ANALYTICAL
BIOCHEMISTRY
188,149-l%
(1990)
Preparation and Characterization of Monoclonal Antibodies to an N-Linked Oligosaccharide Seize Masutani,*,t Takashi Shimano,”
Nobuko Miyazawa,? Shigeru Fujii,? Atsushi Takesada Mori,* and Naoyuki Taniguchit
Nishikawa,?
Hirokazu
Matsukawa,$
*Second Department of Surgery, Osaka University Medical School, l-1-50, Fukushima, Fukushima-ku, Osaka 553; $Oriental Yeast Corporation, 4-4-1, Minamisuita, Suita City, Osaka 564; and TDepartment of Biochemistry, Osaka University Medical School, 4-3-57 Nakanoshima, Kita-ky Osaka 530, Japan
Received
January
23,199O
Two monoclonal antibodies to an N-linked oligosaccharide, MT-5 and MT-g, have been prepared by immunization with a pyridylaminated, asialylated, galactosylated, fucosylated, bisected biantennary sugar. The reactivity of these antibodies was monitored by their reaction with human asialoglycophorin in a solid-phase enzyme-linked immunosorbent assay. Both antibodies reacted with the sugar chains of various human glycoproteins such as immunoglobulin G, transferrin, y-glutamyl transpeptidase, al-acid glycoprotein, and a-fetoprotein. Treatment of asialoglycophorin with & N-acetylhexosaminidase or a-mannosidase resulted in reduction of the binding to these antibodies. The reactivity of MT-5 to asialoglycophorin was slightly inhibited by D-mannOSe and N-acetylglucosamine, whereas that of MT-9 was inhibited by D-mannOSe, N-acetyl-Dglucosamine, chitobiose, and L-fucose. The epitope specificity of MT-5 appears to be a sugar chain containing biantennary N-acetyl-D-glucosamine residues, the bisected N-acetyl-D-glucosamine residue, and a trimannosyl core. The epitope to which MT-9 is directed may be a complex made up of &mannose, chitobiose, and L-fucose. These studies indicate that immunization with pyridylaminated sugars can produce antibodies that recognize N-linked oligosaccharides. Monoclonal/ polyclonal antibodies to the N-linked sugar chains of glycopeptides would be useful in such studies of proteins. 0 1990 Academic Press, Inc.
It is well known that the changes in the extent of glycosylation of glycolipids and glycoproteins are often associated with tumor formation and metastasis (l-8). A number of cancer-associated carbohydrate-containing antigens have been identified by the use of monoclonal antibodies (9,10), but most of these are glycolipids (ll15) which are often related to blood group antigens 0003-2697/90 Copyright All rights
$3.00 0 1990 by Academic Press, of reproduction in any form
(16,17). A monoclonal antibody directed to mucin-type glycoproteins has been prepared (18,19). Several reports have appeared on antisera to defined structural moieties of polysaccharides (20-22), but monoclonal antibodies directed to sugar chains of N-linked glycoproteins have not been established. In the present study antibodies to a Gal-Fuc-bisected GlcNAc-PA1 prepared from human IgG were developed. Two monoclonal antibodies, MT-5 and MT-g, directed to the sugar chains of an N-linked oligosaccharide were prepared by this approach. MATERIALS
AND
METHODS
Reagents. Human IgG, transferrin, and serum albumin (HSA) were kindly supplied by the Green Cross Company, Japan. Asialoglycophorin and q-acid glycoprotein (orosomucoid) were purchased from Sigma Chemical Company (St. Louis, MO). The Fc fragment of IgG was purchased from Jackson Immunoresearch Labs, Inc. a-Fetoprotein was kindly supplied by the Oriental Yeast Company Ltd., Japan. y-GTP was purified from human kidney as described previously (23). Glycosidases. Jack bean sources of P-galactosidase, P-N-acetylhexosaminidase, and cu-mannosidase and fimannosidase from Achatina fulica were purchased from Seikagaku Kogyo Company, Japan. Bovine kidney a-~-
i Abbreviations used: IgG, immunoglobulin G; IgM, immunoglobulin M; y-GTP, y-glutamyl transpeptidase; GlcNAc, N-acetyl-Dglucosamine; PA, pyridylamino; Gal-Fuc-bisected GlcNAc-PA, pyridylaminated, asialylated, galactosylated, fucosylated, bisected biantennary sugar chain; ELISA, enzyme-linked immunosorbent assay; BSA, bovine serum albumin; HSA, human serum albumin; chitobiose, di-N-acetylchitobiose; PBS, phosphate-buffered saline (0.9% NaCl in 0.05 M phosphate buffer, pH 7.4); PBS-Tween, phosphate-buffered saline containing 0.05% Tween 20; SDS, sodium dodecyl sulfate; AFP, a-fetoprotein. 149
Inc. reserved.
