- Email: [email protected]
[18] Streptavidin-biotinylglycopeptide-lectin complex in detection of glycopeptides and determination of lectin specificity
[18]
STREPTAVIDIN--BIOTINYLGLYCOPEPTIDE--LECTIN COMPLEX
253
ing site (D. R. Bundle et al., Auzanneau and Siguskjold). Mild elution conditions are most desirable, and for one case, Se155.4 histidine residues in the binding site render this antibody binding particularly sensitive to pH changes. Elution with an inexpensive, readily available ligand is a preferred strategy when bound antibody may be economically displaced by a small structure.
[18] S t r e p t a v i d i n - B i o t i n y l g l y c o p e p t i d e - L e c t i n C o m p l e x in Detection of Glycopeptides and Determination of Lectin Specificity
By MING-CHUAN SHAO and CHRISTOPHERC. Q. CHIN Introduction Streptavidin-biotinylglycopeptide-lectin complexes can be used in the detection of glycopeptides and the determination of lectin specificity at the picomole level. The method is based on the high affinity between streptavidin and biotin derivatives, 1,2 and between lectins and their complementary sugars, 3 and on the ability of streptavidin to adhere to microtiter plate wells 4'5 (Fig. 1). For the detection of glycopeptides, each unknown (glyco)peptide [from a high-performance liquid chromatography (HPLC) run, for example] is first biotinylated and complexed with streptavidin, each conjugate is then coated to the well of a titer plate, and lectin coupled to horseradish peroxidase (HRP) is added. After incubation, the wells are thoroughly washed and probed for the presence of the peroxidase. A positive test shows the presence of a glycan complementary to the lectin u s e d . 4 Based on the same principle, when a series of well-characterized biotinylglycans complexed with streptavidin is precoated in the wells of a titer plate, the sugar-binding specificity of unknown lectins, conjugated to peroxidase, can also be conveniently screened. 5
i N. M. Green, Adv. Protein Chem. 29, 85 (1975). 2 V. J. Chen and F. Wold, Biochemistry 25, 939 (1986). 3 I. J. Goldstein and C. E. Hayes, Adv. Carbohydr. Chem. Biochem. 35, 127 (1978). 4 M. C. Shao and C. C. Q. Chin, Anal. Biochem. 207, 100 (1992). 5 M. C. Shao, Anal. Bioehem. 205, 77 (1992).
METHODS IN ENZYMOLOGY,VOL. 247
Copyright © 1994by Academic Press, Inc. All rights of reproduction in any form reserved.
254
ENZYMATIC AND AFFINITY METHODS
[18]
ABTS ABTS reduced ' N . ~¢oxidized colorless '~..~ j ~ green
°reradish1 peroxidase
ectin
~-~
_
Oligosacchande Asn, Ser, Thr or peptide
Hexanoyl group -- Biotin
Streptavidin Polystyrene surface
l
FIG. 1. Principle for the detection of glycopeptides and the determination of lectin specificity with streptavidin-biotinylglycopeptide-lectin complex as a tool. The (glyco)peptides from HPLC peptide maps can be biotinylated, complexed with streptavidin, immobilized on a titer plate, and then detected by the activity of HRP coupled covalently to a lectin, which in turn can bind to specific glycans. Similarly, if a series of well-characterized biotinylglycans complexed with streptavidin are precoated in the wells of a titer plate, the sugar-binding specificity of a given lectin can be screened.
P r e p a r a t i o n of the L e c t i n - H o r s e r a d i s h Peroxidase Conjugate
Reagents L e c t i n , 5 nmol by subunit H R P , 20 nmo] N a I O 4 , 60 m M D i e t h y l e n e glycol, 160 m M N a B H 4 , 0.5% S a t u r a t e d a m m o n i u m sulfate Procedure. Briefly, 20 nmol o f H R P , dissolved in 75 p.l o f water, is r e a c t e d with 7 5 / z l o f 60 m M NaIO4 (freshly prepared) at 4 ° for 30 min; the r e a c t i o n is t e r m i n a t e d b y the addition o f 75/zl o f 160 m M diethylene glycol at r o o m t e m p e r a t u r e and i n c u b a t e d for 30 min. Five n a n o m o l e s o f lectin by subunit (the m o l a r ratio o f the lectin subunit to H R P is a b o u t 1 : 4), dissolved in 150/zl o f water, is mixed with the a b o v e solution and
[18]
S T R E P T A V I D I N - - B I O T I N Y L G L Y C O P E P T I D E - L E C T I N COMPLEX
255
dialyzed against 50 mM sodium carbonate, pH 9.6, at 4° overnight with gentle stirring. After the dialysis, 30/A of 0.5% NaBH4 is added to the dialyzed mixture, and the reaction is allowed to proceed at 4° for 2 hr. An equal volume of saturated ammonium sulfate is added to precipitate the lectin-HRP conjugate, leaving unreacted HRP in the supernatant. The lectin-HRP is collected by centrifugation, then suspended in 50% saturated ammonium sulfate or desalted by dialysis against deionized water and lyophilized for storage. The molar ratio of HRP to the lectin in the conjugates prepared usually is 1.5-1.8.
