Glycosidase Activity 403
403
33
Glycosidase Activity
Anthony P. Corfield and Neil Myerscough
1. Introduction
The glycosidases and associated hydrolytic enzymes acting on glycoconjugate oli-
gosaccharides form part of the total mucinase activity. This chapter describes some
assay methods for the determination of these enzymes. Our knowledge of the number
of enzymes required for mucin degradation and their regulation in physiological situ-
ations is scanty (1). The degradation of both protein and carbohydrate domains require
specific enzymes which are able to degrade mucin structure. Carbohydrate degrada-
tion may be dependent on prior or concomitant peptide cleavage in mucins. The issues
that need to be addressed include the following:
1. Being able to demonstrate individual substrate specificity in relation to the known mucin
structure (both protein and carbohydrate).
2. The pathogenic or nutritional role of bacterial degradation leading to the loss (degrada-
tion to create receptor binding sites as part of bacterial colonization or infection of the
host) or recycling (utilization of the released products for energy production)
3. The need to consider the mode of growth of bacteria at the mucosal surface, in particular
the role of biofilms (see Chapter 36).
4. The role of “additional” enzymes such as sulfatases, phosphatases, and lipases, which are
important for rarer posttranslational modifications to mucin peptide and oligosaccharide
structure (1).
Many studies with glycosidases have been carried out using synthetic substrates,
such as 4-nitrophenyl- and 4-methyl umbelliferyl-glycosides. These substrates only
give information with regard to the anomeric configuration of the glycoside, but not
with respect to the nature of linkage to the next sugar in an oligosaccharide. Thus, the
results may have limited physiologic relevance. When the degradation of specific
glycoconjugates such as mucins is to be assessed, alternative, novel substrates have to
be prepared. Only in this way can the manner in which mucins are degraded in vivo be
studied. The design of substrates has followed two directions. The first, the use of
intact mucin/glycoprotein or glycopeptide substrates. These assays have relied on the
From:
Methods in Molecular Biology, Vol. 125: Glycoprotein Methods and Protocols: The Mucins
Edited by: A. Corfield © Humana Press Inc., Totowa, NJ
404 Corfield and Myerscough
detection of the individual monosaccharide being released. The sensitivity of these
assays depends on the sensitivity of the monosaccharide product detection. Colorimet-
ric, fluorimetric, radioactive, ultraviolet (UV) and pulsed amperometric detection
define the limits of these assays. Often further separation techniques (gel filtration,
high-performance liquid chromatography [HPLC], ion-exchange, etc.) are necessary
to isolate and quantify the products. As a result, many of the relevant substrates for
studies of degradation of mucin are not available commercially and must be prepared,
and in some cases, the design of suitable glycoconjugate substrates has not proved
possible. The second direction is the chemical synthesis of oligosaccharides with rel-
evant structure and the identification of degradation by chromatographic methods.
This chapter addresses the question of glycosidase activity. Description of the use
of mucin-related substrates together with some of the widely available synthetic sub-
strates gives an approach to the identity of the general range of mucin-degrading gly-
cosidase activities present in enzymatic preparations.
2. Materials
2.1. Glycosidase Substrates
2.1.1. Natural Glycosidase Substrates
Many naturally occurring glycoconjugates can be utilized as physiologic substrates
for glycosidases in the assays described in this chapter. In addition to those listed here,
other purified glycoconjugates can be used in the same way.
1. Fetuin type III (Sigma, Poole, UK).
2. Salivary gland glycoproteins from bovine (2) and ovine (3) sources.
3. α
1
-Acid glycoprotein (Sigma).
4. Antifreeze glycoprotein (4).
5. Porcine seminal gel glycoprotein (5).
6. Asialoglycoproteins: Prepare in all cases by incubation of the sialoglycoprotein at 1 mg/mL
in 0.1 M HCl at 80°C for 60 min. The sialic acid content is measured before and after the
hydrolysis and additional hydrolysis carried out under the same conditions if significant
sialic acid remains (6).
7. Saponification of O-acetyl esters: Carry out saponification on sialic acids in bovine sali-
vary gland mucin at 1 mg/mL in 0.1 M NaOH at room temperature for 45 min, and neu-
tralize the solution to approx pH 7.0 with 1 M HCl.
8. α2-3 and α2-6 Sialyllactose (Sigma).
2.1.2. Synthetic Glycosidase Substrates
Synthetic substrates form the basis of rapid and sensitive colorimetric (4-nitro–
phenyl-glycosides) and fluorimetric (4-methyl umbelliferyl-glycosides) assays. These
substrates are available through several suppliers, including Sigma; Oxford Glyco-
sciences, Abingdon, UK; Dextra, Reading, UK; Boehringer Mannheim, Lewes, UK;
and Chemica Alta, Edmonton, Canada.
1. 4-Nitrophenyl β-galactose: Dissolve in 1/10 volume of methanol and make up to a final
concentration of 4 mM in 100 mM sodium acetate, pH 6.0.
2. 4-Nitrophenyl α-galactose: Dissolve in 1/10 volume of methanol and make up to a final
concentration of 4 mM in 100 mM sodium acetate, pH 6.0.
Glycosidase Activity 405
3. 4-Nitrophenyl β-N-acetylglucosamine: Dissolve in 1/10 volume of methanol and make
up to a final concentration of 4 mM in 100 mM sodium acetate, pH 6.0.
