[8] Glycosyltransferase probes

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above, and 200 #1 of concentrated HC1 is added. Alternatively, the dry sample can be dissolved in 400/~l of constant boiling HC1. The sample is hydrolyzed at 100 ° for 4 - 6 hr, and the acid is removed by evaporation. Recovery ofglucosamine from glycopeptides containing N-linked oligosaccharides has been shown to be quantitative, l° Approximately 50% of the neutral sugars present in the sample are destroyed under these hydrolysis conditions. Sensitivity. Sample and matrix composition will greatly influence the sensitivity of HPAE-PAD for biological samples. Routine analyses at 1000 nA full scale in the 300-3000 pmol range are accurate and reproducible. Analysis of 10- 100 pmol of hydrolyzates may be performed with a carefully maintained working electrode. ~°

[8] G l y c o s y l t r a n s f e r a s e

Probes

B y SIDNEY W . W H I T E H E A R T , A N T O N I N O PASSANITI, J O N A T H A N S.

REICHNER, GORDON D. HOLT,ROBERT S. HALTIWANGER,and GERALD W. HART

Introduction Glycosyltransferases catalyze the transfer of a monosaccharide from an activated sugar nucleotide donor to a specific acceptor such as monosaccharides, oligosaccharides, lipids, or amino acid residues Sugar Nucleotide + Acceptor glycosyltran~erase,sugar acceptor ÷ nucleotide

The enormous structural diversity of acceptor oligosaccharides, lipids, or proteins require that glycosyltranferases be very specific enzymes that can distinguish the spatial orientation of a single hydroxyl moiety on even the largest and most complicated glycoconjugates. 1 In fact, available data suggest that there probably exist distinct glycosyltransferases for virtually every type of glycosidic linkage known. 2 Since saccharide structures are not directly encoded in the genome, the high specificity of these enzymes is i T. A. Beyer, J. E. Sadler, J. I. Rearick, J. C. Paulson, and R. L. Hill, Adv. Enzymol. 52, 23 (1981). 2 S. Roseman, Chem. Phys. Lipids 5, 270 (1970).

METHODS IN ENZYMOLOGY, VOL. 179

Copyright© 1989by AcademicPress,Inc. All fightsof reproductionin any formreserved.

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crucial to maintaining the fidelity of site-specific glycosylation, 3,4 which appears to be important in many biological processes. 5 The exquisite substrate specificities of glycosyltransferases used as structural probes offer the closest analogy to restriction endonucleases that is available to the glycoconjugate biochemist? It has become feasible to use these enzymes as structural probes because of the recent development of rapid and facile affinity purification methods. 7 Glycosyltransferases offer many advantages over lectins or chemical methods as probes of carbohydrate structure: (1) Transferases are more specific, and their specificities are very well known. (2) They catalyze the formation of a covalent bond, allowing highly sensitive product characterization when used with radioactive sugar nucleotides. For example, purified glycosyltranferases have been used to detect and characterize specific O-linked or N-linked saccharides on purified proteins, 8,9 viruses, ~° or intracellular organelles, t~ (3) As cell surface probes, both sugar nucleotide donor and enzymes are impermeant to the plasma membrane, allowing their use in the study of intracellular trafficking of surface molecules12a3 and in the determination of the role of a specific saccharide linkage in recognition processes. 14-~6 (4) Since the specific activity of the sugar nucleotide is known, under saturation conditions glycosyltransferases can also provide an accurate estimate of the numbers of specific terminal or penultimate saccharide structures accessible on the surface of a living cell or on a purified g l y c o p r o t e i n . 6,H,14,17,18 (5) When combined with specific exoglycosidases, glycosyltransferases can yield important information about the substitution state and polypeptide

