- Email: [email protected]
[15] Affinity chromatography of oligosaccharides on Psathyrella velutina lectin column
228
ENZYMATIC AND AFFINITY METHODS
[15]
[15] A f f i n i t y C h r o m a t o g r a p h y o f O l i g o s a c c h a r i d e s o n Psathyrella velutina L e c t i n C o l u m n By AKIRA KOBATA, NAOHISA KOCHIBE,
and
TAMAO ENDO
Introduction Immobilized lectins have been used successfully for many years to isolate glycoconjugates with specific sugar structures. Researchers in a number of laboratories have begun to realize the potential oflectin columns for the analysis of sugar chains in glycoconjugates; serial lectin column chromatography in particular affords a simple and very sensitive method for fractionating various oligosaccharides. 1'z This chapte r describes the use ofa mushroom-lectin column (derived from Psathyrella velutina lectin, PVL) that recognizes and interacts with terminal N-acetylglucosamine residue. 3,4 Methods
Purification of Psathyrella velutina Lectin Fruiting bodies of P. velutina (100 g) collected in the region of Gunma Prefecture (Japan) are homogenized with 1 liter of 145 mM NaCI in 5 mM sodium phosphate buffer, pH 7.2 (PBS), containing 5 mM ethylenediaminetetraacetic acid (EDTA) and 0.5 mM phenylmethylsulfonyl fluoride. The homogenate is centrifuged at 20,000 g for 20 min at 4°, and the pellet is extracted with 500 ml of fresh extraction buffer. To the combined extracts is added dropwise 20% (v/v) acetic acid with stirring, to adjust the solution to pH 4.0. After standing for several hours, the insoluble material is removed by centrifugation at 20,000 g for 20 min at 4°, and the supernatant is neutralized with 1 N NaOH. The acid-treated extract is passed through a chitin column (3.5 x 30 cm) at a flow rate of 60 ml/ hr, and the column is washed with extraction buffer thoroughly. The bound material is then eluted with 50 mM N-acetylglucosamine in PBS containing 10% (v/v) glycerol. The eluate is concentrated by ultrafiltration through an Advantec Q0100 membranes (Toyo Roshi, Tokyo, Japan). I R. K. Merkle and R. D. C u m m i n g s , this series, Vol. 138, p. 232. 2 A. K o b a t a and T. Endo, J. Chromatogr. 597, 111 (1992). 3 N. Kochibe and K. L. Matta, .I. Biol. Chem. 264, 173 (1989). 4 T. Endo, H. Ohbayashi, K. K a n a z a w a , N. Kochibe, and A. Kobata, J. Biol. Chem. 267, 707 (1992).
METHODS IN ENZYMOLOGY,VOL. 247
Copyright © 1994by AcademicPress, Inc. All rights of reproduction in any form reserved.
[15]
Psathyrella velutina LECTIN AFFINITYCOLUMN
229
The concentrate is sequentially applied to serially connected columns of DEAE-cellulofine (Chisso, Tokyo, Japan, 1.7 x 25 cm) and CM-Sepharose CL-6B (Pharmacia LKB Biotechnology, Uppsala, Sweden, 1.7 x 25 cm) equilibrated with 10 mM sodium phosphate buffer, pH 7.0, containing 10% (v/v) glycerol. After washing thoroughly with the same solution the columns are disconnected, and the material bound to the CM-Sepharose CL-6B column is eluted with 0.4 M NaCI. Finally, the lectin is rechromatographed on the chitin column (1.5 x 20 cm) which has been equilibrated with PBS-glycerol. The bound lectin is eluted with 50 mM N-acetylglucosamine.
Preparation of Lectin Column Purified PVL thus obtained is coupled to Affi-Gel 10 (Bio-Rad Laboratories, Richmond, CA) as follows. The activated gel, which has been washed with 0.1 M NaHCO3, is added to a coupling mixture containing the purified PVL (5 mg/ml), 0.15 M NaC1, 10% (v/v) glycerol, and 20 mM N-acetylglucosamine. The suspension is stirred gently overnight at 4° and then filtered. The gel is resuspended in 1 M ethanolamine, allowed to stand for 2 hr at room temperature, and then washed with 10 mM TrisHCI buffer, pH 7.4, containing 140 mM NaCI. The amount of PVL bound to 1 ml of Affi-Gel 10 is estimated to be approximately 4.4 mg.
Affinity Chromatography on Lectin Column Tritium-labeled oligosaccharides [5-10 x 102 counts/min (cpm), 25-50 pmol] dissolved in 100 t~l of water are applied to a column (8 mm inner diameter) containing 1 ml of PVL-Affi-Gel 10, previously equilibrated with 10 mM Tris-HCl buffer, pH 7.4, containing 0.1 M NaCI, 1 mM CaCI2, 1 mM MgC1z , and 1 mM MnCI2, and allowed to stand at room temperature for 30 min. Oligosaccharides bound to the column are then eluted with 10 ml of the same buffer, followed by buffer containing 1 mM N-acetylglucosamine. Fractions of 1.0 ml are collected at a flow rate of 15 ml/hr, and the radioactivity in each fraction is determined by a liquid scintillation method. Recoveries of oligosaccharides based on radioactivities ranges from 98 to 100%.
