A simple and rapid microplate assay for glycoprotein-processing glycosidases

ANALYTICALBIOCHEMISTRY

181,

log-112

(1989)

A Simple and Rapid Microplate Assay for Glycoprotein-Processing Glycosidases Mohinder Merrell

Received

S. Kang,

John H. Zwolshen,

Dow Research Institute,

February

Brenda

2110 East Galbraith

S. Harry,

Road, Cincinnati,

and Prasad

S. Sunkara

Ohio 45215

22,1989

A simple and convenient microplate assay for glycosidases involved in the glycoprotein-processing reactions is described. The assay is based on specific binding of high-mannose-type oligosaccharide substrates to concanavalin A-Sepharose, while monosaccharides liberated by enzymatic hydrolysis do not bind to concanavalin A-Sepharose. By the use of radiolabeled substrates ([3H]glucose for glucosidases and [3H]mannose for mannosidases), the radioactivity in the liberated monosaccharides can be determined as a measure of the enzymatic activity. This principle was employed earlier for developing assays for glycosidases previously reported (B. Saunier et al. (1982) J. Biol. Chem. 257, 1415514161; T. Szumilo and A. D. Elbein (1985) Anal. Biothem. 151, 32-40). These authors have reported the separation of substrate from the product by concanavalin A-Sepharose column chromatography. This procedure is handicapped by the fact that it cannot be used for a large number of samples and is time consuming. We have simplified this procedure and adapted it to the use of a microplate (96-well plate). This would help in processing a large number of samples in a short time. In this report we show that the assay is comparable to the column assay previously reported. It is linear with time and enzyme concentration and shows expected kinetics with castanospermine, a known inhibitor of a-glucosidase I. Q 1~8s Academic PWSS, IN.

The major pathway of asparagine-linked glycosylation in most eukaryotic cells involves the transfer of an oligosaccharide precursor (Glc,MangGlcNAc,)’ from dolichol pyrophosphate to the asparagine residues of acceptor proteins (1,2). The oligosaccharide undergoes a 1 Abbreviations used: Glc3MangGlcNA, (G3M9N2), glucose,mannosea-acetylglucosamine,; Endo-H, endo-@N-acetylglucosaminidase H; BHK, baby hamster kidney; PBS, phosphate-buffered saline; Con A, concanavalin A; fc, final concentration. 0003-2697/89 $3.00 Copyright 0 1989 by Academic Press, All rights of reproduction in any form

Inc. reserved.

number of processing reactions. The processing of oligosaccharide precursor begins with the removal of glucose residues. At least two distinct glucosidases have been described (3-6). Glucosidase I removes terminal o-1,2linked glucose unit, while glucosidase II removes the next a-1,3-linked glucose residues. After the removal of glucose residues in a variety of cells, this intermediate oligosaccharide has the structure MangGlcNAc, . This glycoprotein may proceed to become high mannose type or it may be processed to form the precursor to complextype oligosaccharides. The additional processing reactions involve the removal of four a-1,2-linked mannoses by mannosidase I (7). A GlcNAc residue is then added to give oligosaccharide GlcNAcMan5GlcNAcz. Addition to this GlcNAc is apparently the signal for mannosidase II which removes two branched mannoses. Further processing occurs by addition of other sugars (see Ref. (8) for review). Modifications of oligosaccharides by processing enzymes have been implicated in a number of reactions which include cell-cell recognition, hormone-receptor binding, interactions between microorganisms and their hosts, glycoprotein targeting, malignant transformation, viral infections, and phagocytosis. Understanding the mechanisms of these enzymes is of major interest in determining how these altered oligosaccharides are synthesized. These enzymes are difficult to assay because of limited availability of the substrates and difficulty in separation of neutral monosaccharides from the neutral substrates. Most of the assays involve tedious and timeconsuming gel filtration and chromatographic analysis. Thus these procedures are not suitable for analysis of large number of samples. We report here a simple and rapid procedure for the assay of these enzymes. The assay involves the use of Con A-Sepharose to bind the substrate in the reaction mixture and the counting of the radioactivity released as free monosaccharide. We have adapted this assay procedure to microtiter plates so that up to 96 different assays can be performed in a single 109

110

KANG

plate in about 2 h as compared to 2 days by column chromatography procedure. MATERIALS

Concanavalin A-Sepharose (10 mg/ml of packed gel) was purchased from Sigma Chemical Co. (St. Louis, MO). Castanospermine was isolated from Custunospermum au&rule seeds at the Merrell Dow Research Institute (9). [2-3H]Mannose (sp act 20-30 Ci/mmol and [l3H]galactose (sp act lo-25 Ci/mmol) were purchased from New England Nuclear (Boston, MA). Pronase was purchased from Calbiochem (San Diego, CA). Endo-PN-acetylglucosaminidase H was from Genzyme. Biogel (Polyacrylamide) was purchased from Bio-Rad Laboratories (Richmond, CA). Cell culture media and other media components were purchased from GIBCO Laboratories (Grand Island, NY). BHK cells were a gift from Dr. Ken Yamada (National Institutes of Health, Bethesda, MD). Pig kidney was purchased from PelFreez biologicals (Rogers, AK). Microtiter 96-well plates (Corning No. 25860) were purchased from Corning (New York). All other chemicals were obtained from commercial sources and were of the highest purity. EXPERIMENTAL