Detection of Glycopeptides in Chromatographic Peptide Maps
Reagents Buffer A, 50 mM sodium carbonate, pH 9.6, containing 0.02% sodium azide Buffer B, 50 mM sodium phosphate, pH 7.4, containing 0.05% Tween 20 Buffer C, 50 mM Tris-HC1 buffer, pH 7.4, containing 150 mM NaC1, 1 mM MgCI2, 1 mM MnCI2, and 1 mM CaCI 2 Buffer D, 50 mM citrate buffer, pH 4.4, containing 0.012% H202 Glycoprotein (asialofetuin, 3 mg) Streptavidin (Sigma Chemical Co., St. Louis, MO), 100/zg/ml (1.5 /zM) in buffer A Sulfo-NHSH-biotin, sulfosuccinimidyl 6-(biotinamido)hexanoate (Pierce Chemical Co., Rockford, IL), 3/zM in buffer A Lectins coupled to HRP: Con A (concanavalin A)-HRP, DSA (Datura stramonium agglutinin)-HRP, and PNA (peanut agglutinin)HRP, 1-2 units of HRP activity in I ml of buffer C (the total amount will vary, depending on the HRP activity in each lectin-HRP complex Bovine serum albumin (BSA), 1% in buffer A Sodium dodecyl sulfate (SDS), 5% solution 2,2'-Azinobis(3-ethylbenzthiazoline sulfonate) (ABTS) (Sigma), 0.3 mg in 1 ml of buffer D
Procedure Step 1: Preparation and Assay of Tryptic Peptides of Proteins. The approach is illustrated with the actual peptide mixture from asialofetuin (3 mg).4 The glycoproteins are reduced with dithiothreitol in 6 M guanidine hydrochloride, reacted with vinylpyridine, dialyzed, and digested with
256
ENZYMATIC AND AFFINITY METHODS
[18]
TPCK-treated trypsin. The digestion is stopped by adjustment of the reaction mixture to pH 3 and heating. An aliquot corresponding to 100 pmol of protein is applied to C~8 column and eluted. Fractions are collected either manually or automatically. The samples are lyophilized, dissolved in 20% acetonitrile, and stored at - 2 0 °. Step 2: Biotinylation of Proteolytic (Glyco)peptides. Ten-microliter aliquots of the HPLC fractions (containing 1-10 pmol sample) are diluted with buffer A and added to wells of a titer plate, mixed carefully with a 10/zl of sulfo-NHSH-biotin (30 pmol) solution, and incubated for 30 min at 25 °. The excess of sulfo-NHSH-biotin is used to maximize the yield of biotinylated (glyco)peptides.
Step 3: Coating of Streptavidin-Biotinylated (Glyco)peptide Complex. After biotinylation, 80/.d of streptavidin (2/zg, 120 pmol of subunits) is added to bind both biotinylated (glyco)peptides and free biotin derivatives, and the resulting conjugates are incubated for 3 hr at 25 ° to affect adherence to the wall of the wells. A molar ratio of streptavidin subunit to sulfoNHSH-biotin of 4 : 1 is used to make all the biotin derivatives complex to streptavidin. Step 4: Blocking with Bovine Serum Albumin. The plate is rinsed 3 times with buffer B, and 350/xl of 1% BSA solution is added to each well. Plates are incubated at 25 ° for 1.5 hr to coat any remaining sites on the wells. Step 5: Incubation with Lectin. The plate is again washed 3 times with buffer B; then 100/zl of lectin-HRP solution (1-2 units/ml at an HRP activity) is added to each well, and the plate is incubated at 25 ° for 1.5 hr. Step 6: Peroxidase Reaction. The plate is washed 5 times with buffer B, 100 /zl of substrate (ABTS) solution is added to each well, and the plate is incubated at 25 ° C for 10-60 min (longer incubations may be needed to detect low levels of binding). The reaction is stopped by the addition of 100 t~l of 5% SDS solution.
Step 7: Measurement of Extent of Binding of Glycopeptides to Lectin-Peroxidase. The absorbance at a wavelength of 405 nm is quantified with the use of a microplate reader.
Comments A representative assay for the glycopeptides in HPLC maps of the tryptic peptides from asialofetuin is presented in Fig. 2. It is well documented that there are three N-linked glycosylation sites designated N1, N2, and N3 on asialofetuin. 6 All of the N-linked oligosaccharides at the 6 M. G. Yet, C. C. Q. Chin, and F. Wold, J. Biol. Chem. 263, 111 (1988).
[18]
STREPTAVI DIN-BIOTINYLGLYCOPEPTIDE-LECTIN A
HPLC
10
27
COMPLEX
257
50
60
41
<
10
20
30
40
Elution Time (min) 1.5
B
+ DSA-HRP
N3 NI 24 N2
,,,,,,,,,,,,,,, <
0.6 - C
41 3
I~81
,,,,,I,,,,,,,,i,I
,11,,
37
+ PNA-HRP
o
43
. ~o
,I 5
10
15
20
25
30
35
I 40
45
Peak Number FIG. 2. (A) Reversed-phase HPLC of the tryptic peptides from asialofetuin (peaks 1-47 were collected). (B), (C) Analyses of the HPLC fractions for glycopeptides by the standard procedure described in the text with DSA-HRP (at an HRP activity of about 100 mU/weU) (B) or P N A - H R P (at an HRP activity of about 200 mU/well) (C) as probes. Each HPLC fraction was diluted 200-fold with 20% acetonitrile for the assay, and the enzyme reaction was carried out for 15 min in case B and for 60 min in case C. The peaks (24 and 37-41) that reacted positively with DSA-HRP are labeled with an " N , " the number to the right of " N " referring to the classification of the glycopeptide identified by amino acid sequence analyses (see text); those peaks (33, 35-38, 43, and 44) that reacted positively with P N A - H R P are labeled with an " O " ; the number above " N " and " O " refers to the number of the peak. The other peaks did not react either with DSA-HRP or PNA-HRP.
three sites contain tri- and tetraantennary structures which can react strongly with DSA after removal of sialic acid. 7 It was found in Fig. 2A,B that HPLC peaks 24, 37, 38, 39, 40, and 41 of the tryptic digest of asialofetuin gave positive reactions with DSA-HRP. Based on sequence 7 K. Yamashita, K. Totani, T. Ohkura, S. Takasaki, I. J. Goldstein, and A. Kobata, J. Biol. Chem. 262, 1602 (1987).
