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A microplate assay for analysis of solution-phase glycosyltransferase reactions: Determination of kinetic constants
ANALYTICAL
BIOCHEMISTRY
199,286-292
(1991)
A Microplate Assay for Analysis of Solution-Phase Glycosyltransferase Reactions: Determination of Kinetic Constants Brenda Jo Mengeling,* Peter L. Smith,* Nancy L. Stults,? David F. Smith,? and Jacques U. Baenziger**’ *Department Biochemistry
Received
of Pathology, Washington University School of Medicine, St. Louis, Missouri 63110; and TDepartment and the Complex Carbohydrate Research Center, University of Georgia, Athens, Georgia 30602
September
10, 1991
We have developed a sensitive and simple method for assaying glycosyltransferase activities. This method makes use of solution-phase transferase reactions followed by capture to a microplate well coated with a substrate-specific monoclonal antibody. Sugar incorporation is quantitated by binding a saccharide-specific lectin and using bioluminescent aequorin for a reporter molecule. We demonstrate this method using the glycoprotein hormone-specific GalNAc-transferase and its acceptor substrate, agalacto-hCG. As little as 20 ng of agalacto-hCG with 32 nU of GalNAc-transferase gives a detectable signal with less than 10% of the acceptor sites substituted. In addition to this high sensitivity, by doing the transferase reactions in solution, we can assay up to 10 pg of agalacto-hCG. We show that this allows the determination of K,,, and V,, kinetic constants that compare well to those obtained with radiolabeled 0 1991 Academic Press, Inc. nucleotide sugars.
Traditionally, glycosyltransferase activities have been assayed by quantitating the incorporation of radiolabeled sugar into acceptor substrate. Such assays produce excellent kinetic determinations, but are usually work and time intensive, expensive in terms of radiolabel, and require at least microgram amounts of purified acceptor substrate and sufficient glycosyltransferase to obtain detectable levels of radiolabel incorporation. Recently, several assays for various glycosyltransferases utilizing substrates immobilized on microtiter plates and detection with saccharide-specific antibodies or lectins have been reported (l-3). These
’ To whom
of
correspondence
should
he addressed.
assays are expedient and eliminate the cost of radiolabel; however, because the transferase reactions are carried out on the solid phase of the microplate well, the opportunity to measure kinetic parameters is severely limited. We have developed a method for assaying glycosyltransferase activities that represents a hybrid of the radiolabeled assays and the solid-phase microplate assays. Basically, we carry out the transferase reaction in solution as in the radiolabeled assay and then capture an aliquot of reaction product to a microplate well. We determine the amount of sugar incorporation into the product captured onto the solid phase using a saccharide-specific lectin or antibody and bioluminescence with aequorin. In so doing, we are able to combine the versatility of the radiolabeled assay with the ease and practicality of a microplate assay. To demonstrate the capabilities of this approach, we present data using the bovine pituitary glycoprotein hormone-specific GalNAc2-transferase and the acceptor substrate, agalacto-hCG (human chorionic gonadotropin). We show that as little as 20 ng of agalacto-hCG and 32 nU of GalNAc-transferase are adequate to obtain a detectable level of incorporation with less than 10% substitution of the available acceptor sites, and that the wide range of substrate levels that can be assayed (1 ng to 10 pg of agalacto-hCG) makes it possible to determine kinetic constants (K, and VW) that are in good agreement with those determined using radiolabeled nucleotide sugars. This assay offers many unique advantages over previous glycosyltransferase assays. 2 Abbreviations used: hCG, human chorionic GalNAc, uridine diphosphate N-acetylgalactosamine; floribunda agglutinin; PBS, phosphate-buffered serum albumin.
gonadotropin; UDPWFA, W&aria saline; BSA, bovine
0003-2697/91
286 Copyright All rights
0 1991 by Academic
of reproduction
in any form
$3.00
Press, Inc. reserved.
GLYCOSYLTRANSFERASE
MATERIALS
AND
METHODS
Materials. An anti-hCG monoclonal antibody, clone 019, was purchased from Ventrex Laboratories, Inc. (Portland, ME). hCG was obtained from the National Hormone and Pituitary Program. Asialo- and agalactohCG were prepared by enzymatic removal of terminal sialic acid and Gal from hCG as described (4). GalNAchCG, bearing P-1,4-linked GalNAc on its Asn-linked oligosaccharides, was prepared from agalacto-hCG by enzymatic addition of GalNAc using bovine P-1,4-galactosyltransferase (Sigma, St. Louis, MO) as described (5). W&aria fioribunda agglutinin was purchased from E-Y Laboratories, Inc. (San Mateo, CA), aminohexanoylbiotin N-hydroxysuccinimide from Zymed Laboratories, Inc. (San Francisco, CA), streptavidin from Boehringer-Mannheim (Indianapolis, IN), and UDPGalNAc from Sigma. Biotinylation of WFA. Pure WFA in phosphate-buffered saline (PBS) at 1 mg/ml was dialyzed overnight at 4°C against 0.1 M NaHCO,. Aminohexanoylbiotin Nhydroxysuccinimide (5 mg/ml in dimethyl sulfoxide) was added to the WFA solution (12%, v/v). Following overnight incubation at 4”C, excess biotinylation reagent was removed by dialysis against PBS. The resulting WFA-biotin was diluted to 250 pglml and stored at -80°C. Preparation and biotinylation of aequorin. quorin was prepared, activated, and biotinylated viously described (3).