Behaoior of Human Milk Oligosaccharides Although oligosaccharide I in Table I is passed through the column (Fig. 1A), oligosaccharide II remains bound to the column and is eluted with the buffer containing 1 mM N-acetylglucosamine (Fig. 1B). In contrast, isomeric oligosaccharide lI! is only slowed in the column (Fig. 1C).
230
ENZYMATIC AND AFFINITY METHODS
[15]
TABLE I STRUCTURESOF RADIOACTIVEOLIGOSACCHARIDES Oligosaccharide I
11 III IV
Structure a Gal/31--~4GlcNAc/31---~3Gal/31---~4GlCoT GIcNAc/31--~3Gal/31---~4GlCoT GlcNAc/31----~6Gal/3l----~4GlCoT GlcNAc/31 6GalB 1---~4GICoT
V
GIcNAcB1 Gal¢l1---~4GlcNAcB1----~2Mano~l N ManB I--~4GlcNAc~1--~4GIcNAcoT
V1
GalB1---~4GlcNAcB1---~2Man~1 GIcNAcB1---~2Man~1 6ManB 1---~4GlcNAc/31----~4GIcNAcoT
VII
GIcNAcBI--~2Man~I Man~ 1 6 ManB 1----~4GIcNAcBI---~4GIcNACoT Mano~l ManB 1--~4GIcNAcB1---~4GIcNACoT GIcNAc¢tI----~4GIcNACoT
VIII IX X
GalB I---~4GIcNAcB1---~2Man~ GalBI---~4GIcNAcB 1---~2Man~11 6 ManB1---~4GlcNAc~I----~4GIcNAcoT
Xl
XlI
GIcNAcB 1---~2Mano~l GIcNAcB l---~2Manc~l ~ManB 1---~4GlcNAcB1---~4GlcNAcor S GalB1----~4GlcNAc¢I1--~2Man~1 GIcNAc~ GlcNAcB 1----~2Mano~l
4 ManB 1---~4GlcNAcBI--~4GIcNACoT
GIcNAc/~1--*2Man~1
Psathyrella velutina LECTIN AFFINITY COLUMN
[15]
231
TABLE I (continued) Oligosaccharide
Structure a
XIII
GlcNAcfl 1 Gal/3l--*4GlcNAcfl l--*2Mana 1
XIV
4
IMan¢l 1--*4GlcNAcB1-~4GlcNAcox 7 GlcN Ac~ l---->2Mana1 GlcNAcfl~ GlcNAcfll--~2Manal
4 ~ManB l-->4GlcNAcBl---~4GlcNAcoT
XV
Galfl 1---*4GlcNAcfll---~2Mana1 GlcNAc/3 l--*2Mana 1 ~Man~ 1--+4GlcNAcfl1---~4GIcNAcoT Man~l Manc~1
XVl
IManfl l~4GlcNAcfl 1--~4GIcNAcoT XVII
S GlcNAcfl l---,2Man~1 Mant~ 1 ~Manfl I--~4GIcNAcl31---*4GIcNAcoT /i
XVlll
GlcNAcfll--~4Man~l GIcNAcfl1---~6ManotI I Manfl 1-->4GlcNAcfl1----~4GIcNAcoT
XlX
Mana 1
Man~l
S
6Manal Man~l/~3 "~63Manfl 1---~4GIcNAcB1--~4GIcNACoT S GlcNAcI31--, 2Manaa 1
(continued)
232
ENZYMATIC AND AFFINITY METHODS
[15]
TABLE I (continued) Oligosaccharide XX
Structure a Galfl1--~4GlcNAcfl1---~2 Mana 1 ,~ 36 Manfl 1--~4GlcNAcfl l--~4GlcNAco~r
Galfl1--~4GIcNAcB1N 4 2 Mana 1 XXI
Galfll--*4GlcNAcfll Galfl 1-~4GlcNAcfl1
XXII
6Manal 72 "~6 Galfll--~4GlcNAcfl l" 3Manfl 1---~4GlcNAcfll--~4GlcNAcoT S Galfl 1-~4GlcNAcfl1---~2 Manaa 1 Galfl 1---~4GlcNAc/31 Manal Galfll--~4GlcNAcB1
6
Galfll--~4GlcNAcfllN 4Man M .,~3 L XXIII
Manfl l----~4GlcNAcfl1---~4GlcNAcoz
Gal¢l1---*4GIcNAcB1 GlcNAcfl1-~ 2 Man~ 1 GIcNAcfll.. 4
7 36 Manfl 1---~4GlcNAcfl1--~4GlcNAcoT
2 Manal' XXIV
GlcNAcfll GlcNAcfll 6ManM ,~2 "~6 GlcNAcfll" 3Manfll--~4GlcNAcfll---~4GlcNAcoT
XXV
GlcNAcfl1---~2Manac~1 GlcNAcfll 6Manal GlcNAcfll
6
GlcNAcfll ..~4ManM ..~3 2
Manfl 1---~4GlcNAcflI--*4GIcNAcoT
GlcNAcfll The subscript OT indicates NaB3H4-reduced oligosaccharides.