3 S. J. Swiedler, J. H. Freed, A. L. Tarentino, T. H. Plummer, Jr., and G. W. Hart, J. Biol. Chem. 260, 4046 (1985). 4 p. Hsieh, M. Rosner, and P. Robbins, J. Biol. Chem. 258, 2555 (1983). 5 T. W. Rademacher, R. B. Parekh, and R. A. Dwek, Annu. Rev. Biochem. 57, 785 (1988). 6 S. W. Whiteheart and G. W. Hart, Anal. Biochem. 163, 123 (1987). 7 j. E. Sadler, T. A. Beyer, and R. L. Hill, J. Chromatogr. 215, 181 (1981). 8 C. E. Machamer and P. Cresswell, Proc. Natl. Acad. Sci. U.S.A. 81, 1287 (1984). 9 G. D. Holt, R. S. Haltiwanger, C.-R. Torres, and G. W. Hart, J. Biol. Chem. 262, 14847 (1987). lOD. M. Benko, R. S. Haltiwanger, G. W. Hart, and W. Gibson, Proc. Natl. Acad. Sci. U.S.A. 85, 2573 (1988). 11 G. D. Holt and G. W. Hart, J. Biol. Chem. 261, 8049 (1986). 12 j. Reichner, S. W. Whiteheart, and G. W. Hart, J. Biol. Chem. 263, 16316 (1988). 13j. R. Duncan and S. Kornfeld, J. Cell Biol. 106, 617 (1988). 14 L. D. Powell, S. W. Whiteheart, and G. W. Hart, J. Immunol. 139, 262 (1987). 15 j. E. Sadler, J. C. Paulson, and R. L. Hill, J. Biol. Chem. 254, 2112 (1979). ~6G. N. Rogers, G. Herrler, J. C. Paulson, and H.-D. Klenk, J. Biol. Chem. 261, 5947 (1986). t~ C.-M. Torres and G. W. Hart, J. Biol. Chem. 259, 3308 (1984). ~s A. Passaniti and G. W. Hart, J. Biol. Chem. 263, 7591 (1988).

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1234

FIG. 1. SDS-PAGE of sialidase-treated AKTB-lb B cell lymphoma after resialylation by three different sialyltransferases. The GalNAc ct-2,6- (lane 1), fl-galactoside t~-2,3- (lane 2), and fl-galactoside ot-2,6- (lane 3) sialyltransferases were used with CMP[3H]NeuAc to label the surfaces of AKTB-lb lymphoma cells that had been pretreated with Vibrio cholerae sialidase. The "no enzyme" lane (lane 4) contains cells that had been treated only with CMp[3H]NeuAc.

distribution of different penultimate saccharides structures (Fig. 1) on biologically important receptors (Fig. 2). Several studies on the use of glycosyltransferases as structuralfunctional probes have been published, 6,t5,19-21 and recent reviews in this 19L. Thilo, this series, Vol. 98, p. 415. 20j. C. Paulson and G. N. Rogers, this series, Vol. 138, p. 162. 2J R. L. Hill, T. A. Beyer, J. C. Paulson, J.-P. Prieels, J. I. Rearick, and J. E. Sadler, in "Frontiers of Bioorganic Chemistry and Molecular Biology" (S. N. Ananchenko, ed.), p. 63, Pergamon, Oxford, 1980.

[8]

GLYCOSYLTRANSFERASE PROBES

85

,,m

214 111

T200

gPTO

68 45

H2

24

Thyl

18 15 FIG. 2. SDS-PAGE analysis of exosialylated cell surface proteins from EL-4 thymoma and C3H He/Sn/J thymocytes. Lanes marked [3H]NeuAc: cells were treated with V. cholerae sialidase prior to sialylation with the fl-galactoside c~-2,6-sialyltransferase and CMP[3H]NeuAc. Lane marked t2SIodine: cells were surface iodinated by the lactoperoxidase method. All other lanes represent total cellular proteins metabolically labeled with either [3H]glucosamine or [35S]methionine or stained with Coomassie after SDS-PAGE. Note that the sialyltransferase substrates are previously identified glycoproteins that represent a subset of the total cellular or cell surface proteins. In addition, these glycoproteins are also antigenically important markers on thymocytes.

series have discussed their purification. 22 While currently only three glycosyltransferases are commercially available, and radiolabeled sugar nucleotides are quite expensive and difficult to synthesize, 23 more of these valu22 j. E. Sadler, T. A. Beyer, C. L. Oppenheimer, J. C. Paulson, J.-P. Prieels, J. I. Rearick, and R. L. Hill, this series, Vol. 83, p. 458. 23 O. Gabriel, this series, Vol. 83, p. 332.