258
ENZYMATIC AND AFFINITY METHODS
[18]
analyses, 4 peaks 37, 38, and 40 correspond to glycopeptide N1, peak 24 to N2, and peaks 39 and 41 to N3. When the tryptic peptides were probed with PNA, which binds tightly to O-linked sugars, containing Gal(/313)GalNAc, seven positive peptides were found (Fig. 2C). Sequence analyses showed that all of them share a common peptide fragment, the Nterminal sequence of which was VTCTLFQTQPV, consistent with that of the known tryptic O-linked glycopeptide of asialofetuin on which there are three potential O-linked glycosylation sites. 8'9 For routine detection of glycopeptides in HPLC peptide maps of an unknown protein, the use of the set of lectins (Con A, DSA, and PNA) should permit the detection in a single experiment of a large number of different glycan structures known to be present in glycoproteins. 4 Con A is quite nonspecific in binding both oligomannose, hybrid, and even biantennary complex glycans. It does not bind tri- and tetraantennary structures, and therefore DSA, which recognizes the Gal(fll-4)GlcNAc structure, is included to cover the latter two types of glycans. PNA, which is specific for GaI(/31-3)GalNAc, a common component of O-linked glycans, is included to be able to screen for both O- and N-linked structures without sialic acid residues. For this reason the test described here should be used only with asialoglycoproteins. Pretreatment of the unknown protein to be assayed for the presence of carbohydrate with neuraminidase 3 and/or acid ~° to remove sialic acid is thus required. The nature of the reaction of the lectin with the N- or O-linked glycopeptides being detected by this method can be confirmed by inhibiting the reaction with an excess amount of a sugar inhibitor appropriate for the lectin, or by digestion of the biotinylated glycopeptides with N- or Oglycosidases.4 The assay is primarily a qualitative one. Because the extent of the biotinylation of the individual peptides may well be quite different and furthermore the lectins may have different affinities for the different oligosaccharide structures on different glycopeptides, quantitative conclusions are quite problematic. This system can also be used in the detection of glycoprotein and the investigation of glycan structures using several lectins of known specificity and glycosidases. An example is shown in Fig. 3. It is known that the glycoproteins ovalbumin, fetuin, asialofetuin, transferrin, and carboxypeptidase Y contain oligosaccharide structures which can bind to Con A 8 R. G. Spiro and V. D. Bhoyroo, J. Biol. Chem. 249, 5704 (1974). 9 K. M. Dziegielewska, W. M. Brown, S.-J. Casey, D. L. Christie, R. C. Foreman, R. M. Hill, and N. R. Saunders, J. Biol. Chem. 265, 4354 (1990). to R. G. Spiro, J. Biol. Chem. 235, 2860 (1960).
[18]
STREPTAVIDIN--BIOTINYLGEYCOPEPTIDE-LECTIN COMPLEX
~
0.6 t A
0.4 =
0.2
~
259
+ Con A-HRP 1. BSA 2. Ovalbumin 3. Glycopeptide (Fr. 27) 4. co-Acid Glycoprotein 5. Fetuin 6. Asialofetuin 7. Transferrin 8. Carboxypeptidase Y
0.4
0.2
0.0 1
2
3
4
5
6
7
8
Sample FIG. 3. (A) Detection of glycoproteins by the standard procedure described in the text. Approximately 1 pmol of each sample was used to coat the well of the plate. Con A - H R P at an HRP activity of 100 mU/well was used. The enzyme reaction was carried out for 15 min. It was observed that the glycoprotein~ ovalbumin, fetuin, asialofetuin, transferrin, and carboxypeptidase Y reacted with Con A - H R P significantly. The glycopeptide (fraction 27) (from HPLC tryptic maps of ovalbumin) 4 was used as a positive control, BSA as a negative control, and a-acid glycoprotein, which has no glycan structures that react with Con A, as another negative control. (B) Determination of the structures of the glycoprotein carbohydrate chain with G N A - H R P (GNA, Galanthus nivalis agglutinin) as a probe. The experiments were carried out under the conditions used in (A) with G N A - H R P instead of Con A-HRP. As shown, only carboxypeptidase Y reacted positively with GNA-HRP.
to various extents. 4'1°-12 It was found (Fig. 3A) that all of them reacted significantly with Con A - H R P . It was noted that nonbiotinylated glycoproteins can also be coated to the well of a plate, but this nonspecific binding differs greatly for different glycoproteins (data not shown). It has been also established that GNA (Galanthus nivalis agglutinin) only recognizes highly branched yeast mannose-type oligosaccharides and does not react with most other glycans.13 As is shown in Fig. 3B, only carboxypeptidase II K. Yamashita, N. Koide, T. Endo, Y. Iwaki, and A. Kobata, J. Biol. Chem. 264, 2415 (1989). t: p. K. Tsai, J. Frever, and C. E. Ballou, J. Biol. Chem. 259, 3805 (1984). t3 N. Shibuya, I. J. Goldstein, E. M. Van Damme, and W. J. Peumans, J. Biol. Chem. 263, 728 (1988).