150
MASUTANI
ET
AL.
fucosidase was a Sigma Chemical Company product. Neuraminidase was kindly supplied by Dr. Y. Ohta.
Saccharides. D-Galactose, D-mannose, GlcNAc, and chitobiose were Seikagaku Kogyo Company products. LFucose was obtained from Fluka AG, Chem. Other products. Complete and incomplete Freund’s adjuvants were Difco Laboratories products. BSA and biotin N-hydroxysuccinimide ester were obtained from Sigma Chemical Company. Peroxidase-conjugated goat anti-mouse IgG + IgM and peroxidase-conjugated goat anti-mouse IgM were obtained from TAGO, Inc. (Burlingame, CA). Peroxidase-conjugated streptavidin was obtained from Zymed Company. Sodium cyanoborohydride was an Aldrich Chemical Company Inc. product. oPhenylenediamine and dimethylsulfoxide were obtained from Wako Pure Chem. Ind. Ltd. Pronase was a Boehringer-Mannheim product and bovine pancreas trypsin was obtained from Sigma Chemical Company. Preparation of asialylated, galuctosyluted, fucosylated, bisected GlcNAc-PA. IgG was lyophilized following dialysis against distilled water. It was then subjected to hydrazinolysis as described previously (24). The oligosaccharides released were reductively aminated with 2aminopyridine by use of sodium cyanoborohydride and a mixture of pyridylamino derivatives separated by gel filtration on a Toyopearl HW-40F (Tosoh, Japan) column (25). The purifiedpyridylamino derivatives were digested with neuraminidase. Separation and identification of the Gal-Fuc-bisected GlcNAc-PA were carried out by HPLC using a Shimadzu LC-3A column (4.6 X 150 mm) packed with ODS-80 TM (Tosoh Co.). Elution was performed at 55°C with 20 mM ammonium acetate buffer, pH 4.0, containing 0.04-l% butanol. The structure of each oligosaccharide eluted was determined by ‘H NMR as reported previously (25). The elution position of the Gal-Fuc-bisected GlcNAc-PA is seen as fraction XII in Fig. 1. Immunizations and preparation of hybridomas. GalFuc-bisected GlcNAc-PA (see structure in Fig. 1) was sonicated with an equal volume of complete or incomplete Freund’s adjuvant, and 5 pg was administered once a week for 5 weeks to Balb/c mice by the intraperitoneal route. Three days after the last injection, spleen cells were harvested and fused with mouse myeloma P3Ul cells. After three times limiting dilutions, the presence of antibodies was detected by a 96-well microtiter plate (Nunc-immunoplate Maxisorp) method. The screening was performed as follows. Fifty microliters of asialoglycophorin (1 pg/ml) was coated on the microtiter plate for 2 h at 37°C. After being washed three times with PBSTween, each well was blocked by reaction with 300 ~1 of 1% BSA for 1 h at 37°C. The wells were then incubated with 50 ~1 of hybridoma culture supernatants for 1 h at 37°C. After being washed three times with PBS-Tween, 50 ~1 of 1:2000 diluted peroxidase-conjugated goat antimouse IgG + IgM was added followed by incubation for
Elution
Time
(min)
FIG. 1.
(Top) The structure of Gal-Fuc-bisected GlcNAc-PA used in this study as the antigen. (Bottom) HPLC profile of the IgG sugar chains. Twelve components (I-XII) were clearly separated on the HPLC column. The arrow (peak XII) indicates the elution position of the Gal-Fuc-bisected GlcNAc-PA.
1 h at 37°C. The wells were then washed six times with PBS-Tween, and incubated with 100 ,a1of substrate (0.6 mg/ml o-phenylenediamine and 0.02% H202 in 100 mM sodium citrate, pH 5.0). The reaction was stopped by addition of 50 ~1 of 1 N H2S04 and the 490-nm absorption of the reaction mixture was determined. Purification and biotinylution of monoclonal antibodies. The IgM monoclonal antibodies produced were purified from the ascitic fluid by precipitation with 3050% ammonium sulfate followed by gel filtration on a Sephacryl S-300 column. Two hundred microliters of biotin N-hydroxysuccinimide ester dissolved in dimethyl sulfoxide (1 mg/ml) was added to 1 ml of the isolated antibody (1 mg/ml). After a 4-h incubation at room temperature, the mixture was dialyzed against PBS overnight at 4°C.