4. 4-Nitrophenyl α-N-acetylgalactosamine: Dissolve in 1/10 volume of methanol and make
up to a final concentration of 4 mM in 100 mM sodium acetate, pH 6.0.
5. 4-Nitrophenyl Galβ1-3GalNAc: Dissolve the substrate directly in 100 mM citrate-phos-
phate, pH 6.0, to give a final concentration of 2 mM.
6. 4-Nitrophenyl α-fucose: Dissolve in 1/10 volume of methanol and make up to a final
concentration of 4 mM in 100 mM sodium acetate, pH 6.0.
7. 4-Methyl umbelliferyl sialic acid: Dissolve the substrate directly in 0.4 M sodium acetate,
pH 4.2, to give a final concentration of 2 mM.
2.1.3. Galactose/
N
-acetyl-D-Galactosamine-Labeled Glycoproteins
Galactose Oxidase and Tritiated Borohydride (
see
Notes 1 and 2)
1. Dissolve 5 mg of the glycoprotein, e.g., antifreeze glycoprotein, asialofetuin, asialo-ovine
submandibular gland mucin, or α
1
-acid glycoprotein, in 500 µL of 50 mM sodium phos-
phate and 5 mM NaCl, pH 7.0.
2. Add 5 U of galactose oxidase (Sigma) to each glycoprotein solution, and incubate for 24 h
at 37°C.
3. Dilute the products five times with 50 mM sodium phosphate and 50 mM NaCl, pH 7.8.
4. Add 12.5 MBq of sodium boro-[
3
H]-hydride (NaB[
3
H]
4
), typically 10 GBq/mmol
(Amersham, UK) to each, and stir the solutions for 60 min at room temperature (Hazard:
radioactive; see Note 1).
5. Add 1.5 mg of solid NaBH
4
(Sigma) to each incubation and stir for a further 30 min
(Hazard: attacks respiratory tract mucous membranes).
6. Add 10-µL aliquots of glacial acetic acid (analytical grade; BDH/Merck, Poole, UK), to
destroy borohydride, until no more bubbles are formed.
7. Add 2 mL of analytical grade methanol, and evaporate to dryness under reduced pressure
below 30°C in a rotary evaporator. Repeat this extraction five times to remove borate.
8. Adjust the pH of the solution to approx 6.5.
9. Desalt the products by repeated runs on a column of Sephadex G25 (30 × 1 cm; Pharmacia,
Milton Keynes, UK) in 0.2 M NaCl with a final run in distilled water.
10. Store the products as 100- to 500-µL aliquots in 0.02% sodium azide (BDH/Merck) at 4°C.
1.4. Sialyl GalNAc [
3
H]-ol from Boar Seminal Gel Mucin (
see
Note 1)
1. Dissolve 25 mg of porcine seminal gel glycoprotein in 4 mL of 0.05 M NaOH.
2. Add 190 mg of solid NaBH
4
(Hazard: attacks respiratory tract mucous membranes).
3. Add 925 MBq of NaB[
3
H]
4
(typically 10 GBq/mmol) (Amersham) in 1 mL of 0.05 M
NaOH, and incubate at 45°C with stirring, for 16 h (see Note 1).
4. Add glacial acetic acid (analytical reagent grade; BDH/Merck) dropwise until no more
bubbles are formed, to destroy excess borohydride (see Note 1).
5. Pass the solution through a column (20 mL) of Dowex 50 H
+
(200–400 mesh) (Bio-Rad,
Hemel Hempstead, UK), and wash with 100 mL of distilled water.
6. Evaporate to dryness under reduced pressure below 30°C using a rotary evaporator. Add
2 mL of methanol and repeat this evaporation five times to remove borate.
7. Dissolve the sample in 5 mL of 0.1 M pyridine acetate, pH 5.0, apply to a column of Bio-
Gel P4 (200–400 mesh, 150 × 2 cm) (Bio-Rad), and elute in the same buffer.
8. Collect 5-mL of fractions and measure radioactivity. Pool the major radioactive peak
(Neu5Acα2-6GalNAc-[
3
H]-ol) (see Note 3).
406 Corfield and Myerscough
9. Evaporate to dryness under reduced pressure below 30°C using a rotary evaporator.
Redissolve in 5 mM pyridine acetate, pH 5.0, and apply to a column (18 × 1 cm) of
Dowex 1 × 2 (–400 mesh, acetate form) (Bio-Rad). Elute with a gradient of 2–350 mM
pyridine acetate pH 5.0 (2 × 250 mL) and collect 5-mL fractions.
10. Pool the radioactive peak and remove the pyridine acetate by rotary evaporation.
11. Convert the oligosaccharide to the sodium salt by titration with Dowex 50 Na
+
(50–100
mesh) (Bio-Rad).
12. Store at 4°C in 2% aqueous ethanol.
2.1.5. Sialyl-[
3
H]-Labeled Sialoglycoproteins
Any sialoglycoprotein can be labeled with tritium after periodate oxidation (see
Note 4). Typically, α
1
-acid glycoprotein and bovine submandibular gland mucin have
been used. The sialic acid content of the glycoprotein must be determined first in order
to make up the correct ratio of sialic acid to periodate in the mild oxidation step. In the
case of some mucin substrates, e.g., bovine submandibular gland mucin, in which
sialic acid O-acetyl esters are expected or suspected, a mild saponification step should
be included before the periodate oxidation (see Subheading 2.1.1., item 7; Note 5).