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able enzymes are becoming available each year. Eventually, when many of these enzymes are more readily available, it is likely that they will become essential tools widely used by nearly all glycobiologists. In this chapter, we outline methods and provide examples applicable to the use of virtually any soluble or membrane-bound glycosyltransferase as a probe of oligosaccharide structure. Methods General Controls

Since glycosyltransferase probes are enzymes, it is necessary to determine optimal parameters for each protein, cell type, or purified organelle to be studied. (1) Dependence of the reaction on enzyme concentration, time, sugar nucleotide concentration, and acceptor concentration should be determined (Fig. 3). (2) For whole cell or organelle labeling studies, the extent of sugar nucleotide degradation should be measured, and the possibility of extensive metabolic reutilization of the labeled saccharide must be eliminated. (3) It is now evident that many glycosyltransferases show oligosaccharide branch specificity24 or are strongly influenced by protein structure 25,26 and may not react with a protein-bound saccharide even though the individual saccharide is a very effective substrate. 6,24 Therefore, product characterization is required to determine the presence or absence of specific structures after the reaction. An advantage of using glycosyltransferases as surface probes is that they selectively detect accessible substrates that may be important in extracellular interactions with other cells or ligands (Fig. 2). Usually, a particular glycosyltransferase will reglycosylate only a small percentage of the penultimate sites uncovered by a promiscuous glycosidase.6,12,~5,1s For some experiments, it is essential to determine if this level of reglycosylation is due to enzyme specificity or crypticity of the substrate) s Beyer et al. 27 have shown that sialyltransferases can sialylate virtually all (> 90%) of their specific free oligosaccharide substrates in vitro. When the fl-galactoside a-2,6-sialyltransferase is used to sialylate intact cells that have been treated with sialidase, about 73% of the sialic acid that can be transferred to exhaustively digested pronase fragments in solution is replaced (Fig. 4). 18 24 D. A. Jozaisse, W. E. C. M. Schiphorst, D. H. van den Eijnden, J. A. van Kuik, H. van Halbeek, and J. F. G. Vliegenthart, J. Biol. Chem. 260, 714 (1985). 25 L. Lang, M. Reitman, J. Tang, R. M. Roberts, and S. Kornfeld, J. Biol. Chem. 259, 14663 (1984). 26 G. Savvidou, M. Klein, A. Grey, K. Dorrington and J. P. Carver, Biochemistry 23, 3736 (1984). 27 T. A. Beyer, J. I. Rearick, J. C. Paulson, J.-P. Prieels, J. E. Sadler, and R. L. Hill, J. Biol. Chem. 262, 14847 (1988).

ÂO-

A

B

x n )

) 20

10

,

I 30

ENZYME

I0

,

30

(0

(U)

60

TIME

90

1 :'0

(min)

D o

Y

5"

0-0

:

:

: 3

:

:

; 6

CMP-NANA

:

:

-

I

(nmole)

C E L L S X IE]6

FIo. 3. Kinetic parameters of the p-galactoside a-2,6-sialyltransferase-mediated exosialylation of living thymocytes. Labeling as a function of enzyme concentration (A), time (B), concentration of CMP[3H]NeuAc (C), and number of cells (D) was examined. For each parameter certain variables were held constant as needed: 108 thymocytes, 6.25 nmol sugar nucleotide, 31 units enzyme, or 30 min incubation time. Macromolecular eounts/min were recovered from the void volume o f a Sephadex G-50 column after solubilization of the cells. 60

•

~

e+pro

i 4° 30 20

10 0 0

5

I0

15

20

Transferase (mU) FIo. 4. Quantification of the sialyltransferase-accessible sites on the surfaces of melanoma cells. In one experiment, cells were either treated (+ sialidase) or not treated ( - sialidase) with V. cholerae sialidase prior to incubation with the fl-galactoside a-2,6-sialyltransferase and 6-fold the K m of the sugar nucleotide CMP[3H]NeuAc. In a separate experiment, intact cells were treated with sialidase, boiled, and exhaustively digested with pronase prior to incubation with the sialyltransferase and sugar nucleotide (+ sialidase + pro). It is clear that up to 73% of the sites accessible to the tmnsferase on total pronase glycopeptides (+ sialidase + pro) are accessible to the transferase when incubated with intact cells (+ sialidase).