Apoaeas pre-
GalNAc-transferase reactions. Each reaction contained partially purified bovine pituitary glycoprotein hormone-specific GalNAc-transferase, agalacto-hCG, and UDP-GalNAc at the specified concentrations. In addition, each reaction (50 ~1 final volume) contained 25 mM Hepes, 0.1% Triton X-100, 10 mM adenosine triphosphate, 1 mg/ml BSA, 15% (v/v) glycerol, 10 mM MnCl,, 5.75 millitrypsin inhibitor units of aprotinin, 1 pg leupeptin, 1 pg antipain, 1 pg pepstatin, and 1 pg chymostatin at pH 7.5. Incubations were at 37°C and the reactions were stopped by placing the tubes in dry ice. Microplate assay for p-1,4-GalNAc addition to hCG. The GalNAc-transferase microplate assay, illustrated in Fig. 1, was performed as follows: (Step 1) GalNActransferase reactions were done as described above. (Step 2) Opaque white Microlite 2 plates (Dynatech) were coated with 1 pg/well anti-hCG monoclonal antibody in 100 pi/well binding buffer (15 mM Na,CO,, 35 mM NaHCO,, pH 9.6) for 3 h at 37°C wrapped in plastic wrap to prevent evaporation. All subsequent steps were performed at room temperature unless indicated otherwise. The wells were then washed 8X with PBS/0.05% Tween 20, blocked for 30 min with PBS/5% BSA, and finally washed 8X with PBS/0.05% Tween 20. The Gal-
MICROPLATE
287
ASSAY
NAc-transferase reactions were diluted to 0.2 ng hCG/ ~1 in PBS/l% BSA. Aliquots of each reaction containing l-8 ng of hCG were captured per well in a final volume of 100 ~1 PBS/l% BSA. hCG was allowed to bind for 45 min and then the wells were washed 8X with PBS/ 0.05% Tween 20. (Step 3) Each well was incubated with 500 ng of biotinylated WFA (75 nM) in 100 ~1 of PBS/l% BSA for 45 min, followed by washing 8X with PBS/ 0.05% Tween 20. (Step 4) Each well was incubated with 50 ng of streptavidin (8.3 nM) in 100 ~1 of PBS/l% BSA for 30 min. The wells were then washed 8X with AEQWash (PBS/5 mM EDTA/O.l% Tween 20). (Step 5) Each well was incubated with 50 ng biotinylated aequorin (23 nM) in 100 ~1 of Tris-buffered saline/2 mM EDTA/O.l% BSA for 30 min. The wells were again washed 8X with AEQ-Wash, and then 50 ~1 Tris-buffered saline/2 mM EDTA/O.l% BSA was added to each well. (Step 6) The assay was developed in an ML1000 microplate luminometer (Dynatech) by addition of 200 kl/well of 50 mM Ca(OAc),, 50 mM Tris-acetate, pH 8. The Peak Program (field test version 5.02) was used to quantitate light output. A window time of 2 s was chosen with before and after peak integral times set at 0.3 s. These settings were empirically shown to give the best signal-to-noise ratios. RESULTS
The microplate assay that we have developed for the characterization of glycosyltransferases such as the glycoprotein hormone-specific GalNAc-transferase is illustrated schematically in Fig. 1. The six steps of the assay are as follows: Step 1. Solution-phase enzyme reaction: The reaction contains substrate (agalacto-hCG, 1 ng to 10 fig), nucleotide sugar (UDP-GalNAc), and transferase (partially purified GalNAc-transferase) and is carried out in solution as described previously (4). Step 2. Capture product (hCG) onto microplate: Reaction aliquots containing product (l-8 ng hCG bearing terminal GalNAc) are incubated in wells coated with an anti-hCG monoclonal antibody which captures the hCG to the microplate. Step 3. Probe for sugar incorporation (GalNAc): The wells are incubated with WFA, which binds to terminal, P-linked GalNAc3 (5), the product of the GalNAc-transferase. The WFA is biotinylated in order to be able to quantitate the amount bound using a “streptavidin sandwich.” Step 4. Bind streptavidin to WFA-biotin: The wells are incubated with tetravalent streptavidin, which binds to the WFA-biotin.
3 S. Kumar
and J. U. Baenziger,
unpublished
observation.
288
MENGELING
1) Solution-phase
ET
enzyme reaction aliquot
agal-hCG
AL.
2) Capture
containing
hCG in microplate
l-8 ng hCG n
(lng to lolg)
terminal GalNAc
p1,4
GalNAc-transferase anti-hCG coated
3) Incubate with biotinylated-Wistaria
mAb well
4) Incubate with streptavidin
Incubate with biotinylated-aequorin in presence of EDTA
Ca2+ Develop assay ’ by Ca*‘addition. Light emitted; measured in microplate luminometer.
FIG.
1.
Scheme
for the GalNAc-transferase
microplate
assay.
Details
Step 5. Bind aequorin-biotin to streptavidin: The streptavidin sandwich is completed by incubating the wells with biotinylated aequorin, which serves as the indicator molecule for quantifying bound WFA. Step 6. Calcium activation of aequorin: Calcium acetate is added to the wells to induce aequorin oxidation of bound coelenterate luciferin. This produces a flash of light over -1 s that is measured in an ML1000 microplate luminometer. Prior to examining the glycoprotein hormone-specific GalNAc-transferase using this assay, it was necessary to optimize each of the steps following the solutionphase reaction (Step 1) and determine that none of these steps would be limiting. A number of monoclonal antibodies specific for hCG were screened to find one that displayed both good affinity and high capture capacity. We determined the amount of monoclonal antibody required to saturate the microplate and used that amount for all subsequent studies. GalNAc-hCG was prepared using UDP-GalNAc and bovine P-1,4-galactosyltransferase as described (6), and this was used to establish the maximum amount of hCG that could be captured (Fig. 2). The amount of hCG captured increased linearly with input up to a maximum of 8 ng. Therefore, in all subsequent assays ~8 ng of hCG was captured per well. We then established the amounts of biotinylated WFA, strepavidin, and biotinylated aequorin required
of the individual
steps
are described
under
Materials
and Methods.
