor nucleotide sugar substrate. The ion-exLACTOSAMINYLTRANSFERASE ACTIVITY change chromatographic characteristics of [3H]GM2 formed these latter compounds were explored further Reaction component (cpm) as described below. Under the conditions of the glycosyltransComplete” 1621 ferase assay, the nucleotide sugar (radiolabeled Minus GM3 120 in the sugar moiety) is susceptible to phosMinus MnCI, 210 phodiesterases and phosphatases resulting in Minus detergents 349 Heat-inactivated enzymeb 127 formation of radiolabeled sugar phosphate and free sugar in addition to the transfer of sugar ’ Complete reaction mixtures contained 50 nmol GM3 to the GM3 acceptor molecule to form GM2 and 0.2 mg brain microsomal protein in 55 ~1 of solution (17). To test where each of these species elutes containing 50 mM Tris buffer (pH 7.3). 5 mM MnQ, 0.36 mM UDP-[‘H]GalNAc (adjusted to a specific activity of 9 mCi/mmol with unlabeled UDP-GalNAc), and detergents (Cutscum and Triton X-100) as described under Materials and Methods. All reactions were incubated for 30 min at 37°C and then processed as described under Materials and Methods. b The enzyme was heated at 95°C for 2 min before addition to the reaction.

4 Although Fredman reports elution of ganghosides from DEAE-Sepharose using 20-500 mM potassium acetate in methanol (18). we have repeatedly found (9) that monosialogangliosides elute completely using IO mM potassium acetate in methanol, while higher order (up to pen&&lo-) gangliosides elute using 20-50 mM potassium acetate in methanol.

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procedure except that after elution with 10 mM potassium acetate in methanol, higher salt concentration fractions were eluted (see Materials and Methods). In a separate experiment, 30 nmol of authentic GM2 was added to a reaction mixture which was then subjected to the same ion-exchange chromatography. The various fractions were evaporated to dryness, resuspended in a small volume of water, and dialyzed overnight at 4°C and the amount of GM2 in each fraction was determined by 2 4 6 6 IO 12 solvanl FVJd quantitative TLC (9). The resulting values MIGRATION DISTANCE km) were compared to those obtained when a FIG. 3. Identification of radiolabeled product. A comknown amount of GM2 was subjected to the plete reaction mixture was incubated for 30 min, processed, and the 10 mM potassium acetate eluate desalted and con- same evaporation, resuspension, and dialysis centrated as described under Materials and Methods. The without having been chromatographed. sample was chromatographed on silicic acid TLC in chloThe results from these studies are shown in roform:methanol:0.25% aqueous potassium chloride (60: Fig. 4. The free sugar eluted in the salt-free 35%). the TLC was scraped in 0.5-cm segments into ! ml of water and sonicated, 6 ml of scintillant was added, and run-through fraction, GM2 (and other monosialogangliosides) eluted in the 10 tnM potasradioactivity was determined. The mobility of standard GM* (arrow) was determined by chromatography in an sium acetate fraction, and the nucleotide sugar adjoining lane after visualization using a resorcinol spray and sugar phosphate eluted partially in higher reagent (9). salt fractions, the rest remaining on the column. Thus, in the same reaction mixture, both in our ion-exchange chromatography method, the transferase activity and the breakdown to free sugar can be measured simply by collectwe prepared assay mixtures which contained [3H]GalNAc or [3H]GalNAc-l-P in place of ing the run-through as well as the 10 mM pothe radiolabeled nucleotide sugar. The reaction tassium acetate fractions. A large percentage mixtures were treated as in our standard assay of substrate counts appearing in the runa--

-

I

Q,+J 25 8% dg

1234 0

1234 lOllhI POTASSIUM

1234 20mM ACETATE

1234 30mM CONCENTRATION

1234 50mM

1234 IM

FIG. 4. Elution of GMz, UDP-GalNAc, GalNAc-l-P, and GalNAc from DEAE-Sepharose. Complete reaction mixtures were prepared containing either UDP-[‘H]GalNAc, [3H]GalNAc-I-P, or [3H]GalNAc as the sole radiolabeled moiety. Chloroform:methanol(2: I) was added (1.1 ml) and the resulting mixtures were added to 0.5 ml DEAE-Sepharose columns. The radiolabel was eluted with increasing concentrations of potassium acetate in methanol as indicated, and radioactivity determined. In a separate experiment, the elution profile of unlabeled GM2 was determined by addition of standard GM2 to a reaction mixture, application to and elution from the column with increasing salt concentrations as indicated, dialysis and concentration of the resulting fractions, and quantitation of GM2 by quantitative TLC (9).

GANGLIOSlDE

GLYCOSYLTRANSFERASE

through would indicate phosphodiesterase and phosphatase levels incompatible with accurate transferase determinations. The above data indicate that this rapid and simple column chromatographic procedure is accurate for the measurement of UDPGalNAc:GM3 N-acetylgalactosaminyltransferase. Based on chromatographic properties of neutral sugars, monosialogangliosides, and nucleotide sugars, this assay should also be applicable to those glycosyltransferases and glycosidases which are involved in the metabolism of monosialogangliosides. ACKNOWLEDGMENTS We are grateful to Dr. L. Hoffman and Dr. L. Schneck for kindly providing the extract of Tay-Sachs tissue, to Dr. S. Basu for advising us on the preparation of GMr from bovine spleen, and to Paula Manzuk for manuscript preparation.

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18. Fredman, P. (1980) in Structure and Function of Gangliosides (Svennerholm, L., Mandel, P., Dreyfus, H., and Urban, P.-F., eds.), pp. 23-3 1, Plenum Press, New York. 19. Roseman, S., and Ludoweig, J. (1954) J. Amer. Chem. Sot. 76,30 I-302. 20. Lowry, 0. H., Rosebrough, N. J., Farr, A. L., and Randall, R. J. (195 1) J. Biol. Chem. 193,265-275. 2 I. Svennerholm, L. (1980) in Structure and Function of Gangliosides (Svennerholm, L., Mandel, P., Dreyfus, H., and Urban, P.-F. eds.), p. 11, Plenum Press, New York.