88

ANALYTICAL METHODS

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Therefore, the exogenous resialylation of these cells appears to be efficient, suggesting that enzyme specificity and not steric hindrance is the major factor in determining the extent of reglycosylation. Membrane-Bound Glycosyltransferases

Many of the known glycosyltransferases are membrane-bound enzymes which must be purified using nonionic detergents. However, to use these transferases as probes of living cells, it is necessary to remove these detergents. The method described below is applicable to any type of membranebound glycosyltransferase, provided the appropriate sugar nucleotide and affinity matrix are available. 7 Our laboratory routinely uses this method to radiolabel a variety of live cells with UDP-N-acetyl[3H]glucosamine and N-acetylglucosaminyltransferase I, 2s or CMp[3H]sialic acid and p-galactoside ~-2,3-, 29 fl-galactoside a-2,6-, 3° or ~-N-acetylgalactosaminide a-2,6sialyltransferases.3~ In the example described below, sialyltransferases are used in combination with CMP-N-[3H]acetylneuraminic acid to probe for specific terminal or penultimate saccharide substrates on the surfaces of live cells. Reagents Sialyltransferases are purified using CDP-hexanolamine-Sepharose affinity chromatography. 32 Rat liver p-galactoside ~-2,6- and porcine submaxillary p-galactoside ~-2,3-sialyltransferases are now commercially available (Genzyme Corporation, Boehringer Mannheim). The rat liver enzyme may be purified according to Weinstein et al. 3° The two porcine enzymes are purified according to Sadler et a/. 29,31 The rat liver enzyme is stored at - 2 0 ° in 36 m M sodium cacodylate, pH 6.5, 0.4 M NaC1, 50% glycerol (v/v), and 0.1% Triton CF-54 (v/v). The porcine enzymes are also stored at - 2 0 ° but are in 7 m M sodium cacodylate, pH 6.5, 7.1 m M NaC1, 0.7% Triton X-100, and 50% glycerol. Under these storage conditions, the enzymes are stable for over 1 year. Enzyme activity is defined and assayed as described. 29-3~ Sialyltransferase buffer (STB): 25 m M HEPES-NaOH, pH6.5, 75 m M NaCI, 100 rnM glucose, and 10 mg/ml bovine serum albumin Fraction V, fatty acid free (Sigma). Column buffer: STB with 0.25% octyl-//-glucopyranoside (w/v). 2s C. L. Oppenheimer and R. L. Hill, J. Biol. Chem. 256, 799 (1981). 29 j. I. Rearick, J. E. Sadler, J. C. Paulson, and R. L. Hill, J. Biol. Chem. 254, 4434 (1979). 3oj. Weinstein, U. de Souza-e-Silva, and J. C. Paulson, J. Biol. Chem. 257, 13845 (1982). 31 j. E. Sadler, J. I. Rearick, J. C. Paulson, and R. L. Hill, J. Biol. Chem. 254, 4434 (1979). 32 j. C. Paulson, W. E. Beranek, and R. L. Hill, J. Biol. Chem. 252, 2356 (1977).

[8]

GLYCOSYLTRANSFERASE PROBES

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Elution buffer: STB with 0.25% octyl-fl-glucopyranoside (w/v), and 1.5 M NaC1. Elution marker buffer: STB with 0.25% (w/v) octyl-fl-glucopyranoside, 1.5 M NaC1, and 5 mg/ml Blue Dextran. Enzyme preparation column: A siliconized 0.3 × 30 cm glass column containing 25 cm of Sephadex G-50-80 (fine) overlaid with 1.3 cm of CDP-hexanolamine Sepharose (13/zmol/ml of bed). The column is equilibrated in column buffer. Cytidine 5'-monophospho[9-3H]sialic acid: Packaged and stored in 70% ethanol at - 2 0 ° (New England Nuclear and American Radiolabeled Chemicals Inc.; 15-35 Ci/mmol). Immediately before using, the radiolabel is first lyophilized to dryness and then resuspended in STB. Lysis buffer: 0.1 M Tris-HCl, pH 7.2, 0.15 M NaC1, 1.5 m M MgC12, 1% aprotinin (v/v), 1 m M phenylmethylsulfonyl fluoride, and 0.5% Nonidet P-40 (v/v).