to produce the maximal response with 8 ng of heavily substituted GalNAc-hCG. Finally, we determined at what level of light units the photomultiplier no longer remained linearly responsive. All of the assays described below were carried out under conditions where the response of the assay was well within its linear range. These conditions must be established empirically
100 1
a; ?60 .E ; .? J 80
4020-
OJ 0
2
4 GalNAc-hCG
6
8 (ng input)
10
12
FIG. 2. Linear range of hCG capture using the microplate assay. hCG, bearing Asn-linked oligosaccharides terminating with GalNAc in place of Gal (GalNAc-hCG), was prepared using o-1,4-galactosyltransferase and UDP-GalNAc as described (5). Increasing amounts of GalNAc-hCG were added to wells coated with anti-hCG mAb in order to establish the linear range of capture and detection at maximal levels of GalNAc incorporation.
GLYCOSYLTRANSFERASE
MICROPLATE
289
ASSAY
800
2 0/ 0
4
8
GalNAc-Tf Time
(min)
FIG. 3. Time course for transfer of GalNAc to agalacto-hCG. Agalacto-hCG, UDP-GalNAc, and partially purified GalNAc-transferase were incubated at 37°C for differing periods of time. For each time point a 6-ng aliquot of hCG was captured and GalNAc incorporation determined. GalNAc-transferase reactions (50 al reaction volume) contained 100 ng agalacto-hCG, 1500 pM UDP-GalNAc, and 5 ~1 (0) or 10 al (0) partially purified GalNAc-transferase.
for each different glycosyltransferase or detection method. Our microplate assay for GalNAc-transferase is linear with respect to reaction time (Fig. 3), transferase concentration (Fig. 4), and substrate concentration (Fig. 5). Previously, utilizing UDP-[3H]GalNAc, we established reaction conditions to characterize the kinetic parameters for the transfer of GalNAc to agalacto-hCG oligosaccharide acceptors by the glycoprotein hormonespecific GalNAc-transferase (4,7). We have examined these same parameters using the microplate assay. As shown in Fig. 3, transfer of GalNAc to agalacto-hCG increased linearly with time for at least 60 min. The loss of linear incorporation after 60 min at the higher transferase concentration reflects consumption of the acceptor sites under these conditions. In addition, GalNAc incorporation increased linearly with increasing amounts of GalNAc-transferase (Fig. 4). The transfer of GalNAc also increased linearly with increasing amounts of agalacto-hCG at concentrations below 0.5 pg/50 ~1 assay volume (Fig. 5). Utilizing biotinylated aequorin and the ML1000 luminometer, we were able to obtain a linear response over a range of lo5 light units. This was not possible using peroxidasebased color reactions. Having established the microplate assay conditions that generate linear light output with respect to time and transferase and substrate concentrations, we next determined kinetic constants for GalNAc incorporation
12
16
20
(pllrxn)
FIG. 4. Incorporation of GalNAc is proportionate to GalNActransferase concentration. Each .50+1 enzyme reaction contained 2 pg agalcto-hCG, 1500 pM UDP-GalNAc, and the indicated amount of partially purified GalNAc-transferase. Following a 30-min incubation at 37”C, 6-ng aliquots of hCG were tested for GalNAc incorporation. Assays were performed in triplicate and averaged.
into agalacto-hCG at multiple UDP-GalNAc concentrations. Previously, we demonstrated that the K,,, for the hormone acceptor varies with respect to UDP-GalNAc concentration (4,7), and these results were borne out with the microplate assay. Figure 6A depicts agalacto-hCG saturation curves obtained at two different
500
r
0
100
200 agal-hCG
300 (nglrxn)
400
500
FIG. 5. Incorporation of GalNAc is linear with respect to agalactohCG concentration at concentrations below the K,. Each enzyme reaction contained 0.2 pl of partially purified GalNAc-transferase, 1500 PM UDP-GalNAc, and the indicated amounts of agalacto-hCG in a 50-al reaction volume. Following incubations for 5 min at 37”C, 2-ng aliquots of hormone were tested for GalNAc incorporation. The amount of signal obtained in all cases represented less than 15% of the maximum for 2 ng of agalacto-hCG. The signal-to-noise ratio ranged from 4.5 to 7.1.
290
MENGELING
25-
ET
AL.
B
67 b
0.10
0.08
i
i
4 hCG
1
6
J
1
-8
-4
r
t
12
8
(I.cglrxn)
FIG. 6. Determination of kinetic constants for the GalNAc-transferase. (A) Saturation curve for GalNAc incorporation into agalacto-hCG. Each enzyme reaction contained the specified amount of agalacto-hCG, 20 ~1 partially purified GalNAc-Tf, and either 334 pM (0) or 1000 pM (0) UDP-GalNAc in a 50-~1 reaction volume. Incubations were at 37°C for 30 min. The amount of GalNAc incorporated per 6 ng of hCG was determined in triplicate and standard deviations are indicated. (B) Double-reciprocal plot of the data shown in (A): 334 pM UDP-GalNAc (0) and 1000 pM UDP-GalNAc (0). Lines were fit by least-squares regression analysis.