1. Dissolve 10–50 mg sialoglycoprotein in 100 mM sodium acetate, 150 mM sodium chlo-
ride buffer, pH 5.5, to give a final concentration of 1 mM with respect to sialic acid and
equilibrate at 4°C.
2. Add ice cold 10 mM sodium metaperiodate (Sigma) in water to give a final concentration
of 1 mM periodate (approx 1/10 of the volume of the sialoglycoprotein solution) and stir.
3. Incubate for 10 min at 4°C with stirring.
4. Add glycerol (0.2 mL for each 10 mL of sialoglycoprotein solution) and stir for a further
10 min.
5. Dialyze the solution against three changes of 2.5 L of 0.05 M sodium phosphate, 0.15 M
sodium chloride, pH 7.4, for 24 h at 4°C.
6. Add 500–1000 Mbq of NaB[
3
H]
4
(typically 10 GBq/mmol) (Amersham) in 0.05 M NaOH
adjusted to a final concentration of approx 0.1 M borohydride with unlabeled NaBH
4
in a
volume of 1 mL and stir for 30 min at room temperature.
7. Add 1 mL of 0.1 M NaBH
4
and incubate for a further 30 min.
8. Dialyze the product against two changes of 3 L of 0.1 M sodium acetate, pH 5.5, and then
against two changes of 3 L of distilled water.
9. Concentrate the sialoglycoprotein solution if necessary (see Note 6).
2.2. Buffers and Reagents for Enzyme Assay
1. General 4-nitrophenyl-glycoside assay buffer: 100 mM sodium acetate, pH 6.0.
2. General 4-nitrophenyl-glycoside stop solution: 10% trichloroacetic acid (TCA) in dis-
tilled water.
3. 0.5 M Sodium carbonate.
4. Ovalbumin (Sigma), 80 mg/mL in 0.1 M sodium phosphate, pH 7.0, buffer.
5. 5% phosphotungstic acid (PTA)/15% TCA: 5% (w/v) PTA (BDH/Merck) and 15% (w/v)
TCA (BDH/Merck) in distilled water.
6. O-glycanase incubation buffer: 100 mM citrate-phosphate, pH 6.0. Prepare 100 mM citric
acid and 200 mM disodium phosphate. Take 1 vol of citric acid and titrate to pH 6.0 with
phosphate, and make up to a final volume equivalent to 1:1 of starting volume.
7. Sialidase incubation buffer (colorimetric assay): 100 mM sodium acetate, 20 mM CaCl
2
,
150 mM NaCl, pH 5.5.
Glycosidase Activity 407
8. Reagents for the Warren assay of sialic acids:
a. 0.25 M Periodic acid: 5.7 g of periodic acid (Sigma) in 75 mL phosphoric acid (BDH/
Merck) make up to 100 mL with distilled water. Stock solutions will keep for approx
12 mo at room temp.
b. 0.38 M sodium arsenite in 0.5 M sodium sulfate: 5 g of sodium arsenite (BDH/Merck),
7.1 g of anhydrous sodium sulphate (BDH/Merck) make up to 100 mL with distilled
water. Solution is stable for several months at room temperature.
c. Thiobarbituric acid in 0.5 M sodium sulfate: 0.9 g of thiobarbituric acid (Aldrich,
Gillingham, UK), 7.1 g of anhydrous sodium sulfate (BDH/Merck) made up to 100 mL
with distilled water. Solution is stable for approx 1 wk only (see Note 7).
d. Cyclohexanone analytical reagent (AR) (Aldrich, Gillingham, UK).
e. N-Acetyl neuraminic acid (sialic acid; Sigma): Standard solution for calibration of
the assay is 0.31 mg/mL, use 10 or 20 µL of this solution in a final volume of 100 µL
for the assay (see Subheading 3.7.1.).
9. Sialidase incubation buffer (fluorimetric assay): 400 mM sodium acetate pH 4.2.
10. Sialidase stop buffer (fluorimetric assay): 85 mM glycine/sodium carbonate buffer, pH 10.0.
11. Sialate O-acetyl esterase buffer: 100 mM triethanolamine, pH 7.8.
12. Acetic acid detection assay kit (Boehringer) (see Note 8).
13. Acylneuraminate pyruvate lyase incubation buffer: 200 mM potassium dihydrogen phos-
phate adjusted to pH 7.2 with KOH.
14. Acylneuraminate pyruvate lyase substrate: 10 mM sialic acid (Sigma) in potassium phos-
phate buffer, pH 7.2, containing 0.5–1.0 kBq/mL of [
14
C]-N-acetylneuraminic acid
(Amersham).
15. Acylneuraminate pyruvate lyase from Clostridium perfringens (Sigma).
3. Methods
3.1.
β
-Galactosidase (
see
Notes 9 and 10)
Several different assays are possible for β-galactosidase; synthetic substrates are
widely available for either colorimetric or fluorimetric assay. Radioactive assays with
glycoproteins give data on physiologically significant molecules (see Notes 9–11).
3.1.1. Synthetic Substrate
1. Mix 25 µL of 4 mMp-nitrophenyl β-galactose in 100 mM sodium acetate, pH 6.0 (see
Subheading 2.1.2., item 1), with 25 µL of extract/enzyme.