Antibody binding studies using glycoproteins and sugar peptides. In the case of binding studies using glycoproteins, a 96-well Nunc microtiter plate was coated overnight at 4°C with 50 ng of each glycoprotein in 50 ~1. For binding studies using sugar peptides, to provide for the greater reactivity of the sugar chains with the microtiter plate, the proteins were digested with Pronase for 24 h at 37°C and the sugar portions were isolated by gel filtration on a Bio-Gel P4 column. Transferrin was first treated with neuraminidase prior to digestion. These sugar peptides were coated on MM Inc. plates with 1 pg peptides/ml overnight at 4°C and the reactivity with two monoclonal antibodies was checked as described before. Enzyme treatment. All enzymatic digestions were carried out overnight at 37°C in a 96-well plate in an incubator. Asialoglycophorin-coated plates was sequentially digested with, P-galactosidase, /3-N-acetylhexosaminidase, Lu-mannosidase,and P-mannosidase. P-Galac-
ANTIBODIES
TO
AN
N-LINKED
151
OLIGOSACCHARIDE
0
2. .e .> t; I [1:
0 8
500 -
al .e 2
BIOCHEMISTRY
188,149-l%
(1990)
Preparation and Characterization of Monoclonal Antibodies to an N-Linked Oligosaccharide Seize Masutani,*,t Takashi Shimano,”
Nobuko Miyazawa,? Shigeru Fujii,? Atsushi Takesada Mori,* and Naoyuki Taniguchit
Nishikawa,?
Hirokazu
Matsukawa,$
*Second Department of Surgery, Osaka University Medical School, l-1-50, Fukushima, Fukushima-ku, Osaka 553; $Oriental Yeast Corporation, 4-4-1, Minamisuita, Suita City, Osaka 564; and TDepartment of Biochemistry, Osaka University Medical School, 4-3-57 Nakanoshima, Kita-ky Osaka 530, Japan
Received
January
23,199O
Two monoclonal antibodies to an N-linked oligosaccharide, MT-5 and MT-g, have been prepared by immunization with a pyridylaminated, asialylated, galactosylated, fucosylated, bisected biantennary sugar. The reactivity of these antibodies was monitored by their reaction with human asialoglycophorin in a solid-phase enzyme-linked immunosorbent assay. Both antibodies reacted with the sugar chains of various human glycoproteins such as immunoglobulin G, transferrin, y-glutamyl transpeptidase, al-acid glycoprotein, and a-fetoprotein. Treatment of asialoglycophorin with & N-acetylhexosaminidase or a-mannosidase resulted in reduction of the binding to these antibodies. The reactivity of MT-5 to asialoglycophorin was slightly inhibited by D-mannOSe and N-acetylglucosamine, whereas that of MT-9 was inhibited by D-mannOSe, N-acetyl-Dglucosamine, chitobiose, and L-fucose. The epitope specificity of MT-5 appears to be a sugar chain containing biantennary N-acetyl-D-glucosamine residues, the bisected N-acetyl-D-glucosamine residue, and a trimannosyl core. The epitope to which MT-9 is directed may be a complex made up of &mannose, chitobiose, and L-fucose. These studies indicate that immunization with pyridylaminated sugars can produce antibodies that recognize N-linked oligosaccharides. Monoclonal/ polyclonal antibodies to the N-linked sugar chains of glycopeptides would be useful in such studies of proteins. 0 1990 Academic Press, Inc.
It is well known that the changes in the extent of glycosylation of glycolipids and glycoproteins are often associated with tumor formation and metastasis (l-8). A number of cancer-associated carbohydrate-containing antigens have been identified by the use of monoclonal antibodies (9,10), but most of these are glycolipids (ll15) which are often related to blood group antigens 0003-2697/90 Copyright All rights
$3.00 0 1990 by Academic Press, of reproduction in any form
(16,17). A monoclonal antibody directed to mucin-type glycoproteins has been prepared (18,19). Several reports have appeared on antisera to defined structural moieties of polysaccharides (20-22), but monoclonal antibodies directed to sugar chains of N-linked glycoproteins have not been established. In the present study antibodies to a Gal-Fuc-bisected GlcNAc-PA1 prepared from human IgG were developed. Two monoclonal antibodies, MT-5 and MT-g, directed to the sugar chains of an N-linked oligosaccharide were prepared by this approach. MATERIALS
AND
METHODS
Reagents. Human IgG, transferrin, and serum albumin (HSA) were kindly supplied by the Green Cross Company, Japan. Asialoglycophorin and q-acid glycoprotein (orosomucoid) were purchased from Sigma Chemical Company (St. Louis, MO). The Fc fragment of IgG was purchased from Jackson Immunoresearch Labs, Inc. a-Fetoprotein was kindly supplied by the Oriental Yeast Company Ltd., Japan. y-GTP was purified from human kidney as described previously (23). Glycosidases. Jack bean sources of P-galactosidase, P-N-acetylhexosaminidase, and cu-mannosidase and fimannosidase from Achatina fulica were purchased from Seikagaku Kogyo Company, Japan. Bovine kidney a-~-
i Abbreviations used: IgG, immunoglobulin G; IgM, immunoglobulin M; y-GTP, y-glutamyl transpeptidase; GlcNAc, N-acetyl-Dglucosamine; PA, pyridylamino; Gal-Fuc-bisected GlcNAc-PA, pyridylaminated, asialylated, galactosylated, fucosylated, bisected biantennary sugar chain; ELISA, enzyme-linked immunosorbent assay; BSA, bovine serum albumin; HSA, human serum albumin; chitobiose, di-N-acetylchitobiose; PBS, phosphate-buffered saline (0.9% NaCl in 0.05 M phosphate buffer, pH 7.4); PBS-Tween, phosphate-buffered saline containing 0.05% Tween 20; SDS, sodium dodecyl sulfate; AFP, a-fetoprotein. 149
Inc. reserved.