Protocol Removal of Detergent from Sialyltransferase. In order to use the sialyltransferases on live cells, it is necessary to remove the Triton detergent. Using a modification of the method of Sadler et al., ~5the purified transferase (0.5 ml) is diluted 1:4 in column buffer and applied slowly to the preparation column (all procedures at 4 °). After the enzyme is loaded, the column is washed with 4 ml of STB to remove all of the detergent remaining in the column. The transferase is then eluted from the CDPhexanolamine-Sepharose by first adding 60/zl of the elution marker buffer, followed by 5 ml of the elution buffer. Fractions (250 gl) are collected, and those marked by the Blue Dextran contain the sialyltransferase (recovery >95%) free of detergent and salt. Enzyme activity activity is assayed to determine recovery as described, z9-3~ In this detergent-depleted state the enzymes lose no more than 20% of their activity over the course of 24 hr when stored on ice. Incubation of Cells with Sialyltransferase. Cells are prepared as a single cell suspension and washed several times in STB. After washing, the cells are resuspended in a small volume (50/zl) of labeling buffer. To this is added the desired amount of sialyltransferase [0-20 miliunits (mU) in a final volume of 100gl]. It is important to include a control without transferase since significant amounts of endogenous cell surface transferases appear to be present on many cell types) 8'33 In addition, a control 33 L. C. Lopez, E. M. Bayna, D. Litoff, N. L. Shaper, J. H. Shaper, and B. D. Shur, J. Cell Biol. 101, 1501 (1985).

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ANALYTICAL METHODS

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containing no cells should be routinely performed to determine the levels of enzyme autosialylation. The reaction is started by adding 1/tCi of CMp[aH]sialic acid and incubating the cells at 4 °. While the enzyme is more active at 37 °, the sugar nucleotide is more stable and cell membrane recycling is blocked at lower temperatures. To stop the sialylation reaction, the cell suspension is diluted with l ml of ice-cold STB, and the cells are sedimented at 350 g for l0 min. The supernatant should be saved so that degradation of the sugar nucleotide can be examined. 6 The cell pellet is suspended in 100 ~tl of STB and layered onto 1 ml of 30% sucrose in PBS. The cells are centrifuged at 1500 gfor 10 min, and the cell pellet is suspended in 1 ml of lysis buffer, vortexed, and incubated on ice with occasional vortexing. After 45 min, the cells are centrifuged at 1500 g for l0 rain. The pellet fraction is discarded. The supernatant fraction is brought to 2% SDS and boiled for 5 min. The remaining unincorporated radiolabel is separated from the 3H-sialylated macromolecules by chromatography on Sephadex G-50-150 (coarse; 1 × 30 cm) in 50 m M ammonium formate and 0.1% SDS (w/v). Radiolabeled macromoles elute in the void volume of the column and can be pooled for further analysis. Use of Exoglycosidases to Probe Penultimate Saccharide Structures. Sialidase pretreatment of cells, when combined with the sialyltransferase reaction, can be used to determine the sialylation state specific saccharides on the cell surface. By comparing the exosialylation of cellular proteins from siaiidase- and non-sialidase-treated cells one can determine which structures are exposed and which are masked by sialic acids in vivo. There are several different sialidases commercially available. The Vibrio cholerae sialidase, which will cleave most forms of sialic acids in 0t-2,3, a-2,6, and a-2,8 linkage, was used in these studies. This particular enzyme releases sialic acids rapidly and is equally effective at 4 ° or 37 °. Single cell suspensions are treated at 4 ° for 45 min with 200 mU/ml V. cholerae sialidase in 25 m M HEPES-KOH, pH 6.75, 150 m M NaC1, 0.1 m M MgC12, and 1.0 m M CaCl2. After treatment, the cells are sedimented at 350 g for 10 min. The supernatants are saved to measure the amount of sialic acid released by using an HPLC modification of the thiobarbituric acid assay. 34 The pelleted cells are washed an additional 4 times in phosphate-buffered saline (PBS) and once in STB prior to incubation with the sialyltransferase as has been described above. No residual cell-associated sialidase is detected after these washes, and cell viability is generally unaffected by the sialidase.

34 L. D. Pow¢ll and G. W. Hart, Anal. Biochem. 157, 179 (1986).

[8]

GLYCOSYLTRANSFERASE PROBES

91

Soluble Glycosyltransferases Several glycosyltranferases have been purified in a soluble form from bovine colostrum and other sources. These enzymes are well suited for use as probes of soluble molecules,9 glycoproteins transferred to nitrocellulose, 35 or cell surface saccharides. 17The example below describes the use of the bovine milk N-acetylglucosamine p-1,4-galactosyltransferase as a probe for terminal GlcNAc residues on the surfaces of live cells or soluble molecules.