concentrations of UDP-GalNAc (334 and 1000 PM UDP-GalNAc), and Fig. 6B shows the resulting doublereciprocal plots. In all, we generated saturation curves at five different UDP-GalNAc concentrations (200,250, 334, 500, and 1000 PM). Figure 7A illustrates a secondary plot (8) of the l/V,,,,, values, generated from l/u versus l/[UDP-GalNAc] plots (not shown), versus 11 [hCG], from which a Km for hCG of 1.8 FM at saturating concentrations of UDP-GalNAc was determined. As shown in Fig. 7B, a secondary plot of the reciprocal of the slopes (l/n) from the l/[UDP-GalNAc] plots versus l/[hCG] gives a K,,,for agalcto-hCG of 0.98 j.&M at the extinction of UDP-GalNAc. When the reverse set of secondary plots is made to determine the K,,, values of UDP-GalNAc at saturation and extinction of agalactohCG, Km values of 30 and 200 PM, respectively, were obtained (not shown). These values are in good agreement with the Km values that we published for crude preparations of GalNAc-transferase using our radiolabeled assay (4,7), demonstrating the capabilities of the microplate assay for determining kinetic parameters. DISCUSSION
We previously identified and characterized a glycoprotein hormone-specific GalNAc-transferase using an assay in which [3H]GalNAc is incorporated into oligosaccharide acceptors on purified agalacto-hCG (4). Like
most glycosyltransferase assays relying on radiolabel incorporation, the GalNAc-transferase assay requires relatively large amounts of pure substrate and transferase. In addition, only a small number of assays can be performed at one time. These limitations were significant when GalNAc-transferase purification or characterization of hormone constructs differing in peptide sequence and expressed in cultured cells is considered. We therefore undertook the development of an assay for the glycoprotein hormone-specific GalNAc-transferase that would, without compromising specificity, provide a marked increase in both sensitivity and efficiency in the number of assays that could be simultaneously performed. The microplate assay that we have developed fulfills these requirements. Utilizing incorporation of radiolabel, we require 0.5 pg of purified agalacto-hCG, 1 &i of UDP-[3H]GalNAc, and 5.5 /IU of glycoprotein hormone-specific GalNAc-transferase for each data point; furthermore, only 20 analyses can be performed over a 48 to 72-h interval. The current microplate method requires no radiolabel and as little as 1 ng of agalacto-hCG and 32 nU of GalNAc-transferase. In addition, the ease of using a microtiter plate with detection in a luminometer that is able to read 96-well plates makes it possible to carry out large numbers of assays in a matter of hours. The large increase in sensitivity of the microplate assay over the radiolabeled assay reflects primarily the
GLYCOSYLTRANSFERASE A
MICROPLATE
291
ASSAY
0.08 6 6-
0.06 -
I
/
-4 lYLhCG, {M-i x 105,
'2
-'2
FIG.
43
7. Secondary plots to determine the K, for hCG at saturation and extinction ) from the plot of l/u versus l/[UDP-GalNAc] at five hCG concentrations: (l~vmaP, K,,, (1.8 PM) for hCG at saturating concentrations of UDP-GalNAc. (B) Secondary plot l/u vs l/fUDP-GalNAcl at hCG concentrations of 1.9.2.5, 3.8, and 7.5 pg. The x-axis UDP-GaiNAc. .
use of aequorin. Aequorin is a bioluminescent molecule that has been cloned from the hydromedusan Aequorea victoria and expressed at high levels in Escherichia coli (9). It consists of the polypeptide (apoaequorin) and mole per mole bound coelenterate luciferin and 0,. Upon addition of Ca2+, aequorin catalyzes the oxidation of luciferin producing a brief (-1-s) flash of blue light that can be measured in a microplate luminometer (MLlOOO, Dynatech). The sensitivity was further increased by using the “Peak Program,” which integrates the number of light units emitted during a time window encompassing the peak of the light flash. By adjusting the “before” and “after” peak time windows, the background can be significantly reduced. As a result, a linear response could be obtained over a concentration range of 105. In contrast, use of horse radish peroxidase as a reporter molecule in a standard color reaction was not linear over a range greater than lo2 and rarely provided signal-to-noise ratios >3:1. The specificity of the assay can be obtained at one or both of two steps. The capture agent, if highly selective, as with a monoclonal antibody, results in capture only of substrate even from a crude reaction. With such a capture reagent, prior purification of the substrate is not necessary. The saccharide-specific agent can also be highly specific, such as WFA, which binds to terminal, P-linked GalNAc. The amount of GalNAc actually transferred can be quantitated in absolute terms by constructing a standard curve using GalNAc-hCG bearing known amounts of terminal GalNAc.
-:/,,,,,
(L.1 x 1oq4
8
l2
of UDP-GalNAc. (A) Secondary plot of they intercepts 1.5,1.9,2.5,3.8, and 7.5 pg. The x-axis intercept gives the of the slopes obtained from the double-reciprocal plot of intercept gives the K,,, for hCG (0.98 FM) at extinction of
The sensitivity of the bioluminescent detection is essential for obtaining kinetic constants since it is necessary to detect GalNAc incorporation at levels that represent
REFERENCES 1. Stults, C. L. M., Wilbur, Biochem. 174, 151-156.
B. J., and Macher,
B. A. (1988)
Anal.
292
MENGELING
2. Crawley, S. C., Hindsgaul, O., Alton, G., Pierce, M. M. (1990) Anal. Biochem. 185, 112-117. 3. Zatta, P. F., Nyame, K., Cormier, M. J., Mattox, P. A., Smith, D. F., and Cummings, R. D. (1991) 194,185-191. 4. Smith, P. L., and Baenziger, J. U. (1988) Science 5. Nyame, K., Smith, D. F., Damian, (1989) J. Bid. Chem. 264,3235-3243.
M.,
and Palcic,
S. A., Prieto, Anal. Biochem. 242,930-933.
R. T., and Cummings,
R. D.
ET
AL.
6. Palcic, M. M., and Hindsgaul, 0. (1991) Glycobiology, in press. 7. Smith, P. L., and Baenziger, J. U. (1990) Proc. Natl. Acad. Sci. USA 67,7275-7279. 8. Segel, I. H. (1975) Enzyme Kinetics: Behavior and Analysis of Rapid Equilibrium and Steady-State Enzyme Systems, Wiley, New York. 9. Prasher, Biophys.