[15]
Psathyrella velutina LECTINAFFINITY COLUMN
233
AL
A i
BI
:1
i--el
/.....:i:iiilli i 5
10
FRACTION
15
20
25
NUMBER
FIG. 1. Affinity column chromatography of human milk oligosaccharides and partial degradation products on the PVL column. (A) Oligosaccharide I; (B) oligosaccharide If; (C) oligosaccharide Ill; (D) oligosaccharide IV. Arrows indicate the positions where the elution buffer was switched to buffer containing 1 mM N-acetylglucosamine. These results indicate that the GlcNAcfll--*3Gal group interacts more strongly with P V L than the GlcNAc/31---~6Gal group. This unique specificity is useful for discrimnation of the two isomers produced by endo-/3galactosidase digestion of poly-N-acetyllactosaminoglycans found in some O-linked oligosaccharides. 5 On the other hand, the oligosaccharide with two Noacetylglucosamine residues interacts far more strongly as evidenced by the fact that oligosaccharide IV binds to the column and elutes more slowly with the buffer containing l mM N-acetylglucosamine than oligosaccharide II (Fig. 1D).
Behavior of N-Linked Oligosaccharides with Different Outer Chain Structures and Partial Degradation Products Oligosaccharide VI, which binds to the column and elutes with the buffer containing 1 mM N-acetylglucosamine (Fig. 2B), passes through the 5 j. Amano, P. Strahl, E. G. Berger, N. Kochibe, and A. Kobata, J. Biol. Chem. 266, 11461 (1991).
234
[15]
ENZYMATIC AND AFFINITY METHODS A
f~
>-
_P _>
B
: : .
G
C
I---
o
H
I
E
5
10
15
20
FRACTION
5
10
15
20
NUMBER
Fl6.2. Affinity column chromatography of N-linked oligosaccharides on the PVL column. (A) Oligosaccharides V, VII, VIII, XIII, XIV, XX, XX1, and XXII; (B) oligosaccharides VI, XXIII, and XXIV; (C) oligosaccharides X and XI; (D) oligosaccharide IX; (E) oligosaccharide XII; (F) oligosaccharides XV and XV1; (G) oligosaccharides XVII and XVIII; (H) oligosaccharide XIX; (1) oligosaccharide XXV. The arrows have the same meaning as in Fig. 1.
column without any interaction after fl-N-acetylhexosaminidase treatment (oligosaccharide VII) (Fig. 2A). Substitution of the two terminal N-acetylglucosamine residues of oligosaccharide VI by Galfll--*4 residues (oligosaccharide V in Fig. 2A) or Galfll-o3 residues (data not shown) completely abolishes its affinity to the column. On the other hand, isomeric biantennary oligosaccharide X and oligosaccharide XI, which contain one N-acetylglucosamine residue as a nonreducing terminal group, are only retarded by the column (Fig. 2C). These results indicate that substitution of one of the two N-acetylglucosamine residues of oligosaccharide VI with galactose reduces its affinity to the lectin column. The lectin interacts with nonreducing terminal fl-linked N-acetylglucosamine residues, but does not show any affinity for nonreducing terminal N-acetylneuraminic acid and N-acetylgalactosamine residues (data not shown). N,N'-Diacetylchitobiitol (oligosaccharide IX) binds to the column and elutes slowly with the buffer containing I mM N-acetylglucosamine (Fig. 2D), but N-acetylglucosaminitol passes through the column without any interaction (data not shown). Addition offl-mannosyl residues to the N,N'-
[15]
Psathyrella velutina LECTIN AFFINITY COLUMN
235
diacetylchitobiitol completely abolishes the affinity of oligosaccharide IX to the lectin column (oligosaccharide VIII in Fig. 2A). These results also confirm that the presence of the terminal B-N-acetylglucosamine residue is essential for an oligosaccharide to interact with the column. Addition of an o~-fucosyl residue at the C-6 position of the proximal N-acetylglucosamine moiety does not affect the behavior of oligosaccharide VI at all. Oligosaccharide XII, however, shows much weaker interaction than oligosaccharide VI (Fig. 2E), indicating that the affinity of oligosaccharides is reduced by the addition of the bisecting N-acetylglucosamine residue. The negative effect of the bisecting N-acetylglucosamine residue is also observed in the case of oligosaccharide XIII and oligosaccharide XIV (Fig. 2A). These results are quite interesting because the addition of a B-linked N-acetylglucosamine residue is expected to enhance the binding of oligosaccharides to the lectin column. This unexpected effect probably arises because the bisecting N-acetylglucosamine residue cannot bind to the lectin column and sterically interferes with access of the lectin to the GIcNAcB1---~2Man group. Although all isomeric N-linked oligosaccharides containing a terminal N-acetylglucosamine residue are retarded in the column, those with the GlcNAcB1---~2Man group (oligoosaccharides XV and XVI) (Fig. 2F) interact more strongly than those with the GlcNAcBI~4Man group (oligosaccharide XVII) or the GlcNAcB1---~6Man group (oligosaccharide XVIII) (Fig. 2G). These results clearly indicate that the binding affinity to PVL is influenced by the B-N-acetylglucosamine linkages and is quite different from that of other N-acetylglucosamine-binding lectins from higher plants, which act preferentially at the GlcNAcBI~4 residue. 6 Although distribution of an GlcNAcB1---~2 residue on the Manal--*6 arm or Man~l---~3 arm is not discriminated by the affinity to the lectin column, addition of two c~-mannosyl residues on the Manal---~6 arm reduces the interaction of an oligosaccharide with the lectin (compare oligosaccharide XIX in Fig. 2H to oligosaccharide XVI in Fig. 2F), probably owing to steric hindrance by the mannosyl residues. All galactosylated tri- and tetraantennary oligosaccharides (XX, XXI, and XXII) do not show any affinity to the lectin column, similar to the case of biantennary ones (Fig. 2A). Both nongalactosylated triantennary oligosaccharides XXIII and XXIV bind to the column and elute with buffer containing 1 mM N-acetylglucosamine (Fig. 2B). However, the nongalactosylated tetraantennary oligosaccharide XXV does not bind to 6 I. J. Goldstein and R. D. Poretz, in "'The Lectins: Properties, Function, and Applications in Biology and Medicine" (I. E. Liener, N. Sharon, and I. J. Goldstein, eds.), p. 33. Academic Press, Orlando, Florida, 1986.