2. Incubate for 20 min at 37°C.
3. Add 50 µL of 10% trichloracetic acid (see Subheading 2.2., item 2) to stop the reaction
4. Add 1 mL of 0.5 M sodium carbonate.
5. Centrifuge to remove any solid, and read the supernatant at 400 nm.
6. Prepare blank incubations by adding TCA to enzyme extract incubated alone for 20 min,
and then add substrate incubated alone for 20 min. Add 1 mL of 0.5 M sodium carbonate,
centrifuge and read at 400 nm as above (see Note 12).
3.1.2. General Mucin Galactosidase Assay Radioactive Substrates
(
see
Note 13)
Radioactive asialoglycoproteins labeled in their terminal galactosyl residues (see
Subheading 2.1.3.) can be used as substrates in this precipitation assay. Depending on
the nature of the radioactive substrate, this assay can be specific for a particular glyco-
sidic linkage (see Note 14).
408 Corfield and Myerscough
1. Mix 50 µL of 0.85 kBq/mL asialo-glycoprotein substrate in 100 mM sodium acetate, pH
6.0, with 50 µL of extract/enzyme.
2. Incubate for 1 h at 37°C.
3. Stop the reaction by the addition of 100 µL of ice-cold ovalbumin (see Subheading 2.2.,
item 4).
4. Add 500 µL of 5% PTA/15% TCA (see Subheading 2.2., item 5) and mix.
5. Stand for 15 min at room temperature.
6. Centrifuge for 5 min at 15,000g (benchtop microcentrifuge).
7. Take 500 µL of the clear supernatant and count the radioactivity.
3.1.3. Glycoprotein Assay with
β
1-3 Linked- and
β
1-4 Linked-Galactose
Radioactive Substrates (
see
Notes 9, 10, and 14)
The detection of β1-3- and β1-4-specific galactosidase activity can be achieved
using antifreeze glycoprotein and asialo-α
1
-acid glycoprotein, respectively (see Sub-
heading 2.1.3.). In these assays, identification of the galactose product is made by gel
filtration.
1. Mix 50 µL of 0.42–0.83 kBq/mL asialo-glycoprotein substrate in 100 mM sodium acetate,
pH 6.0, with 50 µL of extract/enzyme (see Note 10).
2. Incubate for 1–24 h at 37°C.
3. Stop the reaction by adding of 1mL of 0.1 M pyridinium acetate, pH 5.0.
4. Apply the total incubation to a column of Bio-Gel P2 (200–400) (Bio-Rad), and elute
with 0.1 M pyridinium acetate, pH 5.0, collecting 2-mL fractions.
5. Measure the radioactivity in the collected fractions and determine the proportion of tri-
tium label migrating as free galactose (Fig. 1).
3.2.
α
-Galactosidase (
see
Notes 9–12, and 15)
Assay for α-galactosidase can be carried out rapidly using synthetic substrates.
Commercial enzyme is available to use as a positive control. However, several differ-
ent α-galactose linkages are known to occur, and further examination may be neces-
sary to identify the specificity (see Notes 9, 10, and 15).
1. Mix 25 µL of 4 mM 4-nitrophenyl α-galactose (see Subheading 2.1.2., item 2) in 100
mM sodium acetate, pH 6.0, with 25 µL of extract/enzyme.
2. Incubate for 20 min at 37°C.
3. Add 50 µL of 10% TCA to stop the reaction.
4. Add 1 mL of 0.5 M sodium carbonate.
5. Centrifuge to remove any solid, and read the supernatant at 400 nm.
6. Prepare blank incubations by adding TCA to enzyme extract incubated alone for 20 min,
and then add substrate incubated alone for 20 min. Add 1 mL of 0.5 M sodium carbonate,
centrifugem and read at 400 nm as in step 5 (see Note 12).
3.3.
β
-
N
-acetylglucosaminidase (
see
Note 16)
Assay for β-N-acetylglucosaminidase can be carried out easily using synthetic sub-
strates. Commercial enzyme is available to use as a positive control (see Notes 9 and 10).
1. Mix 25 µL of 4 mM 4-nitrophenyl β-N-acetylglucosamine in 100 mM sodium acetate, pH
6.0 (Subheading 2.1.2., item 3), with 25 µL of extract/enzyme.
Glycosidase Activity 409
2. Incubate for 20 min at 37°C.
3. Add 50 µL of 10% TCA to stop the reaction.
4. Add 1 mL of 0.5 M sodium carbonate.
5. Centrifuge to remove any solid, and read the supernatant at 400 nm.
6. Prepare blank incubations by adding trichloracetic acid to enzyme extract incubated alone
for 20 min, and then add substrate incubated alone for 20 min. Add 1 mL of 0.5 M sodium
carbonate, centrifuge, and read at 400 nm as in step 5 (see Note 12).
3.4.
α
-
N
-acetylgalactosaminidase (
see
Note 17)
Assay for α-N-acetylgalactosamine can be carried out easily using synthetic
substrates. Commercial enzyme is available to use as a positive control (see Notes
9 and 10).
3.4.1. Synthetic Substrate
1. Mix 25 µL of 4 mM 4-nitrophenyl α-N-acetylgalactosamine in 100 mM sodium acetate,
pH 6.0, residues (see Subheading 2.1.2., item 4) with 25 µL of enzyme extract.
2. Incubate for 20 min at 37°C.
3. Add 50 µL of 10% TCA to stop the reaction.
4. Add 1 mL of 0.5 M sodium carbonate.
5. Centrifuge to remove any solid, and read the supernatant at 400 nm.
6. Prepare blank incubations by adding trichloracetic acid to enzyme extract incubated alone
for 20 min, and then add substrate incubated alone for 20 min. Add 1mL of 0.5 M sodium
carbonate, centrifuge, and read at 400 nm as in step 5 (see Note 12).