PROCEDURES

Preparation of [3H]GZucose-Labeled Substrate The [3H]glucose-labeled oligosaccharide substrate (G3M9N) for glucosidase I was prepared by metabolically labeling exponentially growing BHK cells with [3H]galactose in the presence of 200 pg/ml of castanospermine (10). BHK cells grown as a monolayer were treated with 200 pg/ml of castanospermine in DMEM (No. 4301600) supplemented with 10% heat-inactivated fetal calf serum, 2 mM L-glutamine, and 1X of PSN antibiotic mixture. After a 3-h incubation with castanospermine, [l3H]galactose (10 &i/ml of medium) was added to label the glycoproteins and cells were allowed to grow to confluency for an additional 48 h. At the end of the labeling period, the cells were washed with cold PBS and scraped with a rubber policeman. Cell pellet was heated for 10 min at 100°C and exhaustively treated with Pronase (usually 72 h) in 50 mM Tris, pH 7.5, containing 10 mM CaCl, and 1% Pronase under toluene atmosphere to obtain glycopeptides. The glycopeptides were separated on columns of Bio-Gel P-4. The glycopeptide peak produced by castanospermine treatment was pooled and treated with Endo-H to release the oligosaccharides. The oligosaccharides obtained by Endo-H hydrolysis were bound to a Con A column previously washed with buffer A (50 mM Tris, pH 7.5, containing 500 mM NaCl) and equilibrated with buffer B (5 mM sodium acetate buffer, pH 5.5, containing 2 mM each of CaCl,, MgCl,, and MnClJ. The oligosaccharides were then eluted with buffer B containing 100 mM a-methylmannoside. The

ET AL.

oligosaccharides eluted from the Con A column were further purified and characterized on a calibrated Bio-Gel P-4 column (1.5 X 200 cm, (-) 400 mesh). The purified oligosaccharides having the Glc3Man,GlcNAc structure were used as substrates in these studies. Purification

of a-Glucosidase I

cu-Glucosidase I was purified to homogeneity (unpublished data) from pig kidney by modification of the AffiGel affinity chromatography procedure described earlier (11). Assay Procedure for a-Glucosidase I (a) Column chromatography procedure. We essentially used the Con A-Sepharose procedure as previously described (10) with the modification that the reaction was stopped with acetic acid and no deproteinization of samples was found to be necessary before chromatographing of the samples on the columns. Briefly, Con ASepharose (0.25 ml of packed gel) in a Pasteur pipet was washed first with 5 ml of buffer A and then with 5 ml of buffer B. After incubation of the reaction mixture (as described below), the reaction was stopped by addition of acetic acid (20% final concentration). The reaction mixture was adjusted to 1 ml with water and was passed through the Con A-Sepharose column. The column was eluted with buffer B and three (1 ml each) fractions were collected. The liberated monosaccharides were obtained in the first two fractions. These fractions were counted to obtain the total monosaccharide radioactive counts. (b) Microtiter plate assay. We compared the above column procedure with the microtiter plate assay described below. Con A-Sepharose was washed first with buffer A, then with buffer B as described above, and resuspended in buffer B (gel:buffer, 1:l) before use. Different amounts of Con A-Sepharose in buffer B (0.05, 0.1, and 0.15 ml) were used to find the optimum amount of gel needed to bind more than 95% of the substrate. We then determined the amount of acetic acid needed to stop the reaction by adding glacial acetic acid to the reaction mixture to a final concentration (fc) of 10 to 44%. The enzymatic assays were performed in a 96-well microplate in a total volume of 80 ~1, which contained 10,000 cpm of [3H]G3MgN substrate, 100 mM potassium phosphate buffer, pH 6.8, and purified cu-glucosidase I. The reaction mixture was incubated at 37°C for the times indicated for each experiment and the reaction was stopped by adding 20 ~1 of glacial acetic acid. Half of this reaction mixture was chromatographed on concanavalin A-Sepharose columns as described above. To the remaining half of the mixture, 150 ~1of concanavalin A-Sepharose in buffer B was added and the microplate was spun at 500g for 5 min. An aliquot of supernatant was removed and counted. At the end of these experi-

MICROPLATE

Time

ASSAY

ments, Con A-Sepharose can be easily recovered from microplate wells, regenerated with a-methylmannoside, and reused (10). AND