236
ENZYMATIC AND AFFINITY METHODS
[15]
A
IpO
B
O
N ¢r
5 FRACTION
10
15
NUMBER
FIG. 3. Elution profiles of immunoglobulin G oligosaccharides by the PVL column. Radioactive oligosaccharide fractions obtained by hydrazinolysis (S. Takasaki, T. Mizuochi, and A. Kobata, this series, Vol. 83, p. 263) of immunoglobulin G from a healthy individual (A) and from a patient with rheumatoid arthritis (B) were subjected to PVL affinity column chromatography. The arrows have the same meaning in Fig. 1.
the column but is retarded in the column (Fig. 2I). These results indicate that the binding affinity to PVL is not simply determined by the number of N-acetylglucosamine residues but is influenced by the steric arrangement of the N-acetylglucosamine residues. Application of Psathyrella velutina Lectin to Detection of Pathological Conditions It is well known that manifestation of N-linked oligosaccharides containing terminal N-acetylglucosamine in the outer chain moieties can be observed in glycoproteins produced in tissues under diseased states. 7,8 7 R. B. Parekh, R. A. Dwek, B. J. Sutton, D. L. Fernandes, A. Leung, D. Stanworth, T. W. Rademacher, T. Mizuochi, T. Taniguchi, K. Matsuta, F. Takeuchi, Y. Nagano, T. Miyamoto, and A. Kobata, Nature (London) 316, 452 (1985). 8 K. Yamashita, A. Hitoi, N. Taniguchi, N. Yokosawa, Y. Tsukada, and A. Kobata, Cancer Res. 43, 5059 (1983).
[16]
SEPARATION OF SIALYLTRANSFERASES
237
An immobilized PVL column can be used as an effective tool to study such rare or abnormal N-linked oligosaccharides with fl-N-acetylglucosamine residues as nonreducing termini. The column is especially useful for detection of oligosaccharide V I , 4'9 because no other fl-N-acetylglucosaminebinding lectin has been reported to bind this oligosaccharide. As an example of such studies, comparative analysis of the radioactive oligosaccharides liberated from immunoglobulin G (IgG) samples purified from sera of healthy individuals and patients with rheumatoid arthritis is shown. It is found that the galactose contents of the N-linked oligosaccharides of serum IgG from patients with rheumatoid arthritis are much lower than those of healthy individuals.7 In accord with this finding, a clear-cut difference is detected as shown in Fig. 3. This result indicates that an immobilized PVL column can be used as a simple tool to detect the extent of pathological changes in the sugar chains of immunoglobulin G. Quite recently, we have developed a simpler and more sensitive method for detection of the aberrant IgG in the sera of patients with rheumatoid arthritis by using this novel N-acetylglucosamine-binding lectin.10 9 M. Tandai, T. Endo, S. Sasaki, Y. Masuho, N. Kochibe, and A. Kobata, Arch. Biochem. Biophys. 291, 339 (1991). ~0N. Tsuchiya, T. Endo, K. Matsuta, S. Yoshinoya, F. Takeuchi, Y. Nagano, M. Shiota, K. Furukawa, N. Kochibe, K. Ito, and A. Kobata, J. lmmunol. 151, 1137 (1993).
[16] S e p a r a t i o n o f G a l f l l , 4 G l c N A c a - 2 , 6 - a n d Galfll,3(4)GlcNAc a-2,3-Sialyltransferases by Affinity Chromatography B y SUBRAMANIAM SABESAN, JAMES C. PAULSON, a n d JASMINDER WEINSTEIN
Introduction
Glycosyltransferases are important biochemical tools in investigations of carbohydrate structure and biological functions. 1,2 With the development of molecular cloning technology, glycosyltransferases have advanced from being rare, expensive reagents to biochemical reagents of choice for large-scale oligosaccharide synthesis) These enzymes I j. C. Paulson and G. N. Rogers, this series, Vol. 138, p. 162. 2 S. Roth, U.S. Patent 5,180,674 (1992). 3 y. Ito, J. J. Gaudino, and J. C. Paulson, Pure Appl. Chem. 65, 753 (1993).
METHODS IN ENZYMOLOGY, VOL. 247
Copyright © 1994 by Academic Press, Inc. All rights of reproduction in any form reserved.