Reagents Bovine milk GIcNAc fl-l,4-galactosyltransferase (lactose synthase) (Sigma): The enzyme is autogalactosylated prior to use by incubating 25 units in 1 ml of buffer containing 0.4 m M UDPgalactose, 5 m M MgC12, 1 m M mercaptoethanol, 1% aprotinin (v/v, Sigma) in 50 m M Tris-HC1, pH 7.3, at 37 ° for 30 min. The enzyme is then concentrated by precipitation with 85% saturated ammonium sulfate (twice) and assayed by the method of Trayer and Hill? 6 The enzyme is stable for at least 1 year when stored at a concentration of 20 to 30 units/ml in 25 m M HEPES-NaOH, pH 7.3, 5 m M MnC12, 50% glycerol at - 2 0 °. This enzyme is also available commercially from Boehringer Mannheim. Cell preparation buffer: 10 m M HEPES-NaOH, pH 7.3, 24 m M NaHCO3, 137 m M NaC1, and 5 m M sodium pyruvate. Labeling buffer: Cell preparation buffer with the following additions: 7% aprotinin (v/v), 10 mMgalactose, and 5 m M MnC12.5'-Adenosine monophosphate is added to this buffer from a 10× stock to achieve a final concentration of 2.5 mM. Uridine diphospho-D-[6-3H]galactose (Amersham, 5 - 2 0 Ci/mmol; UDP[1-3H]Gal and UDP[4,5-aH]Gal are available from New England Nuclear): Packaged and stored at - 2 0 ° in 50% ethanol. Before using, the radiolabel is first lyophilized to dryness and then suspended in water or labeling buffer. Lysis buffer: 0.1 M Tris-HC1, pH 7.2, 0.15 M NaCI, 1.5 m M MgC12, 1% approtinin (v/v), 1 m M phenylmethylsulfonyl fluoride, and 0.5% Nonidet P-40 (v/v). Gel filtration chromatography: Sephadex G-50-150 (1 × 30 cm) is equilibrated in 50 m M ammonium formate, 0.1% SDS, 0.02% sodium azide. The BioGel P-4 (Bio-Rad, 400 mesh) column (1 × 200 cm) is equilibrated at 50 ° in 0.2 M ammonium acetate. 35 R. E. Parchment, C. M. Ewing, and J. H. Shaper, Anal Biochem. 154, 460 (1986). 36 I. P. Trayer and R. L. Hill, J. Biol. Chem. 246, 6666 (1971).

92

ANALYTICAL METHODS

[8]

Protocol Cells are prepared as a single cell suspension and washed several times in cell preparation buffer. It is important not to use proteases such as trypsin-EDTA (or related techniques) to prepare single cell suspensions, as they will degrade cell surface proteins. After washing, cells are resuspended in a small volume (50/tl) of labeling buffer. To this, the desired amount of galactosyltransferase (0-200 m U to a final volume of 100/tl) is added. It is important to include a control lacking galactosyltransferase since endogenous cell surface transferases have been reported. 33 The reaction is started by addition of the UDp[aH]galactose, and the cells are incubated at 4 °. Typically we use 2 - 5 / i C i for 106 tumor cells or up to l0/zCi for l0 s primary lymphocyte cultures. Live cells can be galactosylated at 4 °, 10 °, 15 °, or 37 ° since the GlcNAc fl-l,4-galactosyltransferase activity is fairly temperature insensitive; low temperatures prevent membrane recycling. To stop the galactosylation reaction, cells are diluted with l ml of ice-cold cell preparation buffer and sedimented at 350 g for l0 min. The supernatant is saved to determine the stability of the sugar nucleotide during the reaction. 37 The cell pellet is suspended in 100/zl of labeling buffer and layered onto 1 ml of 30% sucrose. The cells are centrifuged at 1500g for 10 min. The cell pellet is suspended in 1 ml of lysis buffer, vortexed, and incubated on ice. After 45 min, the cells are centrifuged at 1500 g for 10 min. The pellet fraction is discarded. The supernatant fraction is brought to 2% SDS (w/v) and boiled for 5 min. The remaining unincorporated radiolabel is then separated from the 3H-galactosylated macromolecules by sizing chromatography on Sephadex G-50-150 (1 × 30 cm) in 50 m M ammonium formate and 0.1% SDS (w/v). Soluble, cell-derived substrates can also be analyzed using the bovine milk GIcNAc fl- 1,4-galactosyltransferase. Galactosylation of isolated proteins or organelles is accomplished using the same labeling buffer as listed above with added nonionic detergent, Nonidet P-40 (0.1-2.0%, v/v) if necessary. The reaction is stopped with final concentrations of 0.1% SDS and 50 m M EDTA. The 3H-galactosylated macromolecules are then separated from unincorporated label by gel filtration (Sephadex G-50) chromatography as above. Analysis of 3H-Galactosylated Species. Radiolabeled macromolecules are lyophilized to dryness, solubilized in a small volume of deionized water, divided into portions for each of the analyses below, and precipitated with 8 vol acetone at - 2 0 ° for 5 hr. A portion of the samples is solubilized in SDS sample buffer and fractionated by SDS-PAGE. as The 37 E. F. Porzig, Dev. Biol. 67, 114 (1978). 38 U. K. Laemmli, Nature (London) 227, 680 (1970).