3.4.2. Mucin
α
-
N
-Acetylgalactosaminidase Assay with Radioactive
Substrate (
see
Notes 13 and 16)
Radioactive asialo-ovine salivary gland mucin is labeled in the terminal N-acetyl-
galactosaminyl residues (see Subheading 2.1.3.) and only contains this single sugar
attached to the peptide backbone (see Notes 13 and 14).
1. Mix 50 µL of 0.85 kBq/mL asialo-ovine salivary gland mucin substrate in 100 mM sodium
acetate, pH 6.0, with 50 µL of extract/enzyme.
2. Incubate for 1 h at 37°C.
3. Stop the reaction by adding 100 µL of ice-cold ovalbumin (see Subheading 2.2., item 4).
4. Add 500 µL of 5% PTA/15% TCA (see Subheading 2.2., item 5) and mix.
5. Stand for 15 min at room temperature
6. Centrifuge for 5 min at 15,000g (benchtop microcentrifuge)
7. Take 500 µL of the clear supernatant and count the radioactivity.
3.5.
α
-Fucosidase (
see
Note 18)
Assay for α-fucosidase can be carried out easily using synthetic substrates. Com-
mercial enzyme is available to use as a positive control (see Notes 9 and 10)
1. Mix 25 µL of 4 mM 4-nitrophenyl α-fucose in 100 mM sodium acetate, pH 6.0, (see
Subheading 2.1.2., item 6).with 25 µL of extract/enzyme.
2. Incubate for 30 min at 37°C.
3. Add 50 µL of 10% TCA to stop the reaction.
4. Add 1 mL of 0.5 M sodium carbonate.
410 Corfield and Myerscough
5. Centrifuge to remove any solid, and read the supernatant at 400 nm.
6. Prepare blank incubations by adding trichloracetic acid to enzyme extract incubated alone
for 20 min, and then add substrate incubated alone for 20 min. Add 1 mL of 0.5 M sodium
carbonate, centrifuge, and read at 400 nm as in step 5 (see Note 12).
3.6.
O
-Glycanase (
see
Note 19)
Determination of O-glycanase activity can be made with synthetic or glycoprotein
substrates.
3.6.1. Synthetic Substrate
1. Mix 25 µL of 2 mM 4-nitrophenyl Galβ1-3GalNAc in 100 mM citrate-phosphate, pH 6.0
(see Subheading 2.1.2., item 5), with 25 µL of extract/enzyme.
2. Incubate for 20 min at 37°C.
3. Add 50 µL of 10% TCA to stop the reaction.
4. Add 1 mL of 0.5 M sodium carbonate.
5. Centrifuge to remove any solid, and read the supernatant at 400 nm.
6. Prepare blank incubations by adding trichloracetic acid to enzyme extract incubated alone
for 20 min, and then add substrate incubated alone for 20 min. Add 1 mL of 0.5 M sodium
carbonate, centrifuge, and read at 400 nm as in step 5 (see Note 12).
3.6.2. Tritiated Glycoprotein Substrate
1. Mix 50 µL of 0.42–0.83 kBq/mL antifreeze glycoprotein substrate (see Subheading
2.1.3.) in 100 mM citrate phosphate buffer, pH 6.0, with 50 µL of extract/enzyme (see
Note 9).
Fig. 1. Identification of the products of O-glycanase and β1-3-galactosidase activity on
antifreeze glycoprotein substrate by Bio-Gel P2 chromatography. The positions of elution
of intact [
3
H]-labeled antifreeze glycoprotein (ᮀ), Galβ1-3GalNAc (᭡) and galactose (᭺)
are shown.
Glycosidase Activity 411
2. Incubate for 1–24 h at 37°C.
3. Stop the reaction by adding 1 mL of 0.1 M pyridinium acetate, pH 5.0.
4. Apply the total incubation to a column of Bio-Gel P2 (200–400) (Bio-Rad), and elute
with 0.1 M pyridinium acetate, pH 5.0, collecting 2-mL fractions.
5. Measure the radioactivity in the collected fractions and determine the proportion of tri-
tium label migrating as free Galβ1-3GalNAc (see Fig 1).
3.7. Sialidase (
see
Notes 4, 5, 9, 13, and 20).
Sialidase activity can be measured using several types of assay. The variation in
sialic acid substitution in glycoconjugates indicates the varied range of sialidase
activities that may be expected and underlines the need for examination of different
substrates and assay conditions (see Notes 10 and 20).
3.7.1. Colorimetric Sialidase Assay
Preliminary determination of the sialic acid content of the substrates is necessary to
prepare the substrates for the assays given next.
1. Prepare substrates (bovine salivary gland mucin, saponified; α
1
-acid glycoprotein; α2-3
sialyllactose) to give a final concentration of 1 mM sialic acid in 100 mM sodium acetate,
20 mM CaCl
2
, and 150 mM NaCl, pH 5.5, and keep on ice.
2. Mix 50 µL of substrate with 50 µL of enzyme extract and incubate for 60 min at 37°C.
3. Remove from incubation block and mix with 20 µL of sodium periodate solution (see
Subheading 2.2., item 8a).