ENZYMATIC AND AFFINITY METHODS
[15]
[15] A f f i n i t y C h r o m a t o g r a p h y o f O l i g o s a c c h a r i d e s o n Psathyrella velutina L e c t i n C o l u m n By AKIRA KOBATA, NAOHISA KOCHIBE,
and
TAMAO ENDO
Introduction Immobilized lectins have been used successfully for many years to isolate glycoconjugates with specific sugar structures. Researchers in a number of laboratories have begun to realize the potential oflectin columns for the analysis of sugar chains in glycoconjugates; serial lectin column chromatography in particular affords a simple and very sensitive method for fractionating various oligosaccharides. 1'z This chapte r describes the use ofa mushroom-lectin column (derived from Psathyrella velutina lectin, PVL) that recognizes and interacts with terminal N-acetylglucosamine residue. 3,4 Methods
Purification of Psathyrella velutina Lectin Fruiting bodies of P. velutina (100 g) collected in the region of Gunma Prefecture (Japan) are homogenized with 1 liter of 145 mM NaCI in 5 mM sodium phosphate buffer, pH 7.2 (PBS), containing 5 mM ethylenediaminetetraacetic acid (EDTA) and 0.5 mM phenylmethylsulfonyl fluoride. The homogenate is centrifuged at 20,000 g for 20 min at 4°, and the pellet is extracted with 500 ml of fresh extraction buffer. To the combined extracts is added dropwise 20% (v/v) acetic acid with stirring, to adjust the solution to pH 4.0. After standing for several hours, the insoluble material is removed by centrifugation at 20,000 g for 20 min at 4°, and the supernatant is neutralized with 1 N NaOH. The acid-treated extract is passed through a chitin column (3.5 x 30 cm) at a flow rate of 60 ml/ hr, and the column is washed with extraction buffer thoroughly. The bound material is then eluted with 50 mM N-acetylglucosamine in PBS containing 10% (v/v) glycerol. The eluate is concentrated by ultrafiltration through an Advantec Q0100 membranes (Toyo Roshi, Tokyo, Japan). I R. K. Merkle and R. D. C u m m i n g s , this series, Vol. 138, p. 232. 2 A. K o b a t a and T. Endo, J. Chromatogr. 597, 111 (1992). 3 N. Kochibe and K. L. Matta, .I. Biol. Chem. 264, 173 (1989). 4 T. Endo, H. Ohbayashi, K. K a n a z a w a , N. Kochibe, and A. Kobata, J. Biol. Chem. 267, 707 (1992).
METHODS IN ENZYMOLOGY,VOL. 247
Copyright © 1994by AcademicPress, Inc. All rights of reproduction in any form reserved.
[15]
Psathyrella velutina LECTIN AFFINITYCOLUMN
229
The concentrate is sequentially applied to serially connected columns of DEAE-cellulofine (Chisso, Tokyo, Japan, 1.7 x 25 cm) and CM-Sepharose CL-6B (Pharmacia LKB Biotechnology, Uppsala, Sweden, 1.7 x 25 cm) equilibrated with 10 mM sodium phosphate buffer, pH 7.0, containing 10% (v/v) glycerol. After washing thoroughly with the same solution the columns are disconnected, and the material bound to the CM-Sepharose CL-6B column is eluted with 0.4 M NaCI. Finally, the lectin is rechromatographed on the chitin column (1.5 x 20 cm) which has been equilibrated with PBS-glycerol. The bound lectin is eluted with 50 mM N-acetylglucosamine.
Preparation of Lectin Column Purified PVL thus obtained is coupled to Affi-Gel 10 (Bio-Rad Laboratories, Richmond, CA) as follows. The activated gel, which has been washed with 0.1 M NaHCO3, is added to a coupling mixture containing the purified PVL (5 mg/ml), 0.15 M NaC1, 10% (v/v) glycerol, and 20 mM N-acetylglucosamine. The suspension is stirred gently overnight at 4° and then filtered. The gel is resuspended in 1 M ethanolamine, allowed to stand for 2 hr at room temperature, and then washed with 10 mM TrisHCI buffer, pH 7.4, containing 140 mM NaCI. The amount of PVL bound to 1 ml of Affi-Gel 10 is estimated to be approximately 4.4 mg.
Affinity Chromatography on Lectin Column Tritium-labeled oligosaccharides [5-10 x 102 counts/min (cpm), 25-50 pmol] dissolved in 100 t~l of water are applied to a column (8 mm inner diameter) containing 1 ml of PVL-Affi-Gel 10, previously equilibrated with 10 mM Tris-HCl buffer, pH 7.4, containing 0.1 M NaCI, 1 mM CaCI2, 1 mM MgC1z , and 1 mM MnCI2, and allowed to stand at room temperature for 30 min. Oligosaccharides bound to the column are then eluted with 10 ml of the same buffer, followed by buffer containing 1 mM N-acetylglucosamine. Fractions of 1.0 ml are collected at a flow rate of 15 ml/hr, and the radioactivity in each fraction is determined by a liquid scintillation method. Recoveries of oligosaccharides based on radioactivities ranges from 98 to 100%.