260
[18]
ENZYMATIC AND AFFINITY METHODS
Hybrid Type
High Mannose Type o--q,
o~
o"
•7
o--q,
o~
•
1. Man6-R
2. ManS-R
•/
o"
0
0
3. GIeNAeManS-R
o~
o~
o= g
r~= o= o "
0
4. GleNAc2Man5-R 5. GleNAe3ManS-R
0
6. GalGleNAe3ManS-R
Complex Type 07"
0'4
l+
,0~-~0 f
o
0
l
0
7. GleNAcMan3-R
0
8. GleNAe2Man3-R
9. GIcNAc3-4Man3-R
D~O.t •
D~O/
• )
m'--I
vl~ 0
l
D ~" 0
10. Gal2GleNAc2Man3-R
11. SiaGal2GlcNAe2Man3-R
.r--}
?
E]~' 0
12. Sia2Oal2 GlcNAc2Man3-R
R=6-(biotinamido)hexanoyl-Asn-GleNAc2; e, Mannose; o, N-Acetylglucosamine; [], Galactose;•, Sialicacid. Code:
Bond position
x-4
Configuration
a ~
[~
1Flo. 4. Structures of the 12 biotinylglycans used in the study of oligosaccharide-binding specificity of Con A. All were bound to streptavidin. The anomeric configuration and the linkage position are encoded as indicated.
Y, which contains highly branched yeast mannose-type oligosaccharides,12 reacted positively with G N A - H R P .
Assay of Oligosaccharide-Binding Specificity of Lectin
Reagents Streptavidin, 30 pmol/ml (2/zg/ml) in buffer A Series of biotinylglycans, each at 150 pmol/ml in buffer A (the structures of 12 biotinylglycans used here are shown in Fig. 4) The other reagents are the same as those used in the procedure above.
Procedure Step 1: Preparation and Coating of Streptavidin-Biotinylglycan Complex to Microplates. Apply a 50-/zl aliquot of streptavidin (0.1 /zg, 1.5 pmol) to each well of a microtiter plate, followed by the addition of 50/zl of a selected biotinylglycan (7.5 pmol) solution, and incubate the mixture at 25° for at least 3 hr; overnight is often convenient. As 1 tool of streptavidin can bind 4 mol of biotinylglycans, a molar ratio of streptavidin to
[18]
STREPTAVIDIN-BIOTINYLGLYCOPEPTIDE-LECTIN 1
2
3
4
5
6
7
8
9
COMPLEX 10
11
261 12
A
FIG. 5. Typical picture of binding of Con A - H R P (row A) and Con A - H R P plus amethyl mannoside (row B) to the 12 streptavidin-biotinylglycans with an aminohexanoyl group, coated to wells A I - A I 2 and B1-B12 of a plate. The structures of the 12 glycan derivatives are shown in Fig. 4. The assays were carried out according to the standard procedure described in the text. The inhibitor for Con A sugar binding, 100 mM a-methyl mannoside dissolved in the Con A - H R P solution, was added to each well from B1 to BI2. The enzyme reactions were carried out for 15 min.
biotinylglycan of 1 : 5 is used in order to ensure complete saturation of streptavidin by the biotinylglycan. The subsequent steps are the same as those (steps 4-7) involved in the detection of glycopeptides in HPLC peptide maps (see above). Comments
A representative assay of the oligosaccharide-binding specificity of Con A is presented in Fig. 5. The glycan structures involved are given in Fig. 4. The level of Con A binding to the high-mannose type of oligosaccharides Man6-R and M a n ; R and hybrid GlcNAcMans-R is highest. The extent of binding to the other three hybrid structures is decreased, consistent with the previous finding that when GlcNAcMans-R is bisected with a fll,4-linked N-acetylglucosamine residue to the fl-linked mannose the binding is weakened/TM The level of Con A binding to the complex type GlcNAcMan3-R and GlcNAczMan3-R is similar to that for GIcNAc2 Mans-R. The compounds Gal2GlcNAczMan3-R, SiaGalzGlcNAczMan3-R, SiazGal2GlcNAczMan3-R, and GlcNA%.4Man3-R with a bisecting N-acetylglucosamine residue gave lower color with Con A, showing that the affinity of the lectin for these glycans is very weak. As predicted, the addition of a-methyl mannoside strongly inhibited the color formation (Fig. 5B). This documents the key role of the interaction between Con A-HRP and oligosaccharides in the assay. The sugar-binding specificities of a given lectin can be conveniently screened both qualitatively and quantitatively by using streptavidin14j. U. Baenzinger and D. Fiete, J. Biol. Chem. 254, 2400 (1979).
262
ENZYMATIC AND AFFINITY METHODS
[18]
biotinylglycans as probes. The error of this method usually is less than _+20%. 15
Acknowledgments Part of this work was supported by a research Grant (AU-916) from the Robert A. Welch Foundation to Dr. Finn Wold. We thank Dr. Finn Wold for helpful comments and discussions.