D., McCann, Res. Commun.
R. O., and Cormier, 126, 1259-1268.
M. J. (1985)
Biochem.
BIOCHEMISTRY
199,286-292
(1991)
A Microplate Assay for Analysis of Solution-Phase Glycosyltransferase Reactions: Determination of Kinetic Constants Brenda Jo Mengeling,* Peter L. Smith,* Nancy L. Stults,? David F. Smith,? and Jacques U. Baenziger**’ *Department Biochemistry
Received
of Pathology, Washington University School of Medicine, St. Louis, Missouri 63110; and TDepartment and the Complex Carbohydrate Research Center, University of Georgia, Athens, Georgia 30602
September
10, 1991
We have developed a sensitive and simple method for assaying glycosyltransferase activities. This method makes use of solution-phase transferase reactions followed by capture to a microplate well coated with a substrate-specific monoclonal antibody. Sugar incorporation is quantitated by binding a saccharide-specific lectin and using bioluminescent aequorin for a reporter molecule. We demonstrate this method using the glycoprotein hormone-specific GalNAc-transferase and its acceptor substrate, agalacto-hCG. As little as 20 ng of agalacto-hCG with 32 nU of GalNAc-transferase gives a detectable signal with less than 10% of the acceptor sites substituted. In addition to this high sensitivity, by doing the transferase reactions in solution, we can assay up to 10 pg of agalacto-hCG. We show that this allows the determination of K,,, and V,, kinetic constants that compare well to those obtained with radiolabeled 0 1991 Academic Press, Inc. nucleotide sugars.
Traditionally, glycosyltransferase activities have been assayed by quantitating the incorporation of radiolabeled sugar into acceptor substrate. Such assays produce excellent kinetic determinations, but are usually work and time intensive, expensive in terms of radiolabel, and require at least microgram amounts of purified acceptor substrate and sufficient glycosyltransferase to obtain detectable levels of radiolabel incorporation. Recently, several assays for various glycosyltransferases utilizing substrates immobilized on microtiter plates and detection with saccharide-specific antibodies or lectins have been reported (l-3). These
’ To whom
of
correspondence
should
he addressed.
assays are expedient and eliminate the cost of radiolabel; however, because the transferase reactions are carried out on the solid phase of the microplate well, the opportunity to measure kinetic parameters is severely limited. We have developed a method for assaying glycosyltransferase activities that represents a hybrid of the radiolabeled assays and the solid-phase microplate assays. Basically, we carry out the transferase reaction in solution as in the radiolabeled assay and then capture an aliquot of reaction product to a microplate well. We determine the amount of sugar incorporation into the product captured onto the solid phase using a saccharide-specific lectin or antibody and bioluminescence with aequorin. In so doing, we are able to combine the versatility of the radiolabeled assay with the ease and practicality of a microplate assay. To demonstrate the capabilities of this approach, we present data using the bovine pituitary glycoprotein hormone-specific GalNAc2-transferase and the acceptor substrate, agalacto-hCG (human chorionic gonadotropin). We show that as little as 20 ng of agalacto-hCG and 32 nU of GalNAc-transferase are adequate to obtain a detectable level of incorporation with less than 10% substitution of the available acceptor sites, and that the wide range of substrate levels that can be assayed (1 ng to 10 pg of agalacto-hCG) makes it possible to determine kinetic constants (K, and VW) that are in good agreement with those determined using radiolabeled nucleotide sugars. This assay offers many unique advantages over previous glycosyltransferase assays. 2 Abbreviations used: hCG, human chorionic GalNAc, uridine diphosphate N-acetylgalactosamine; floribunda agglutinin; PBS, phosphate-buffered serum albumin.
gonadotropin; UDPWFA, W&aria saline; BSA, bovine
0003-2697/91
286 Copyright All rights
0 1991 by Academic
of reproduction
in any form
$3.00
Press, Inc. reserved.
GLYCOSYLTRANSFERASE
MATERIALS
AND
METHODS
Materials. An anti-hCG monoclonal antibody, clone 019, was purchased from Ventrex Laboratories, Inc. (Portland, ME). hCG was obtained from the National Hormone and Pituitary Program. Asialo- and agalactohCG were prepared by enzymatic removal of terminal sialic acid and Gal from hCG as described (4). GalNAchCG, bearing P-1,4-linked GalNAc on its Asn-linked oligosaccharides, was prepared from agalacto-hCG by enzymatic addition of GalNAc using bovine P-1,4-galactosyltransferase (Sigma, St. Louis, MO) as described (5). W&aria fioribunda agglutinin was purchased from E-Y Laboratories, Inc. (San Mateo, CA), aminohexanoylbiotin N-hydroxysuccinimide from Zymed Laboratories, Inc. (San Francisco, CA), streptavidin from Boehringer-Mannheim (Indianapolis, IN), and UDPGalNAc from Sigma. Biotinylation of WFA. Pure WFA in phosphate-buffered saline (PBS) at 1 mg/ml was dialyzed overnight at 4°C against 0.1 M NaHCO,. Aminohexanoylbiotin Nhydroxysuccinimide (5 mg/ml in dimethyl sulfoxide) was added to the WFA solution (12%, v/v). Following overnight incubation at 4”C, excess biotinylation reagent was removed by dialysis against PBS. The resulting WFA-biotin was diluted to 250 pglml and stored at -80°C. Preparation and biotinylation of aequorin. quorin was prepared, activated, and biotinylated viously described (3).