Behaoior of Human Milk Oligosaccharides Although oligosaccharide I in Table I is passed through the column (Fig. 1A), oligosaccharide II remains bound to the column and is eluted with the buffer containing 1 mM N-acetylglucosamine (Fig. 1B). In contrast, isomeric oligosaccharide lI! is only slowed in the column (Fig. 1C).
230
ENZYMATIC AND AFFINITY METHODS
[15]
TABLE I STRUCTURESOF RADIOACTIVEOLIGOSACCHARIDES Oligosaccharide I
11 III IV
Structure a Gal/31--~4GlcNAc/31---~3Gal/31---~4GlCoT GIcNAc/31--~3Gal/31---~4GlCoT GlcNAc/31----~6Gal/3l----~4GlCoT GlcNAc/31 6GalB 1---~4GICoT
V
GIcNAcB1 Gal¢l1---~4GlcNAcB1----~2Mano~l N ManB I--~4GlcNAc~1--~4GIcNAcoT
V1
GalB1---~4GlcNAcB1---~2Man~1 GIcNAcB1---~2Man~1 6ManB 1---~4GlcNAc/31----~4GIcNAcoT
VII
GIcNAcBI--~2Man~I Man~ 1 6 ManB 1----~4GIcNAcBI---~4GIcNACoT Mano~l ManB 1--~4GIcNAcB1---~4GIcNACoT GIcNAc¢tI----~4GIcNACoT
VIII IX X
GalB I---~4GIcNAcB1---~2Man~ GalBI---~4GIcNAcB 1---~2Man~11 6 ManB1---~4GlcNAc~I----~4GIcNAcoT
Xl
XlI
GIcNAcB 1---~2Mano~l GIcNAcB l---~2Manc~l ~ManB 1---~4GlcNAcB1---~4GlcNAcor S GalB1----~4GlcNAc¢I1--~2Man~1 GIcNAc~ GlcNAcB 1----~2Mano~l
4 ManB 1---~4GlcNAcBI--~4GIcNACoT
GIcNAc/~1--*2Man~1
Psathyrella velutina LECTIN AFFINITY COLUMN
[15]
231
TABLE I (continued) Oligosaccharide
Structure a
XIII
GlcNAcfl 1 Gal/3l--*4GlcNAcfl l--*2Mana 1
XIV
4
IMan¢l 1--*4GlcNAcB1-~4GlcNAcox 7 GlcN Ac~ l---->2Mana1 GlcNAcfl~ GlcNAcfll--~2Manal
4 ~ManB l-->4GlcNAcBl---~4GlcNAcoT
XV
Galfl 1---*4GlcNAcfll---~2Mana1 GlcNAc/3 l--*2Mana 1 ~Man~ 1--+4GlcNAcfl1---~4GIcNAcoT Man~l Manc~1
XVl
IManfl l~4GlcNAcfl 1--~4GIcNAcoT XVII
S GlcNAcfl l---,2Man~1 Mant~ 1 ~Manfl I--~4GIcNAcl31---*4GIcNAcoT /i
XVlll
GlcNAcfll--~4Man~l GIcNAcfl1---~6ManotI I Manfl 1-->4GlcNAcfl1----~4GIcNAcoT
XlX
Mana 1
Man~l
S
6Manal Man~l/~3 "~63Manfl 1---~4GIcNAcB1--~4GIcNACoT S GlcNAcI31--, 2Manaa 1
(continued)
232
ENZYMATIC AND AFFINITY METHODS
[15]
TABLE I (continued) Oligosaccharide XX
Structure a Galfl1--~4GlcNAcfl1---~2 Mana 1 ,~ 36 Manfl 1--~4GlcNAcfl l--~4GlcNAco~r
Galfl1--~4GIcNAcB1N 4 2 Mana 1 XXI
Galfll--*4GlcNAcfll Galfl 1-~4GlcNAcfl1
XXII
6Manal 72 "~6 Galfll--~4GlcNAcfl l" 3Manfl 1---~4GlcNAcfll--~4GlcNAcoT S Galfl 1-~4GlcNAcfl1---~2 Manaa 1 Galfl 1---~4GlcNAc/31 Manal Galfll--~4GlcNAcB1
6
Galfll--~4GlcNAcfllN 4Man M .,~3 L XXIII
Manfl l----~4GlcNAcfl1---~4GlcNAcoz
Gal¢l1---*4GIcNAcB1 GlcNAcfl1-~ 2 Man~ 1 GIcNAcfll.. 4
7 36 Manfl 1---~4GlcNAcfl1--~4GlcNAcoT
2 Manal' XXIV
GlcNAcfll GlcNAcfll 6ManM ,~2 "~6 GlcNAcfll" 3Manfll--~4GlcNAcfll---~4GlcNAcoT
XXV
GlcNAcfl1---~2Manac~1 GlcNAcfll 6Manal GlcNAcfll
6
GlcNAcfll ..~4ManM ..~3 2
Manfl 1---~4GlcNAcflI--*4GIcNAcoT
GlcNAcfll The subscript OT indicates NaB3H4-reduced oligosaccharides.