15 C. F. Brewer and L. Bhattacharyya, J. Biol. Chem. 261, 7306 (1986).
STREPTAVIDIN--BIOTINYLGLYCOPEPTIDE--LECTIN COMPLEX
253
ing site (D. R. Bundle et al., Auzanneau and Siguskjold). Mild elution conditions are most desirable, and for one case, Se155.4 histidine residues in the binding site render this antibody binding particularly sensitive to pH changes. Elution with an inexpensive, readily available ligand is a preferred strategy when bound antibody may be economically displaced by a small structure.
[18] S t r e p t a v i d i n - B i o t i n y l g l y c o p e p t i d e - L e c t i n C o m p l e x in Detection of Glycopeptides and Determination of Lectin Specificity
By MING-CHUAN SHAO and CHRISTOPHERC. Q. CHIN Introduction Streptavidin-biotinylglycopeptide-lectin complexes can be used in the detection of glycopeptides and the determination of lectin specificity at the picomole level. The method is based on the high affinity between streptavidin and biotin derivatives, 1,2 and between lectins and their complementary sugars, 3 and on the ability of streptavidin to adhere to microtiter plate wells 4'5 (Fig. 1). For the detection of glycopeptides, each unknown (glyco)peptide [from a high-performance liquid chromatography (HPLC) run, for example] is first biotinylated and complexed with streptavidin, each conjugate is then coated to the well of a titer plate, and lectin coupled to horseradish peroxidase (HRP) is added. After incubation, the wells are thoroughly washed and probed for the presence of the peroxidase. A positive test shows the presence of a glycan complementary to the lectin u s e d . 4 Based on the same principle, when a series of well-characterized biotinylglycans complexed with streptavidin is precoated in the wells of a titer plate, the sugar-binding specificity of unknown lectins, conjugated to peroxidase, can also be conveniently screened. 5
i N. M. Green, Adv. Protein Chem. 29, 85 (1975). 2 V. J. Chen and F. Wold, Biochemistry 25, 939 (1986). 3 I. J. Goldstein and C. E. Hayes, Adv. Carbohydr. Chem. Biochem. 35, 127 (1978). 4 M. C. Shao and C. C. Q. Chin, Anal. Biochem. 207, 100 (1992). 5 M. C. Shao, Anal. Bioehem. 205, 77 (1992).
METHODS IN ENZYMOLOGY,VOL. 247
Copyright © 1994by Academic Press, Inc. All rights of reproduction in any form reserved.
254
ENZYMATIC AND AFFINITY METHODS
[18]
ABTS ABTS reduced ' N . ~¢oxidized colorless '~..~ j ~ green
°reradish1 peroxidase
ectin
~-~
_
Oligosacchande Asn, Ser, Thr or peptide
Hexanoyl group -- Biotin
Streptavidin Polystyrene surface
l
FIG. 1. Principle for the detection of glycopeptides and the determination of lectin specificity with streptavidin-biotinylglycopeptide-lectin complex as a tool. The (glyco)peptides from HPLC peptide maps can be biotinylated, complexed with streptavidin, immobilized on a titer plate, and then detected by the activity of HRP coupled covalently to a lectin, which in turn can bind to specific glycans. Similarly, if a series of well-characterized biotinylglycans complexed with streptavidin are precoated in the wells of a titer plate, the sugar-binding specificity of a given lectin can be screened.
P r e p a r a t i o n of the L e c t i n - H o r s e r a d i s h Peroxidase Conjugate
Reagents L e c t i n , 5 nmol by subunit H R P , 20 nmo] N a I O 4 , 60 m M D i e t h y l e n e glycol, 160 m M N a B H 4 , 0.5% S a t u r a t e d a m m o n i u m sulfate Procedure. Briefly, 20 nmol o f H R P , dissolved in 75 p.l o f water, is r e a c t e d with 7 5 / z l o f 60 m M NaIO4 (freshly prepared) at 4 ° for 30 min; the r e a c t i o n is t e r m i n a t e d b y the addition o f 75/zl o f 160 m M diethylene glycol at r o o m t e m p e r a t u r e and i n c u b a t e d for 30 min. Five n a n o m o l e s o f lectin by subunit (the m o l a r ratio o f the lectin subunit to H R P is a b o u t 1 : 4), dissolved in 150/zl o f water, is mixed with the a b o v e solution and
[18]
S T R E P T A V I D I N - - B I O T I N Y L G L Y C O P E P T I D E - L E C T I N COMPLEX
255
dialyzed against 50 mM sodium carbonate, pH 9.6, at 4° overnight with gentle stirring. After the dialysis, 30/A of 0.5% NaBH4 is added to the dialyzed mixture, and the reaction is allowed to proceed at 4° for 2 hr. An equal volume of saturated ammonium sulfate is added to precipitate the lectin-HRP conjugate, leaving unreacted HRP in the supernatant. The lectin-HRP is collected by centrifugation, then suspended in 50% saturated ammonium sulfate or desalted by dialysis against deionized water and lyophilized for storage. The molar ratio of HRP to the lectin in the conjugates prepared usually is 1.5-1.8.