Apoaeas pre-
GalNAc-transferase reactions. Each reaction contained partially purified bovine pituitary glycoprotein hormone-specific GalNAc-transferase, agalacto-hCG, and UDP-GalNAc at the specified concentrations. In addition, each reaction (50 ~1 final volume) contained 25 mM Hepes, 0.1% Triton X-100, 10 mM adenosine triphosphate, 1 mg/ml BSA, 15% (v/v) glycerol, 10 mM MnCl,, 5.75 millitrypsin inhibitor units of aprotinin, 1 pg leupeptin, 1 pg antipain, 1 pg pepstatin, and 1 pg chymostatin at pH 7.5. Incubations were at 37°C and the reactions were stopped by placing the tubes in dry ice. Microplate assay for p-1,4-GalNAc addition to hCG. The GalNAc-transferase microplate assay, illustrated in Fig. 1, was performed as follows: (Step 1) GalNActransferase reactions were done as described above. (Step 2) Opaque white Microlite 2 plates (Dynatech) were coated with 1 pg/well anti-hCG monoclonal antibody in 100 pi/well binding buffer (15 mM Na,CO,, 35 mM NaHCO,, pH 9.6) for 3 h at 37°C wrapped in plastic wrap to prevent evaporation. All subsequent steps were performed at room temperature unless indicated otherwise. The wells were then washed 8X with PBS/0.05% Tween 20, blocked for 30 min with PBS/5% BSA, and finally washed 8X with PBS/0.05% Tween 20. The Gal-
MICROPLATE
287
ASSAY
NAc-transferase reactions were diluted to 0.2 ng hCG/ ~1 in PBS/l% BSA. Aliquots of each reaction containing l-8 ng of hCG were captured per well in a final volume of 100 ~1 PBS/l% BSA. hCG was allowed to bind for 45 min and then the wells were washed 8X with PBS/ 0.05% Tween 20. (Step 3) Each well was incubated with 500 ng of biotinylated WFA (75 nM) in 100 ~1 of PBS/l% BSA for 45 min, followed by washing 8X with PBS/ 0.05% Tween 20. (Step 4) Each well was incubated with 50 ng of streptavidin (8.3 nM) in 100 ~1 of PBS/l% BSA for 30 min. The wells were then washed 8X with AEQWash (PBS/5 mM EDTA/O.l% Tween 20). (Step 5) Each well was incubated with 50 ng biotinylated aequorin (23 nM) in 100 ~1 of Tris-buffered saline/2 mM EDTA/O.l% BSA for 30 min. The wells were again washed 8X with AEQ-Wash, and then 50 ~1 Tris-buffered saline/2 mM EDTA/O.l% BSA was added to each well. (Step 6) The assay was developed in an ML1000 microplate luminometer (Dynatech) by addition of 200 kl/well of 50 mM Ca(OAc),, 50 mM Tris-acetate, pH 8. The Peak Program (field test version 5.02) was used to quantitate light output. A window time of 2 s was chosen with before and after peak integral times set at 0.3 s. These settings were empirically shown to give the best signal-to-noise ratios. RESULTS
The microplate assay that we have developed for the characterization of glycosyltransferases such as the glycoprotein hormone-specific GalNAc-transferase is illustrated schematically in Fig. 1. The six steps of the assay are as follows: Step 1. Solution-phase enzyme reaction: The reaction contains substrate (agalacto-hCG, 1 ng to 10 fig), nucleotide sugar (UDP-GalNAc), and transferase (partially purified GalNAc-transferase) and is carried out in solution as described previously (4). Step 2. Capture product (hCG) onto microplate: Reaction aliquots containing product (l-8 ng hCG bearing terminal GalNAc) are incubated in wells coated with an anti-hCG monoclonal antibody which captures the hCG to the microplate. Step 3. Probe for sugar incorporation (GalNAc): The wells are incubated with WFA, which binds to terminal, P-linked GalNAc3 (5), the product of the GalNAc-transferase. The WFA is biotinylated in order to be able to quantitate the amount bound using a “streptavidin sandwich.” Step 4. Bind streptavidin to WFA-biotin: The wells are incubated with tetravalent streptavidin, which binds to the WFA-biotin.
3 S. Kumar
and J. U. Baenziger,
unpublished
observation.
288
MENGELING
1) Solution-phase
ET
enzyme reaction aliquot
agal-hCG
AL.
2) Capture
containing
hCG in microplate
l-8 ng hCG n
(lng to lolg)
terminal GalNAc
p1,4
GalNAc-transferase anti-hCG coated
3) Incubate with biotinylated-Wistaria
mAb well
4) Incubate with streptavidin
Incubate with biotinylated-aequorin in presence of EDTA
Ca2+ Develop assay ’ by Ca*‘addition. Light emitted; measured in microplate luminometer.
FIG.
1.
Scheme
for the GalNAc-transferase
microplate
assay.
Details
Step 5. Bind aequorin-biotin to streptavidin: The streptavidin sandwich is completed by incubating the wells with biotinylated aequorin, which serves as the indicator molecule for quantifying bound WFA. Step 6. Calcium activation of aequorin: Calcium acetate is added to the wells to induce aequorin oxidation of bound coelenterate luciferin. This produces a flash of light over -1 s that is measured in an ML1000 microplate luminometer. Prior to examining the glycoprotein hormone-specific GalNAc-transferase using this assay, it was necessary to optimize each of the steps following the solutionphase reaction (Step 1) and determine that none of these steps would be limiting. A number of monoclonal antibodies specific for hCG were screened to find one that displayed both good affinity and high capture capacity. We determined the amount of monoclonal antibody required to saturate the microplate and used that amount for all subsequent studies. GalNAc-hCG was prepared using UDP-GalNAc and bovine P-1,4-galactosyltransferase as described (6), and this was used to establish the maximum amount of hCG that could be captured (Fig. 2). The amount of hCG captured increased linearly with input up to a maximum of 8 ng. Therefore, in all subsequent assays ~8 ng of hCG was captured per well. We then established the amounts of biotinylated WFA, strepavidin, and biotinylated aequorin required
of the individual
steps
are described
under
Materials
and Methods.