[15]
Psathyrella velutina LECTINAFFINITY COLUMN
233
AL
A i
BI
:1
i--el
/.....:i:iiilli i 5
10
FRACTION
15
20
25
NUMBER
FIG. 1. Affinity column chromatography of human milk oligosaccharides and partial degradation products on the PVL column. (A) Oligosaccharide I; (B) oligosaccharide If; (C) oligosaccharide Ill; (D) oligosaccharide IV. Arrows indicate the positions where the elution buffer was switched to buffer containing 1 mM N-acetylglucosamine. These results indicate that the GlcNAcfll--*3Gal group interacts more strongly with P V L than the GlcNAc/31---~6Gal group. This unique specificity is useful for discrimnation of the two isomers produced by endo-/3galactosidase digestion of poly-N-acetyllactosaminoglycans found in some O-linked oligosaccharides. 5 On the other hand, the oligosaccharide with two Noacetylglucosamine residues interacts far more strongly as evidenced by the fact that oligosaccharide IV binds to the column and elutes more slowly with the buffer containing l mM N-acetylglucosamine than oligosaccharide II (Fig. 1D).
Behavior of N-Linked Oligosaccharides with Different Outer Chain Structures and Partial Degradation Products Oligosaccharide VI, which binds to the column and elutes with the buffer containing 1 mM N-acetylglucosamine (Fig. 2B), passes through the 5 j. Amano, P. Strahl, E. G. Berger, N. Kochibe, and A. Kobata, J. Biol. Chem. 266, 11461 (1991).
234
[15]
ENZYMATIC AND AFFINITY METHODS A
f~
>-
_P _>
B
: : .
G
C
I---
o
H
I
E
5
10
15
20
FRACTION
5
10
15
20
NUMBER
Fl6.2. Affinity column chromatography of N-linked oligosaccharides on the PVL column. (A) Oligosaccharides V, VII, VIII, XIII, XIV, XX, XX1, and XXII; (B) oligosaccharides VI, XXIII, and XXIV; (C) oligosaccharides X and XI; (D) oligosaccharide IX; (E) oligosaccharide XII; (F) oligosaccharides XV and XV1; (G) oligosaccharides XVII and XVIII; (H) oligosaccharide XIX; (1) oligosaccharide XXV. The arrows have the same meaning as in Fig. 1.
column without any interaction after fl-N-acetylhexosaminidase treatment (oligosaccharide VII) (Fig. 2A). Substitution of the two terminal N-acetylglucosamine residues of oligosaccharide VI by Galfll--*4 residues (oligosaccharide V in Fig. 2A) or Galfll-o3 residues (data not shown) completely abolishes its affinity to the column. On the other hand, isomeric biantennary oligosaccharide X and oligosaccharide XI, which contain one N-acetylglucosamine residue as a nonreducing terminal group, are only retarded by the column (Fig. 2C). These results indicate that substitution of one of the two N-acetylglucosamine residues of oligosaccharide VI with galactose reduces its affinity to the lectin column. The lectin interacts with nonreducing terminal fl-linked N-acetylglucosamine residues, but does not show any affinity for nonreducing terminal N-acetylneuraminic acid and N-acetylgalactosamine residues (data not shown). N,N'-Diacetylchitobiitol (oligosaccharide IX) binds to the column and elutes slowly with the buffer containing I mM N-acetylglucosamine (Fig. 2D), but N-acetylglucosaminitol passes through the column without any interaction (data not shown). Addition offl-mannosyl residues to the N,N'-
[15]
Psathyrella velutina LECTIN AFFINITY COLUMN
235
diacetylchitobiitol completely abolishes the affinity of oligosaccharide IX to the lectin column (oligosaccharide VIII in Fig. 2A). These results also confirm that the presence of the terminal B-N-acetylglucosamine residue is essential for an oligosaccharide to interact with the column. Addition of an o~-fucosyl residue at the C-6 position of the proximal N-acetylglucosamine moiety does not affect the behavior of oligosaccharide VI at all. Oligosaccharide XII, however, shows much weaker interaction than oligosaccharide VI (Fig. 2E), indicating that the affinity of oligosaccharides is reduced by the addition of the bisecting N-acetylglucosamine residue. The negative effect of the bisecting N-acetylglucosamine residue is also observed in the case of oligosaccharide XIII and oligosaccharide XIV (Fig. 2A). These results are quite interesting because the addition of a B-linked N-acetylglucosamine residue is expected to enhance the binding of oligosaccharides to the lectin column. This unexpected effect probably arises because the bisecting N-acetylglucosamine residue cannot bind to the lectin column and sterically interferes with access of the lectin to the GIcNAcB1---~2Man group. Although all isomeric N-linked oligosaccharides containing a terminal N-acetylglucosamine residue are retarded in the column, those with the GlcNAcB1---~2Man group (oligoosaccharides XV and XVI) (Fig. 2F) interact more strongly than those with the GlcNAcBI~4Man group (oligosaccharide XVII) or the GlcNAcB1---~6Man group (oligosaccharide XVIII) (Fig. 2G). These results clearly indicate that the binding affinity to PVL is influenced by the B-N-acetylglucosamine linkages and is quite different from that of other N-acetylglucosamine-binding lectins from higher plants, which act preferentially at the GlcNAcBI~4 residue. 6 Although distribution of an GlcNAcB1---~2 residue on the Manal--*6 arm or Man~l---~3 arm is not discriminated by the affinity to the lectin column, addition of two c~-mannosyl residues on the Manal---~6 arm reduces the interaction of an oligosaccharide with the lectin (compare oligosaccharide XIX in Fig. 2H to oligosaccharide XVI in Fig. 2F), probably owing to steric hindrance by the mannosyl residues. All galactosylated tri- and tetraantennary oligosaccharides (XX, XXI, and XXII) do not show any affinity to the lectin column, similar to the case of biantennary ones (Fig. 2A). Both nongalactosylated triantennary oligosaccharides XXIII and XXIV bind to the column and elute with buffer containing 1 mM N-acetylglucosamine (Fig. 2B). However, the nongalactosylated tetraantennary oligosaccharide XXV does not bind to 6 I. J. Goldstein and R. D. Poretz, in "'The Lectins: Properties, Function, and Applications in Biology and Medicine" (I. E. Liener, N. Sharon, and I. J. Goldstein, eds.), p. 33. Academic Press, Orlando, Florida, 1986.