Detection of Glycopeptides in Chromatographic Peptide Maps
Reagents Buffer A, 50 mM sodium carbonate, pH 9.6, containing 0.02% sodium azide Buffer B, 50 mM sodium phosphate, pH 7.4, containing 0.05% Tween 20 Buffer C, 50 mM Tris-HC1 buffer, pH 7.4, containing 150 mM NaC1, 1 mM MgCI2, 1 mM MnCI2, and 1 mM CaCI 2 Buffer D, 50 mM citrate buffer, pH 4.4, containing 0.012% H202 Glycoprotein (asialofetuin, 3 mg) Streptavidin (Sigma Chemical Co., St. Louis, MO), 100/zg/ml (1.5 /zM) in buffer A Sulfo-NHSH-biotin, sulfosuccinimidyl 6-(biotinamido)hexanoate (Pierce Chemical Co., Rockford, IL), 3/zM in buffer A Lectins coupled to HRP: Con A (concanavalin A)-HRP, DSA (Datura stramonium agglutinin)-HRP, and PNA (peanut agglutinin)HRP, 1-2 units of HRP activity in I ml of buffer C (the total amount will vary, depending on the HRP activity in each lectin-HRP complex Bovine serum albumin (BSA), 1% in buffer A Sodium dodecyl sulfate (SDS), 5% solution 2,2'-Azinobis(3-ethylbenzthiazoline sulfonate) (ABTS) (Sigma), 0.3 mg in 1 ml of buffer D
Procedure Step 1: Preparation and Assay of Tryptic Peptides of Proteins. The approach is illustrated with the actual peptide mixture from asialofetuin (3 mg).4 The glycoproteins are reduced with dithiothreitol in 6 M guanidine hydrochloride, reacted with vinylpyridine, dialyzed, and digested with
256
ENZYMATIC AND AFFINITY METHODS
[18]
TPCK-treated trypsin. The digestion is stopped by adjustment of the reaction mixture to pH 3 and heating. An aliquot corresponding to 100 pmol of protein is applied to C~8 column and eluted. Fractions are collected either manually or automatically. The samples are lyophilized, dissolved in 20% acetonitrile, and stored at - 2 0 °. Step 2: Biotinylation of Proteolytic (Glyco)peptides. Ten-microliter aliquots of the HPLC fractions (containing 1-10 pmol sample) are diluted with buffer A and added to wells of a titer plate, mixed carefully with a 10/zl of sulfo-NHSH-biotin (30 pmol) solution, and incubated for 30 min at 25 °. The excess of sulfo-NHSH-biotin is used to maximize the yield of biotinylated (glyco)peptides.
Step 3: Coating of Streptavidin-Biotinylated (Glyco)peptide Complex. After biotinylation, 80/.d of streptavidin (2/zg, 120 pmol of subunits) is added to bind both biotinylated (glyco)peptides and free biotin derivatives, and the resulting conjugates are incubated for 3 hr at 25 ° to affect adherence to the wall of the wells. A molar ratio of streptavidin subunit to sulfoNHSH-biotin of 4 : 1 is used to make all the biotin derivatives complex to streptavidin. Step 4: Blocking with Bovine Serum Albumin. The plate is rinsed 3 times with buffer B, and 350/xl of 1% BSA solution is added to each well. Plates are incubated at 25 ° for 1.5 hr to coat any remaining sites on the wells. Step 5: Incubation with Lectin. The plate is again washed 3 times with buffer B; then 100/zl of lectin-HRP solution (1-2 units/ml at an HRP activity) is added to each well, and the plate is incubated at 25 ° for 1.5 hr. Step 6: Peroxidase Reaction. The plate is washed 5 times with buffer B, 100 /zl of substrate (ABTS) solution is added to each well, and the plate is incubated at 25 ° C for 10-60 min (longer incubations may be needed to detect low levels of binding). The reaction is stopped by the addition of 100 t~l of 5% SDS solution.
Step 7: Measurement of Extent of Binding of Glycopeptides to Lectin-Peroxidase. The absorbance at a wavelength of 405 nm is quantified with the use of a microplate reader.
Comments A representative assay for the glycopeptides in HPLC maps of the tryptic peptides from asialofetuin is presented in Fig. 2. It is well documented that there are three N-linked glycosylation sites designated N1, N2, and N3 on asialofetuin. 6 All of the N-linked oligosaccharides at the 6 M. G. Yet, C. C. Q. Chin, and F. Wold, J. Biol. Chem. 263, 111 (1988).
[18]
STREPTAVI DIN-BIOTINYLGLYCOPEPTIDE-LECTIN A
HPLC
10
27
COMPLEX
257
50
60
41
<
10
20
30
40
Elution Time (min) 1.5
B
+ DSA-HRP
N3 NI 24 N2
,,,,,,,,,,,,,,, <
0.6 - C
41 3
I~81
,,,,,I,,,,,,,,i,I
,11,,
37
+ PNA-HRP
o
43
. ~o
,I 5
10
15
20
25
30
35
I 40
45
Peak Number FIG. 2. (A) Reversed-phase HPLC of the tryptic peptides from asialofetuin (peaks 1-47 were collected). (B), (C) Analyses of the HPLC fractions for glycopeptides by the standard procedure described in the text with DSA-HRP (at an HRP activity of about 100 mU/weU) (B) or P N A - H R P (at an HRP activity of about 200 mU/well) (C) as probes. Each HPLC fraction was diluted 200-fold with 20% acetonitrile for the assay, and the enzyme reaction was carried out for 15 min in case B and for 60 min in case C. The peaks (24 and 37-41) that reacted positively with DSA-HRP are labeled with an " N , " the number to the right of " N " referring to the classification of the glycopeptide identified by amino acid sequence analyses (see text); those peaks (33, 35-38, 43, and 44) that reacted positively with P N A - H R P are labeled with an " O " ; the number above " N " and " O " refers to the number of the peak. The other peaks did not react either with DSA-HRP or PNA-HRP.
three sites contain tri- and tetraantennary structures which can react strongly with DSA after removal of sialic acid. 7 It was found in Fig. 2A,B that HPLC peaks 24, 37, 38, 39, 40, and 41 of the tryptic digest of asialofetuin gave positive reactions with DSA-HRP. Based on sequence 7 K. Yamashita, K. Totani, T. Ohkura, S. Takasaki, I. J. Goldstein, and A. Kobata, J. Biol. Chem. 262, 1602 (1987).