4. Leave at room temperature for 30 min.
5. Add 200 µL of sodium arsenite solution (see Subheading 2.2., item 8b), mix until yel-
low-brown iodate color appears, and then the solution finally becomes colorless (may
take several minutes, if unsure wait for 5 min).
6. Add 200 µL of thiobarbituric acid solution (see Subheading 2.2., item 8c) and incubate
for 15 min at 95°C.
7. Cool on ice (10 min) and add 700 µL of cyclohexanone (see Subheading 2.2., item 8d).
8. Shake to mix the two layers.
9. Centrifuge at 12,000g for 2 min in a benchtop microcentrifuge to separate the two layers.
10. Read the organic (pink) layer at both 532 and 549 nm.
11. Calculate the ∆OD value for the released sialic acid, allowing for the interference by
compound-forming chromophores at 532 nm using the formula 0.9 OD549–0.3 OD532.
12. Process a standard of 3 µg of Neu5Ac (Sigma) (see Subheading 2.2., item 8e) in 100 µL
through the same assay, and correct with the same formula (0.9 OD549–0.3 OD532) (see
Note 21).
13. Convert the ∆OD value into moles of Neu5Ac to give the activity of the enzyme.
3.7.2. Radioactive Sialidase Assay (
see
Note 13)
1. Mix 50 µL of radioactive substrate (0.8–1.5 kBq/mL; see Subheading 2.1.5., e.g., bovine
salivary gland mucin and α
1
-acid glycoprotein) in incubation buffer (see Subheading
2.2., item 7) with 50 µL of extract/enzyme.
2. Incubate for 60 min at 37°C.
3. Stop the reaction by adding 100 µL of ice-cold ovalbumin (see Subheading 2.2., item 4).
4. Add 500 µL of 5% PTA/15% TCA (see Subheading 2.2., item 5) and mix.
5. Stand for 15 min at room temperature.
412 Corfield and Myerscough
6. Centrifuge for 5 min at 15,000g (benchtop microcentrifuge).
7. Take 500 µL of the clear supernatant and count the radioactivity.
3.7.3. Fluorimetric Sialidase Assay (
see
Note 11)
1. Mix 50 µL of 2 mM 4-methyl umbelliferyl sialic acid (see Subheading 2.1.2., item 7) in
acetate incubation buffer (see Subheading 2.2., item 9) with 50 µL of enzyme extract.
2. Prepare blanks using distilled water in place of enzyme extract (see Note 12).
3. Incubate for 60 min at 37°C.
4. Stop the reaction with 1 mL of stop buffer (see Subheading 2.2., item 10) and mix.
5. Centrifuge (microcentrifuge) for approx 40 s at 14,000g. Keep in the dark before reading
in a fluorimeter.
6. Read in a fluorimeter at excitation 365 nm and emission 448 nm.
7. Calculate fluorescence after subtraction of the blanks from the test values.
8. Calibrate the results using standard curves of 4-methylumbelliferone.
3.8. Sialate
O
-acetyl Esterase (
see
Note 22)
The sialate O-acetyl esterase is assayed using bovine salivary gland mucin (see
Subheading 2.1.1., items 2 and 7) and the released acetic acid is detected using a
quantitative kit assay.
1. Mix 100 µL of bovine salivary mucin substrate in incubation buffer (see Subheading 2.2.,
item 11) with 100 µL of enzyme extract (see Note 23), and incubate for 60 min at 37°C.
2. Stop the reaction by heating at 95°C for 3 min.
3. Cool the incubation mixture on ice or freeze at –20°C until use.
4. Measure acetic acid in the incubation using the NAD/NADH kit assay (see Subheading
2.2., item 12).
5. Calculate the amount of acetic acid released during the initial incubation with the salivary
mucin substrate.
3.9. Acylneuraminate Pyruvate Lyase (
see
Note 24)
1. Mix 50 µL of sialic acid substrate in phosphate buffer, pH 7.2 (see Subheading 2.2.,
items 13 and 14), with 50 µL of enzyme extract.
2. Incubate for 60 min at 37°C.
3. Stop the reaction with 1 mL of ice-cold water.
4. Centrifuge for 2 min in a bench microcentrifuge at 12–15,000g.
5. Apply the supernatant to a column of Dowex 1 × 8, 200–400 mesh, Cl
-
form (1 mL wet
weight ), and wash with 5 mL of water directly into a 20-mL scintillation vial.
6. Place in an oven at 80°C until water has evaporated off.
7. Redissolve the residue in 0.5 mL of water and add scintillation fluid (1 mL).
8. Include a positive control with purified acylneuraminate pyruvate lyase from Clostridium
perfringens (see Subheading 2.2., item 15).
4. Notes
1. It is advisable to carry out labeling experiments in a recognized radioisotope laboratory.
The use of radioactive borotritide (NaB[
3
H]
4
) requires large amounts of [
3
H] and results
in the emission of tritium gas. It is important to check the registration limits for [
3
H] in
the laboratory to be used. Since a hood is required it is necessary that the tritium gas be
properly trapped and that the necessary decontamination procedures be applicable. If regu-
Glycosidase Activity 413
lar (multiple) tritium labeling is envisaged, it is especially important to check the regis-
tration limits and decontamination procedures with the radioactive officer responsible for
the laboratory.
2. Galactose oxidase will act on terminal galactose and N-acetyl-
D
-galactosamine residues
to give oxidation at the primary alcohol group at carbon 6. Glycoproteins with both of
these sugars in terminal nonreducing locations, e.g., asialoseminal gel mucin, will be
labeled in both residues.