236
ENZYMATIC AND AFFINITY METHODS
[15]
A
IpO
B
O
N ¢r
5 FRACTION
10
15
NUMBER
FIG. 3. Elution profiles of immunoglobulin G oligosaccharides by the PVL column. Radioactive oligosaccharide fractions obtained by hydrazinolysis (S. Takasaki, T. Mizuochi, and A. Kobata, this series, Vol. 83, p. 263) of immunoglobulin G from a healthy individual (A) and from a patient with rheumatoid arthritis (B) were subjected to PVL affinity column chromatography. The arrows have the same meaning in Fig. 1.
the column but is retarded in the column (Fig. 2I). These results indicate that the binding affinity to PVL is not simply determined by the number of N-acetylglucosamine residues but is influenced by the steric arrangement of the N-acetylglucosamine residues. Application of Psathyrella velutina Lectin to Detection of Pathological Conditions It is well known that manifestation of N-linked oligosaccharides containing terminal N-acetylglucosamine in the outer chain moieties can be observed in glycoproteins produced in tissues under diseased states. 7,8 7 R. B. Parekh, R. A. Dwek, B. J. Sutton, D. L. Fernandes, A. Leung, D. Stanworth, T. W. Rademacher, T. Mizuochi, T. Taniguchi, K. Matsuta, F. Takeuchi, Y. Nagano, T. Miyamoto, and A. Kobata, Nature (London) 316, 452 (1985). 8 K. Yamashita, A. Hitoi, N. Taniguchi, N. Yokosawa, Y. Tsukada, and A. Kobata, Cancer Res. 43, 5059 (1983).
[16]
SEPARATION OF SIALYLTRANSFERASES
237
An immobilized PVL column can be used as an effective tool to study such rare or abnormal N-linked oligosaccharides with fl-N-acetylglucosamine residues as nonreducing termini. The column is especially useful for detection of oligosaccharide V I , 4'9 because no other fl-N-acetylglucosaminebinding lectin has been reported to bind this oligosaccharide. As an example of such studies, comparative analysis of the radioactive oligosaccharides liberated from immunoglobulin G (IgG) samples purified from sera of healthy individuals and patients with rheumatoid arthritis is shown. It is found that the galactose contents of the N-linked oligosaccharides of serum IgG from patients with rheumatoid arthritis are much lower than those of healthy individuals.7 In accord with this finding, a clear-cut difference is detected as shown in Fig. 3. This result indicates that an immobilized PVL column can be used as a simple tool to detect the extent of pathological changes in the sugar chains of immunoglobulin G. Quite recently, we have developed a simpler and more sensitive method for detection of the aberrant IgG in the sera of patients with rheumatoid arthritis by using this novel N-acetylglucosamine-binding lectin.10 9 M. Tandai, T. Endo, S. Sasaki, Y. Masuho, N. Kochibe, and A. Kobata, Arch. Biochem. Biophys. 291, 339 (1991). ~0N. Tsuchiya, T. Endo, K. Matsuta, S. Yoshinoya, F. Takeuchi, Y. Nagano, M. Shiota, K. Furukawa, N. Kochibe, K. Ito, and A. Kobata, J. lmmunol. 151, 1137 (1993).
[16] S e p a r a t i o n o f G a l f l l , 4 G l c N A c a - 2 , 6 - a n d Galfll,3(4)GlcNAc a-2,3-Sialyltransferases by Affinity Chromatography B y SUBRAMANIAM SABESAN, JAMES C. PAULSON, a n d JASMINDER WEINSTEIN
Introduction
Glycosyltransferases are important biochemical tools in investigations of carbohydrate structure and biological functions. 1,2 With the development of molecular cloning technology, glycosyltransferases have advanced from being rare, expensive reagents to biochemical reagents of choice for large-scale oligosaccharide synthesis) These enzymes I j. C. Paulson and G. N. Rogers, this series, Vol. 138, p. 162. 2 S. Roth, U.S. Patent 5,180,674 (1992). 3 y. Ito, J. J. Gaudino, and J. C. Paulson, Pure Appl. Chem. 65, 753 (1993).
METHODS IN ENZYMOLOGY, VOL. 247
Copyright © 1994 by Academic Press, Inc. All rights of reproduction in any form reserved.