258
ENZYMATIC AND AFFINITY METHODS
[18]
analyses, 4 peaks 37, 38, and 40 correspond to glycopeptide N1, peak 24 to N2, and peaks 39 and 41 to N3. When the tryptic peptides were probed with PNA, which binds tightly to O-linked sugars, containing Gal(/313)GalNAc, seven positive peptides were found (Fig. 2C). Sequence analyses showed that all of them share a common peptide fragment, the Nterminal sequence of which was VTCTLFQTQPV, consistent with that of the known tryptic O-linked glycopeptide of asialofetuin on which there are three potential O-linked glycosylation sites. 8'9 For routine detection of glycopeptides in HPLC peptide maps of an unknown protein, the use of the set of lectins (Con A, DSA, and PNA) should permit the detection in a single experiment of a large number of different glycan structures known to be present in glycoproteins. 4 Con A is quite nonspecific in binding both oligomannose, hybrid, and even biantennary complex glycans. It does not bind tri- and tetraantennary structures, and therefore DSA, which recognizes the Gal(fll-4)GlcNAc structure, is included to cover the latter two types of glycans. PNA, which is specific for GaI(/31-3)GalNAc, a common component of O-linked glycans, is included to be able to screen for both O- and N-linked structures without sialic acid residues. For this reason the test described here should be used only with asialoglycoproteins. Pretreatment of the unknown protein to be assayed for the presence of carbohydrate with neuraminidase 3 and/or acid ~° to remove sialic acid is thus required. The nature of the reaction of the lectin with the N- or O-linked glycopeptides being detected by this method can be confirmed by inhibiting the reaction with an excess amount of a sugar inhibitor appropriate for the lectin, or by digestion of the biotinylated glycopeptides with N- or Oglycosidases.4 The assay is primarily a qualitative one. Because the extent of the biotinylation of the individual peptides may well be quite different and furthermore the lectins may have different affinities for the different oligosaccharide structures on different glycopeptides, quantitative conclusions are quite problematic. This system can also be used in the detection of glycoprotein and the investigation of glycan structures using several lectins of known specificity and glycosidases. An example is shown in Fig. 3. It is known that the glycoproteins ovalbumin, fetuin, asialofetuin, transferrin, and carboxypeptidase Y contain oligosaccharide structures which can bind to Con A 8 R. G. Spiro and V. D. Bhoyroo, J. Biol. Chem. 249, 5704 (1974). 9 K. M. Dziegielewska, W. M. Brown, S.-J. Casey, D. L. Christie, R. C. Foreman, R. M. Hill, and N. R. Saunders, J. Biol. Chem. 265, 4354 (1990). to R. G. Spiro, J. Biol. Chem. 235, 2860 (1960).
[18]
STREPTAVIDIN--BIOTINYLGEYCOPEPTIDE-LECTIN COMPLEX
~
0.6 t A
0.4 =
0.2
~
259
+ Con A-HRP 1. BSA 2. Ovalbumin 3. Glycopeptide (Fr. 27) 4. co-Acid Glycoprotein 5. Fetuin 6. Asialofetuin 7. Transferrin 8. Carboxypeptidase Y
0.4
0.2
0.0 1
2
3
4
5
6
7
8
Sample FIG. 3. (A) Detection of glycoproteins by the standard procedure described in the text. Approximately 1 pmol of each sample was used to coat the well of the plate. Con A - H R P at an HRP activity of 100 mU/well was used. The enzyme reaction was carried out for 15 min. It was observed that the glycoprotein~ ovalbumin, fetuin, asialofetuin, transferrin, and carboxypeptidase Y reacted with Con A - H R P significantly. The glycopeptide (fraction 27) (from HPLC tryptic maps of ovalbumin) 4 was used as a positive control, BSA as a negative control, and a-acid glycoprotein, which has no glycan structures that react with Con A, as another negative control. (B) Determination of the structures of the glycoprotein carbohydrate chain with G N A - H R P (GNA, Galanthus nivalis agglutinin) as a probe. The experiments were carried out under the conditions used in (A) with G N A - H R P instead of Con A-HRP. As shown, only carboxypeptidase Y reacted positively with GNA-HRP.
to various extents. 4'1°-12 It was found (Fig. 3A) that all of them reacted significantly with Con A - H R P . It was noted that nonbiotinylated glycoproteins can also be coated to the well of a plate, but this nonspecific binding differs greatly for different glycoproteins (data not shown). It has been also established that GNA (Galanthus nivalis agglutinin) only recognizes highly branched yeast mannose-type oligosaccharides and does not react with most other glycans.13 As is shown in Fig. 3B, only carboxypeptidase II K. Yamashita, N. Koide, T. Endo, Y. Iwaki, and A. Kobata, J. Biol. Chem. 264, 2415 (1989). t: p. K. Tsai, J. Frever, and C. E. Ballou, J. Biol. Chem. 259, 3805 (1984). t3 N. Shibuya, I. J. Goldstein, E. M. Van Damme, and W. J. Peumans, J. Biol. Chem. 263, 728 (1988).