to produce the maximal response with 8 ng of heavily substituted GalNAc-hCG. Finally, we determined at what level of light units the photomultiplier no longer remained linearly responsive. All of the assays described below were carried out under conditions where the response of the assay was well within its linear range. These conditions must be established empirically
100 1
a; ?60 .E ; .? J 80
4020-
OJ 0
2
4 GalNAc-hCG
6
8 (ng input)
10
12
FIG. 2. Linear range of hCG capture using the microplate assay. hCG, bearing Asn-linked oligosaccharides terminating with GalNAc in place of Gal (GalNAc-hCG), was prepared using o-1,4-galactosyltransferase and UDP-GalNAc as described (5). Increasing amounts of GalNAc-hCG were added to wells coated with anti-hCG mAb in order to establish the linear range of capture and detection at maximal levels of GalNAc incorporation.
GLYCOSYLTRANSFERASE
MICROPLATE
289
ASSAY
800
2 0/ 0
4
8
GalNAc-Tf Time
(min)
FIG. 3. Time course for transfer of GalNAc to agalacto-hCG. Agalacto-hCG, UDP-GalNAc, and partially purified GalNAc-transferase were incubated at 37°C for differing periods of time. For each time point a 6-ng aliquot of hCG was captured and GalNAc incorporation determined. GalNAc-transferase reactions (50 al reaction volume) contained 100 ng agalacto-hCG, 1500 pM UDP-GalNAc, and 5 ~1 (0) or 10 al (0) partially purified GalNAc-transferase.
for each different glycosyltransferase or detection method. Our microplate assay for GalNAc-transferase is linear with respect to reaction time (Fig. 3), transferase concentration (Fig. 4), and substrate concentration (Fig. 5). Previously, utilizing UDP-[3H]GalNAc, we established reaction conditions to characterize the kinetic parameters for the transfer of GalNAc to agalacto-hCG oligosaccharide acceptors by the glycoprotein hormonespecific GalNAc-transferase (4,7). We have examined these same parameters using the microplate assay. As shown in Fig. 3, transfer of GalNAc to agalacto-hCG increased linearly with time for at least 60 min. The loss of linear incorporation after 60 min at the higher transferase concentration reflects consumption of the acceptor sites under these conditions. In addition, GalNAc incorporation increased linearly with increasing amounts of GalNAc-transferase (Fig. 4). The transfer of GalNAc also increased linearly with increasing amounts of agalacto-hCG at concentrations below 0.5 pg/50 ~1 assay volume (Fig. 5). Utilizing biotinylated aequorin and the ML1000 luminometer, we were able to obtain a linear response over a range of lo5 light units. This was not possible using peroxidasebased color reactions. Having established the microplate assay conditions that generate linear light output with respect to time and transferase and substrate concentrations, we next determined kinetic constants for GalNAc incorporation
12
16
20
(pllrxn)
FIG. 4. Incorporation of GalNAc is proportionate to GalNActransferase concentration. Each .50+1 enzyme reaction contained 2 pg agalcto-hCG, 1500 pM UDP-GalNAc, and the indicated amount of partially purified GalNAc-transferase. Following a 30-min incubation at 37”C, 6-ng aliquots of hCG were tested for GalNAc incorporation. Assays were performed in triplicate and averaged.
into agalacto-hCG at multiple UDP-GalNAc concentrations. Previously, we demonstrated that the K,,, for the hormone acceptor varies with respect to UDP-GalNAc concentration (4,7), and these results were borne out with the microplate assay. Figure 6A depicts agalacto-hCG saturation curves obtained at two different
500
r
0
100
200 agal-hCG
300 (nglrxn)
400
500
FIG. 5. Incorporation of GalNAc is linear with respect to agalactohCG concentration at concentrations below the K,. Each enzyme reaction contained 0.2 pl of partially purified GalNAc-transferase, 1500 PM UDP-GalNAc, and the indicated amounts of agalacto-hCG in a 50-al reaction volume. Following incubations for 5 min at 37”C, 2-ng aliquots of hormone were tested for GalNAc incorporation. The amount of signal obtained in all cases represented less than 15% of the maximum for 2 ng of agalacto-hCG. The signal-to-noise ratio ranged from 4.5 to 7.1.
290
MENGELING
25-
ET
AL.
B
67 b
0.10
0.08
i
i
4 hCG
1
6
J
1
-8
-4
r
t
12
8
(I.cglrxn)
FIG. 6. Determination of kinetic constants for the GalNAc-transferase. (A) Saturation curve for GalNAc incorporation into agalacto-hCG. Each enzyme reaction contained the specified amount of agalacto-hCG, 20 ~1 partially purified GalNAc-Tf, and either 334 pM (0) or 1000 pM (0) UDP-GalNAc in a 50-~1 reaction volume. Incubations were at 37°C for 30 min. The amount of GalNAc incorporated per 6 ng of hCG was determined in triplicate and standard deviations are indicated. (B) Double-reciprocal plot of the data shown in (A): 334 pM UDP-GalNAc (0) and 1000 pM UDP-GalNAc (0). Lines were fit by least-squares regression analysis.