[8]

GLYCOSYLTRANSFERASE PROBES

93

radiolabeled species can then be visualized by autofluorography after EnaHance (New England Nuclear) treatment. Macromolecules may also be treated with peptide N-glycosidase F (PNGase F), which will release N-linked oligosaccharides from the protein backbone. The acetone-precipitated material is suspended and digested with PNGase F as described. 39 After digestion, the released oligosacchatides are fractionated away from the resistant material by gel filtration chromatography (Sephadex G-50-150 in 0.1% SDS, 50 mM, ammonium formate). The PNGase F-resistant fraction (void volume) can be analyzed by SDS-PAGE while the labeled oligosaccharides (included volume) can be further analyzed by conventional techniques: gel filtration chromatography, 4° ion-exchange chromatography (amino-bonded HPLC, 4~ Mono Q FPLC, or Dionex HPLC42), or paper chromatography. 37 Another portion of the sample may be incubated in 0.1 N NaOH, 1 M NaBH4 at 37 ° for 24-48 hr 43 for the fl-elimination reaction. The released radiolabeled structures are fractionated by gel filtration and analyzed as described above. O-Linked N-acetylglucosamine will be released by this method and can be identified by BioGel P-4 sizing chromatography and high-voltage, paper electrophoresis in borate 44 or by any of several HPLC methods.~7 Specific Applications

O-Linked N-Acetylglucosamine. The use of the GIcNAc p-1,4-galactosyltransferase to study the distribution of terminal N-acetylglucosamine residues has revealed a new type of protein glycosylation. In early studies ~7 which were designed to look for cell surface N-acetylglucosamine residues, it was observed that permeabilized ceils incorporated over 10-fold more [3H]galactose than intact cells. The [3H]galactose transferred to intraceUular glycoproteins was shown to be added to a previously undesctibed saccharide structure, an O-Linked N-acetylglucosamine (O-GlcNAc). Subsequent studies on the distribution of this structure indicate that proteins beating O-GlcNAc are specifically localized in the cytosol and nucleus. 45-47 39 A. L. Tarentino, C. M. Gomez, and T. H. Plummer, Jr., Biochemistry 24, 4665 (1982). 40 K. Yamashita, T. Mizuochi, and A. Kobata, this series, Vol. 83, p. 105. 41 j. U. Baenziger and M. Natowicz, Anal, Biochem. 112, 357 (1981). 42 M. R. Hardy and R. R. Townsend, Proc. Natl. Acad. Sci. U.S.A. 85, 3289 (1988). 43 R. G. Spiro, this series, Vol. 28, p. 3. 44 E. J. Bourne, A. B. Foster, and P. M. J. Grant, Chem. Soc. (London) p. 4311 (1956). 45 G. D. Holt, C. M. Snow, A. Senior, R. S. Haltiwanger, L. Gerace, and G. W. Hart, J. Cell Biol. 104, 1157 (1987). 46 j. Hanover, C. Cohen, M. Witlingham, and M. Park, J. Biol. Chem. 262, 9887 (1987). 47 C. M. Snow, A. Senior, and L. Gerace, J. CellBiol. 104, 1143 (1987).

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ANALYTICAL METHODS

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Analysis of the [aH]galactosylated, O-linked GlcNAc-containing proteins of the nucleus shows that many of them are nuclear pore constituents. 45 Additional studies have since shown that this novel modification is present on cytosolic red blood cell proteins 9 and chromatin proteins. 4s Therefore, the use of the galactosyltransferase probe has allowed the identification of a novel structure (O-GIcNAc) that was not detected by standard metabolic labeling techniques.