DISCUSSION

Optimal Gel and Acetic Acid Concentrations A total of 0.1 ml of Con A-Sepharose gel (0.05 ml of packed gel) was enough to bind all the substrate (data not shown). We routinely used 0.075 ml of packed gel in 0.15 ml of buffer B in all microplate assays. Less than 10% final concentration of acetic acid in the assay mixture stops the reaction completely; we routinely used 20% (fc) of acetic acid to stop the reaction. Acetic acid concentrations of greater than 40% did not affect binding of the substrate to Con A-Sepharose (data not shown). When 3H-labeled glucose substrate is incubated with pig kidney cY-glucosidaseI, the terminal glucose is liberated. Figure 1 shows the release of free glucose when the reaction was allowed to proceed for the indicated times. As can be seen from the graph, the release of glucose by two concentrations of the enzyme was linear during the early portion of the incubation, with some leveling off toward the end of the reaction period. A maximum of 30% release of the total radioactivity in the substrate was observed with increased concentrations of the enzyme or with longer incubation periods (data not shown). During this incubation (Fig. l), more than half of the label in the substrate, which is susceptible to hydrolysis by glucosidase I, was released. It is clear that both column assay and microplate assay gave similar re-

111

GLYCOSIDASES

(min.)

FIG. 1. Effect of incubation time on the activity of purified pig kidney cy-glucosidase I. Two concentrations (a, A, 10 ~1 or 0, 0, 5 /.d) of the enzyme were incubated in 80 ~1 total volume containing 10,000 cpm of [3H]G3MgN for the indicated time in 96-well plates. The reaction was stopped with 20 ~1 of acetic acid. The radioactivity released as [“HIglucose was determined in half of the sample by column assay (0, a) and in other half by microplate assay (0, A), as described under Experimental Procedures.

RESULTS

FOR

Microliter

of Enzyme

FIG. 2. Effect of different enzyme concentrations on the activity of a-glucosidase 1. Increasing amounts of the enzyme (5 to 20 ~1) were incubated with 10,000 cpm of substrate, as in Fig. 1, for 20 min and [3H]glucose was determined in half of the sample by the column assay (0) and in the remaining half by the microplate assay (0).

leases of radioactivity at both enzyme concentrations and at all the time points studied, showing little difference between the two procedures used. In order to examine the effect of changes in the protein concentration, hydrolysis of radiolabeled substrate in the presence of increasing concentrations of enzyme was studied. As shown in Fig. 2, increasing amounts of radioactivity were released by the increasing concentrations of enzyme. Rate of hydrolysis was linear with up to a four-fold increase in enzyme concentration used. With both assays, similar amounts of radioactivity were released at each concentration of enzyme used.

r?

1

‘0 ‘;

1

Castanospermine

Cont.

(PM)

FIG. 3. Effect of castanospermine concentration on the release of [3H]glucose from [3H]G,M,N from pig kidney cy-glucosidase I. Ten microliters of the enzyme was incubated with the indicated amount of castanospermine in 80 pl of total reaction mixture for 60 min at 37°C. At the end of incubation, reaction was stopped with 20 ~1 of acetic acid and radioactivity was determined in half of the sample by column assay (0) and in the remaining half by microplate assay (0).

112

KANG

In order to compare these assays for examining the effect of inhibitors of glycoprotein processing, we used castanospermine, an inhibitor of a-glucosidase I (12). Figure 3 shows the effect of increasing amounts of castanospermine on the inhibition of release of free glucose from the substrate. It is apparent from the graph that in either of the assay procedures used, identical inhibition of a-glucosidase I activity was observed. The IC& for castanospermine against pig kidney glucosidase I was determined to be 4 X lo-’ M by both assays. This value is similar to the one reported (13) for rat intestinal glycohydrolase (sucrase) . These results demonstrate that the microplate assay and the column assay gave similar results for Ly-glucosidase I activity. The microplate assay is simpler and convenient and a large number of samples can be processed quickly. Since both procedures use Con A-Sepharose for affinity binding of the substrate, agents that do not interfere with binding of substrates to Con A-Sepharose in the column assay would not be expected to interfere in the microplate assay. A particular advantage of this assay is that deproteinization of the samples (10) is not required, thus reducing the number of manipulations needed. Currently, we are using this procedure to test the ability of different compounds as glycosidase inhibitors and to determine their inhibitory activities. It has been previously shown that the column procedure can be used for glucosidases I and II and for mannosidase I (10). It was suggested that the substrate and the oligosaccharide reaction product of mannosidase II should bind to concanavalin A (14). Thus, these can be easily separated from liberated monosaccharides. The microplate assay should thus be adaptable to study the ac-

ET

AL.

tivity of all these enzymes. Because of convenience, reliability, simplicity, and speed, the microplate assay can be used to measure the activity of glucosidases and mannosidases where large numbers of assays are needed, for example, monocolonal antibody production, in cloning experiments or for evaluation of new inhibitors. REFERENCES 1. Robbins,

P. W., Hubbard,

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247,555-562. 12. Pan, Y. T., Hori, H., Saul, R., Sanford, B. A., Molyneux, R. J., and Elbein, A. D. (1983) Biochemistry 22,3975-3984. 13. Rhinehart, B. L., Robinson, K. M., Panye, A. J., Wheatley, M. E., Fisher, J. L., Liu, P. S., and Cheng, W. (1987) Life Sci. 41,2325-

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