concentrations of UDP-GalNAc (334 and 1000 PM UDP-GalNAc), and Fig. 6B shows the resulting doublereciprocal plots. In all, we generated saturation curves at five different UDP-GalNAc concentrations (200,250, 334, 500, and 1000 PM). Figure 7A illustrates a secondary plot (8) of the l/V,,,,, values, generated from l/u versus l/[UDP-GalNAc] plots (not shown), versus 11 [hCG], from which a Km for hCG of 1.8 FM at saturating concentrations of UDP-GalNAc was determined. As shown in Fig. 7B, a secondary plot of the reciprocal of the slopes (l/n) from the l/[UDP-GalNAc] plots versus l/[hCG] gives a K,,,for agalcto-hCG of 0.98 j.&M at the extinction of UDP-GalNAc. When the reverse set of secondary plots is made to determine the K,,, values of UDP-GalNAc at saturation and extinction of agalactohCG, Km values of 30 and 200 PM, respectively, were obtained (not shown). These values are in good agreement with the Km values that we published for crude preparations of GalNAc-transferase using our radiolabeled assay (4,7), demonstrating the capabilities of the microplate assay for determining kinetic parameters. DISCUSSION
We previously identified and characterized a glycoprotein hormone-specific GalNAc-transferase using an assay in which [3H]GalNAc is incorporated into oligosaccharide acceptors on purified agalacto-hCG (4). Like
most glycosyltransferase assays relying on radiolabel incorporation, the GalNAc-transferase assay requires relatively large amounts of pure substrate and transferase. In addition, only a small number of assays can be performed at one time. These limitations were significant when GalNAc-transferase purification or characterization of hormone constructs differing in peptide sequence and expressed in cultured cells is considered. We therefore undertook the development of an assay for the glycoprotein hormone-specific GalNAc-transferase that would, without compromising specificity, provide a marked increase in both sensitivity and efficiency in the number of assays that could be simultaneously performed. The microplate assay that we have developed fulfills these requirements. Utilizing incorporation of radiolabel, we require 0.5 pg of purified agalacto-hCG, 1 &i of UDP-[3H]GalNAc, and 5.5 /IU of glycoprotein hormone-specific GalNAc-transferase for each data point; furthermore, only 20 analyses can be performed over a 48 to 72-h interval. The current microplate method requires no radiolabel and as little as 1 ng of agalacto-hCG and 32 nU of GalNAc-transferase. In addition, the ease of using a microtiter plate with detection in a luminometer that is able to read 96-well plates makes it possible to carry out large numbers of assays in a matter of hours. The large increase in sensitivity of the microplate assay over the radiolabeled assay reflects primarily the
GLYCOSYLTRANSFERASE A
MICROPLATE
291
ASSAY
0.08 6 6-
0.06 -
I
/
-4 lYLhCG, {M-i x 105,
'2
-'2
FIG.
43
7. Secondary plots to determine the K, for hCG at saturation and extinction ) from the plot of l/u versus l/[UDP-GalNAc] at five hCG concentrations: (l~vmaP, K,,, (1.8 PM) for hCG at saturating concentrations of UDP-GalNAc. (B) Secondary plot l/u vs l/fUDP-GalNAcl at hCG concentrations of 1.9.2.5, 3.8, and 7.5 pg. The x-axis UDP-GaiNAc. .
use of aequorin. Aequorin is a bioluminescent molecule that has been cloned from the hydromedusan Aequorea victoria and expressed at high levels in Escherichia coli (9). It consists of the polypeptide (apoaequorin) and mole per mole bound coelenterate luciferin and 0,. Upon addition of Ca2+, aequorin catalyzes the oxidation of luciferin producing a brief (-1-s) flash of blue light that can be measured in a microplate luminometer (MLlOOO, Dynatech). The sensitivity was further increased by using the “Peak Program,” which integrates the number of light units emitted during a time window encompassing the peak of the light flash. By adjusting the “before” and “after” peak time windows, the background can be significantly reduced. As a result, a linear response could be obtained over a concentration range of 105. In contrast, use of horse radish peroxidase as a reporter molecule in a standard color reaction was not linear over a range greater than lo2 and rarely provided signal-to-noise ratios >3:1. The specificity of the assay can be obtained at one or both of two steps. The capture agent, if highly selective, as with a monoclonal antibody, results in capture only of substrate even from a crude reaction. With such a capture reagent, prior purification of the substrate is not necessary. The saccharide-specific agent can also be highly specific, such as WFA, which binds to terminal, P-linked GalNAc. The amount of GalNAc actually transferred can be quantitated in absolute terms by constructing a standard curve using GalNAc-hCG bearing known amounts of terminal GalNAc.
-:/,,,,,
(L.1 x 1oq4
8
l2
of UDP-GalNAc. (A) Secondary plot of they intercepts 1.5,1.9,2.5,3.8, and 7.5 pg. The x-axis intercept gives the of the slopes obtained from the double-reciprocal plot of intercept gives the K,,, for hCG (0.98 FM) at extinction of
The sensitivity of the bioluminescent detection is essential for obtaining kinetic constants since it is necessary to detect GalNAc incorporation at levels that represent
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B. J., and Macher,
B. A. (1988)
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292
MENGELING
2. Crawley, S. C., Hindsgaul, O., Alton, G., Pierce, M. M. (1990) Anal. Biochem. 185, 112-117. 3. Zatta, P. F., Nyame, K., Cormier, M. J., Mattox, P. A., Smith, D. F., and Cummings, R. D. (1991) 194,185-191. 4. Smith, P. L., and Baenziger, J. U. (1988) Science 5. Nyame, K., Smith, D. F., Damian, (1989) J. Bid. Chem. 264,3235-3243.
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and Palcic,
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ET
AL.
6. Palcic, M. M., and Hindsgaul, 0. (1991) Glycobiology, in press. 7. Smith, P. L., and Baenziger, J. U. (1990) Proc. Natl. Acad. Sci. USA 67,7275-7279. 8. Segel, I. H. (1975) Enzyme Kinetics: Behavior and Analysis of Rapid Equilibrium and Steady-State Enzyme Systems, Wiley, New York. 9. Prasher, Biophys.
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