3. The use of porcine seminal gel has the advantage that the sialic acid is found as Neu5Ac
(no Neu5Gc is present). In addition to Neu5Acα2-6GalNAc, the tetrasaccharide
Neu5Acα2-3Galβ1-3[Neu5Acα2-6]GalNAc is also found. The β-eliminated, reduced
oligosaccharides are well separated on Bio-Gel P4 and Dowex 1 × 2 acetate.
4. Lower concentrations of periodate are used to limit the oxidation to the C7–C9 end of the
sialic acid molecule. Higher concentrations of periodate result in the oxidation of other
sugars in glycoconjugate oligosaccharides (7).
5. Mucins in the human colorectum typically have O-acetyl esters located on C7-C9
hydroxyl groups of sialic acids. In order to oxidize these hydroxyl groups with periodic
acid, the esters must be removed. This can be done rapidly and under mild conditions by
saponification. The conditions described (see Subheading 2.1.1., item 7) do not cause
degradation of the glycoproteins used in the protocols given here. In the case of other
glycoproteins, it is best to check the preparation after saponification for the loss of carbo-
hydrate and peptide cleavage.
6. When the final volume containing the glycoconjugate is large and needs to be reduced,
several methods are available for concentration. These include rotary evaporation under
reduced pressure, freeze drying, and concentration cells. Because drying of samples may
affect solubility, especially with mucins, the recovery and integrity of the products should
be routinely checked.
7. The preparation of the thiobarbituric acid stock solution requires that the fresh solution
be heated under hot tap water with shaking to completely dissolve all components. On
storage a precipitate appears that may need to be filtered before use. The stock solution
should be stored in a stoppered bottle at room temperature. Fresh stock solution should be
prepared on a weekly basis.
8. The amount of acetic acid in the esterase incubation is determined using a commercial
UV test kit (Boehringer). This is an NAD-linked assay system converting acetic acid to
acetyl-CoA (acetyl CoA synthetase), acetyl-CoA to citrate (citrate synthase), and finally
using the conversion of NAD to NADH with malate dehydrogenase. The change in absor-
bance is measured at 340 nm. The kit contains all enzymes and buffers and gives details
of the calculation necessary to determine the amount of acetic acid in each sample.
9. It is recommended that each assay be validated using commercially available enzyme
sources with known substrates and to run such incubations routinely as positive controls.
This is especially important when separation techniques such as gel filtration and HPLC
are utilized to identify the released monosaccharide product. Commercial enzymes and
substrates can be obtained from a variety of suppliers including, Oxford Glycosciences;
Sigma; Dextra; ICN Biomedicals, High Wycombe, UK; Boehringer Mannheim; Calbiochem-
Novabiochem (UK), Nottingham, UK; and Seikagaku Kogyku, Tokyo, Japan. Remember
that when an unknown substrate or enzyme is being tested, differences in substrate speci-
ficities exist for glycosidases. A negative result indicates that other possible substrates
should be tested before the absence of enzymatic activity can be concluded. In the case of
novel substrates that are resistant to commercial enzymes, a detailed structural analysis is
necessary to identify the oligosaccharide type and monosaccharide linkage(s).
414 Corfield and Myerscough
10. Individual galactosidases show a range of pH optima using 4-nitrophenyl and glycopro-
tein substrates. The examination of unknown samples for this activity should include a
suitable range of pHs to cover the known ranges described. The commercial sources as
mentioned in Note 9 should be included to provide positive controls for these activities.
Examples are Escherichia coli (200 mM Tris-HCl, pH 7.3); Aspergillus niger (200 mM
sodium acetate, pH 4.0); coffee bean α-galactosidase (200 mM Tris-HCl, pH 6.5); bovine
testes (100 mM citrate phosphate, pH 4.0).
11. In addition to the colorimetric assays given in this chapter, additional sensitivity can be
attained by the use of fluorimetric assays with 4-methyl umbelliferyl-glycosides. Further
information may be derived from the use of individual reducing oligosaccharide sub-
strates that can be tagged radioactively or with fluorescent labels. Products can be sepa-
rated from substrates by HPLC techniques. Remember that the synthetic substrate assays
may not give physiologically relevant information and that oligosaccharides also repre-
sent partial physiologic substrates. Thus, the use of intact natural substrates should be
considered. However, when impure enzyme mixtures are being analyzed, the use of natu-
ral substrates must include direct product (monosaccharide) identification. Depending on
the type of assay used additional enzymatic activities may lead to fragmentation of the
substrate, giving false-positive results (see also Note 13 and Chapter 31 on total mucinase
activity and glycoprotein fragmentation during degradation).
12. Blank incubations can be prepared using enzyme extract incubated under normal condi-
tions but without substrate, stopping the reaction with TCA and combining the stopped
enzyme with substrate also incubated alone. Alternatively, heat-treated (5 min at 95°C)
enzyme extract can be used in a normal incubation. In many cases, the heat treatment of
crude extracts results in interference with the colorimetric or fluorescent reaction and
should be avoided.
13. Assays using precipitation of the substrate must be used with enzyme sources which gen-
erate no alternative soluble product. Crude extracts are likely to contain proteinases and
peptidases that may generate soluble glycopeptide products, and these will give a “false-
positive” result in the precipitation assay. If radioactive substrates are to be used with
crude enzyme preparations separation of the monosaccharide product e.g. galactose, must
be part of the assay procedure. This type of assay is described under Subheading 3.1.3.