260
[18]
ENZYMATIC AND AFFINITY METHODS
Hybrid Type
High Mannose Type o--q,
o~
o"
•7
o--q,
o~
•
1. Man6-R
2. ManS-R
•/
o"
0
0
3. GIeNAeManS-R
o~
o~
o= g
r~= o= o "
0
4. GleNAc2Man5-R 5. GleNAe3ManS-R
0
6. GalGleNAe3ManS-R
Complex Type 07"
0'4
l+
,0~-~0 f
o
0
l
0
7. GleNAcMan3-R
0
8. GleNAe2Man3-R
9. GIcNAc3-4Man3-R
D~O.t •
D~O/
• )
m'--I
vl~ 0
l
D ~" 0
10. Gal2GleNAc2Man3-R
11. SiaGal2GlcNAe2Man3-R
.r--}
?
E]~' 0
12. Sia2Oal2 GlcNAc2Man3-R
R=6-(biotinamido)hexanoyl-Asn-GleNAc2; e, Mannose; o, N-Acetylglucosamine; [], Galactose;•, Sialicacid. Code:
Bond position
x-4
Configuration
a ~
[~
1Flo. 4. Structures of the 12 biotinylglycans used in the study of oligosaccharide-binding specificity of Con A. All were bound to streptavidin. The anomeric configuration and the linkage position are encoded as indicated.
Y, which contains highly branched yeast mannose-type oligosaccharides,12 reacted positively with G N A - H R P .
Assay of Oligosaccharide-Binding Specificity of Lectin
Reagents Streptavidin, 30 pmol/ml (2/zg/ml) in buffer A Series of biotinylglycans, each at 150 pmol/ml in buffer A (the structures of 12 biotinylglycans used here are shown in Fig. 4) The other reagents are the same as those used in the procedure above.
Procedure Step 1: Preparation and Coating of Streptavidin-Biotinylglycan Complex to Microplates. Apply a 50-/zl aliquot of streptavidin (0.1 /zg, 1.5 pmol) to each well of a microtiter plate, followed by the addition of 50/zl of a selected biotinylglycan (7.5 pmol) solution, and incubate the mixture at 25° for at least 3 hr; overnight is often convenient. As 1 tool of streptavidin can bind 4 mol of biotinylglycans, a molar ratio of streptavidin to
[18]
STREPTAVIDIN-BIOTINYLGLYCOPEPTIDE-LECTIN 1
2
3
4
5
6
7
8
9
COMPLEX 10
11
261 12
A
FIG. 5. Typical picture of binding of Con A - H R P (row A) and Con A - H R P plus amethyl mannoside (row B) to the 12 streptavidin-biotinylglycans with an aminohexanoyl group, coated to wells A I - A I 2 and B1-B12 of a plate. The structures of the 12 glycan derivatives are shown in Fig. 4. The assays were carried out according to the standard procedure described in the text. The inhibitor for Con A sugar binding, 100 mM a-methyl mannoside dissolved in the Con A - H R P solution, was added to each well from B1 to BI2. The enzyme reactions were carried out for 15 min.
biotinylglycan of 1 : 5 is used in order to ensure complete saturation of streptavidin by the biotinylglycan. The subsequent steps are the same as those (steps 4-7) involved in the detection of glycopeptides in HPLC peptide maps (see above). Comments
A representative assay of the oligosaccharide-binding specificity of Con A is presented in Fig. 5. The glycan structures involved are given in Fig. 4. The level of Con A binding to the high-mannose type of oligosaccharides Man6-R and M a n ; R and hybrid GlcNAcMans-R is highest. The extent of binding to the other three hybrid structures is decreased, consistent with the previous finding that when GlcNAcMans-R is bisected with a fll,4-linked N-acetylglucosamine residue to the fl-linked mannose the binding is weakened/TM The level of Con A binding to the complex type GlcNAcMan3-R and GlcNAczMan3-R is similar to that for GIcNAc2 Mans-R. The compounds Gal2GlcNAczMan3-R, SiaGalzGlcNAczMan3-R, SiazGal2GlcNAczMan3-R, and GlcNA%.4Man3-R with a bisecting N-acetylglucosamine residue gave lower color with Con A, showing that the affinity of the lectin for these glycans is very weak. As predicted, the addition of a-methyl mannoside strongly inhibited the color formation (Fig. 5B). This documents the key role of the interaction between Con A-HRP and oligosaccharides in the assay. The sugar-binding specificities of a given lectin can be conveniently screened both qualitatively and quantitatively by using streptavidin14j. U. Baenzinger and D. Fiete, J. Biol. Chem. 254, 2400 (1979).
262
ENZYMATIC AND AFFINITY METHODS
[18]
biotinylglycans as probes. The error of this method usually is less than _+20%. 15
Acknowledgments Part of this work was supported by a research Grant (AU-916) from the Robert A. Welch Foundation to Dr. Finn Wold. We thank Dr. Finn Wold for helpful comments and discussions.
15 C. F. Brewer and L. Bhattacharyya, J. Biol. Chem. 261, 7306 (1986).