Comparisons of the Sialylation State of Different Cell Surface Proteins. Cell surface structures have long been associated with metastatic potential in many different tumor systems.49 Using these different sialyltransferases together with CMP-N-[aH]acetylneuraminic acid, it has been possible to study the specific cell surface expression of several different oligosaccharide structures, is It was found that metastatic and nonmetastatic melanoma cells express no terminal exogenously sialylatable Gal(fll-4)GlcNAc, Gal(fll-3)GalNAc, or GalNAc-Ser residues on their cell surfaces. However, when the cells were first desialylated with sialidase and then probed with the sialyltransferases, it was apparent that there are large differences in the expression of these structures on the two cell types. The nonmetastatic cells express 4-fold more Gal(fll-3)GalNAc residues and 2.5-fold more Galfll-4GlcNAc residues than the metastatic cells. ~s By conventional cell surface labeling techniques such as ~25I-iodination or periodate-NaB3H4 labeling, 5° these cells appear essentially indistinguishable. Therefore, use of these transferases has provided information on cell surface structures that are undetectable by conventional, less sensitive methods. Addition of Radiolabeled Markers to Cell Surface Molecules. Glycosyltransferases can be used to mark specific oligosaccharide structures on the cell surface with a radiolabeled saccharide. Since this covalent marker is added at the cell surface, the internalization and subsequent recycling of these labeled oligosaccharides can be followed. Several examples of this approach have been reported. Thilo used galactosyltransferase and UDp[aH]galactose to study the internalization rates of cell surface glycoproteins in a variety of different cell types, from macrophages to Dictyostelium. 51,52 Duncan and Kornfeld have used a similar labeling system to study the internalization of the mannose 6-phosphate receptor and the subsequent reprocessing of its oligosaccharide units. ~3 Reichner et al. 12 have used the fl-galactoside a-2,6-sialyltransferase to measure the extent to 4s W. G. Kelly and G. W. Hart, Cell57, 243 (1989). 49 j. W. Dennis and S. Laferte, Cancer Metastasis Rev. 5, 185 (1987). 50 C. G. Gahmberg and L. C. Andersson, J. Biol. Chem. 252, 5888 (1977). s~ L. Thilo and G. Vogel, Proc. NatL Acad. Sci. U.S.A. 77, 1015 (1980). 52 L. Thilo Proc. Natl. Acad. Sci. U.S.A. 82, 1711 (1985).

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IMMUNOBLOTTING AND BINDING OF ACIDIC GLYCANS

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which desialylated cell surface glycoproteins are internalized and resialylated. All of these studies have made use of glycosyltransferases to study the metabolic pathways of specific cell surface glycoproteins of known biological function. Reconstruction of Biological Activities. The importance of carbohydrate structure in biological recognition5 has long been recognized, but experiments have mostly been of a destructive nature. MN blood group antigens are known to be carried by glycophorin, and removal of the sialic acids by neuraminidase treatment destroys their reactivity. Sadler et al.15 showed, by using sialyltransferases to restore functional agglutination, that the reactivity of erythrocytes is due to sialic acid either 2-3-1inked to Gal(fll-3)GalNAc or 2,6-1inked to GalNAc-Ser. Paulson and colleagues have used similar approaches to eliminate and then restore influenza virus binding sites on red blood cells) 6,5a Glycosyltransferases can be used for the reconstruction of specific saccharide moieties so that their biological reactivities can be more directly defined. s3 S. M. Carroll, H. H. Higa, a n d J. C. Paulson, J. Biol. Chem. 256, 8357 (1981).

[9] I m m u n o b l o t t i n g a n d I m r n u n o b i n d i n g o f A c i d i c Polysaccharides Separated by Gel Electrophoresis By GRADIMIR N. MISEVIC

Introduction The identification and isolation of polysaccharide antigens with monoclonal or polyclonal antibodies is an important method for structurefunction analyses of carbohydrates on the cell surface, in the extracellular matrix, and in blood plasma. Polyacrylamide gel electrophoresis of acidic polysaccharides 1-3 has been used to develop a specific immunological detection system for glycan antigens. Two simple and fast analytical methods are available for the immunoidentification of polysaccharides: (1) Acidic glycans separated on polyacrylamide gels are electrophoretically transferred to DEAE-cellulose sheets. Detection of the antigens is then achieved by the incubation of such blots with a specific first antibody followed by a peroxidase-coupled second antibody directed against the first one. The peroxidase reaction allows visualization of transferred polysaccharide antigen bands on the DEAE-cellulose sheets. (2) A mixture of purified polysaccharides is incubated with an antiglycan antibody. The METHODS IN ENZYMOLOGY, VOL. 179

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