14. The preparation of galactose oxidase/borotritide-labeled substrates, as detailed in Sub-
heading 2.1.3. can be carried out with glycoproteins that have an exclusive glycosidic
linkage type. Antifreeze glycoprotein contains only β1-3 linked-galactose and asialo-α
1
-
acid glycoprotein only β1-4 linked-galactose. The use of these substrates automatically
gives a linkage-specific enzymatic assay. Glycoproteins that have terminal GalNAc resi-
dues are also labeled (see Note 2); hence, asialo-ovine salivary gland mucin contains only
GalNAc linked to the peptide backbone and is a good substrate for the enzyme removing
GalNAc from the peptide. Glycoproteins with terminal GalNAc as blood group A,
(GalNAc α1-3Gal-) or Sda or Cad antigen (GalNAc β1-4Gal-) are also potential sub-
strates. However, such glycoproteins may also have terminal galactose residues and need
to be carefully analyzed before use in specific assays.
15. Blood group B activity is associated with the expression of terminal α1-3 galactose and is
commonly associated with mucins. If mucin degradation is being studied, the blood group
of the source material should be determined. In addition, the secretor status of individuals
is relevant since only secretor-positive individuals will express blood group activity in
mucins. There are also examples of galactose in other α-linkages, and examination of
additional oligosaccharide substrates (see Note 9) by HPLC is an alternative way to iden-
tify the presence of α1-4 or α1-6 galactosidase activities.
Glycosidase Activity 415
16. The detection of N-acetyl-
D
-glucosaminidase activity is generally carried out with the
4-nitrophenyl- or 4-methyl umbelliferyl-synthetic substrates. Preparation of glycoprotein
substrates labeled specifically in GlcNAc residues is difficult to achieve. The availability
of GlcNAc-terminated oligosaccharides provides a source of substrates for HPLC based
assays after radioactive or fluorescent tagging.
17. Glycoproteins with terminal GalNAc as blood group A (GalNAc α1-3Gal-) or as Tn antigen
(GalNAc α-O-ser/thr) are substrates for different enzymes. The use of synthetic substrates
must be compared with substrates containing GalNAc in either of these two arrangements.
Oligosaccharide substrates are only possible with the blood group A structure. The pres-
ence of β-linked GalNAc has also been found in the Sda or Cad antigens (GalNAc β1-
4Gal-). These are substrates for different enzymes and have not been examined in detail.
18. Fucose is present in glycoproteins in a number of different linkages, including α1-2, α1-3,
α1-4, and α1-6, and as with other glycosidases, there are a number of different fucosidases
that have varying specificity for these linkages in different oligosaccharides. Many of these
oligosaccharides are available commercially (see Note 8) and can be used to examine the
detailed specificity of fucosidase activity detected with synthetic substrates.
19. O-glycanase (endo-α-N-acetylgalactosaminidase) is an enzyme that removes the disaccha-
ride Galβ1-3GalNAc from peptide-carbohydrate substrates by cleavage of the O-glycosidic
linkage. Several of these enzymes have been identified and purified from bacteria. Commer-
cial sources of O-glycanase enzyme are available and can be used to validate the assay. Al-
most all O-glycanases described to date will not act on Galβ1-3GalNAc substituted by any
other sugars. It is possible to “generate” substrate by prior treatment of a glycoprotein with
sialidase and fucosidase to strip the O-linked oligosaccharides to the basic Galβ1-3GalNAc
disaccharide. In the enzyme assay it is important to identify the product as a disaccharide and
in the radioactive assay described this is achieved by gel filtration. A clear discrimination
between Galβ1-3GalNAc and Gal products should be made. In the case of the synthetic sub-
strate Galβ1-3GalNAc-4-nitrophenyl it is possible that the joint action of β1-3-galactosidase
and O-acetylgalactosaminidase could generate a false-positive result. Where such doubt may
arise an alternative assay with direct disaccharide product identification is needed.
20. Sialidases represent a group of enzymes which act on the wide range of sialoglyco-
conjugates with a variety of linkages of sialic acids to different sugars (8). Although the
sialidases from microorganisms often show a broad substrate specificity there are also
examples of limited substrate activity and new enzyme sources need to be evaluated using
several substrates to determine its properties (see ref. 8 for examples). In the case of
sialidases acting on mucin, sialic acids on O-linked oligosaccharides are the main targets.
However, each mucin contains a characteristic mixture of sialo-oligosaccharides. Exami-
nation of individual oligosaccharide substrates analogous to those present in mucins, e.g.,
α2-3- or α2-6-sialyl lactose isomers may give further information concerning sialidase
specificity. However, it will not predict the activity of enzymes on the intact mucin mol-
ecules where oligosaccharide structure and their arrangement on the polypeptide will play
a role. It is thus important to use intact mucin substrates to examine sialidase activity.
21. The use of the correction formula for the colorimetric assay is valid for values of OD532
up to 0.80 × OD549. Above this value the correction becomes inaccurate.
22. Acylneuraminate O-acetyl esterase acts on ester groups in sialic acids. As these O-acetyl
ester groups reduce the activity of sialidase activity the esterase is proposed to have a
regulatory role in the rate of colonic mucin degradation where these ester forms of sialic
acids are abundant (9). Mucins in the human colon contain sialic acids with O-acetyl
esters on the hydroxyl groups at carbons C7, C8, and C9 and these may be present as
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