150
MASUTANI
ET
AL.
fucosidase was a Sigma Chemical Company product. Neuraminidase was kindly supplied by Dr. Y. Ohta.
Saccharides. D-Galactose, D-mannose, GlcNAc, and chitobiose were Seikagaku Kogyo Company products. LFucose was obtained from Fluka AG, Chem. Other products. Complete and incomplete Freund’s adjuvants were Difco Laboratories products. BSA and biotin N-hydroxysuccinimide ester were obtained from Sigma Chemical Company. Peroxidase-conjugated goat anti-mouse IgG + IgM and peroxidase-conjugated goat anti-mouse IgM were obtained from TAGO, Inc. (Burlingame, CA). Peroxidase-conjugated streptavidin was obtained from Zymed Company. Sodium cyanoborohydride was an Aldrich Chemical Company Inc. product. oPhenylenediamine and dimethylsulfoxide were obtained from Wako Pure Chem. Ind. Ltd. Pronase was a Boehringer-Mannheim product and bovine pancreas trypsin was obtained from Sigma Chemical Company. Preparation of asialylated, galuctosyluted, fucosylated, bisected GlcNAc-PA. IgG was lyophilized following dialysis against distilled water. It was then subjected to hydrazinolysis as described previously (24). The oligosaccharides released were reductively aminated with 2aminopyridine by use of sodium cyanoborohydride and a mixture of pyridylamino derivatives separated by gel filtration on a Toyopearl HW-40F (Tosoh, Japan) column (25). The purifiedpyridylamino derivatives were digested with neuraminidase. Separation and identification of the Gal-Fuc-bisected GlcNAc-PA were carried out by HPLC using a Shimadzu LC-3A column (4.6 X 150 mm) packed with ODS-80 TM (Tosoh Co.). Elution was performed at 55°C with 20 mM ammonium acetate buffer, pH 4.0, containing 0.04-l% butanol. The structure of each oligosaccharide eluted was determined by ‘H NMR as reported previously (25). The elution position of the Gal-Fuc-bisected GlcNAc-PA is seen as fraction XII in Fig. 1. Immunizations and preparation of hybridomas. GalFuc-bisected GlcNAc-PA (see structure in Fig. 1) was sonicated with an equal volume of complete or incomplete Freund’s adjuvant, and 5 pg was administered once a week for 5 weeks to Balb/c mice by the intraperitoneal route. Three days after the last injection, spleen cells were harvested and fused with mouse myeloma P3Ul cells. After three times limiting dilutions, the presence of antibodies was detected by a 96-well microtiter plate (Nunc-immunoplate Maxisorp) method. The screening was performed as follows. Fifty microliters of asialoglycophorin (1 pg/ml) was coated on the microtiter plate for 2 h at 37°C. After being washed three times with PBSTween, each well was blocked by reaction with 300 ~1 of 1% BSA for 1 h at 37°C. The wells were then incubated with 50 ~1 of hybridoma culture supernatants for 1 h at 37°C. After being washed three times with PBS-Tween, 50 ~1 of 1:2000 diluted peroxidase-conjugated goat antimouse IgG + IgM was added followed by incubation for
Elution
Time
(min)
FIG. 1.
(Top) The structure of Gal-Fuc-bisected GlcNAc-PA used in this study as the antigen. (Bottom) HPLC profile of the IgG sugar chains. Twelve components (I-XII) were clearly separated on the HPLC column. The arrow (peak XII) indicates the elution position of the Gal-Fuc-bisected GlcNAc-PA.
1 h at 37°C. The wells were then washed six times with PBS-Tween, and incubated with 100 ,a1of substrate (0.6 mg/ml o-phenylenediamine and 0.02% H202 in 100 mM sodium citrate, pH 5.0). The reaction was stopped by addition of 50 ~1 of 1 N H2S04 and the 490-nm absorption of the reaction mixture was determined. Purification and biotinylution of monoclonal antibodies. The IgM monoclonal antibodies produced were purified from the ascitic fluid by precipitation with 3050% ammonium sulfate followed by gel filtration on a Sephacryl S-300 column. Two hundred microliters of biotin N-hydroxysuccinimide ester dissolved in dimethyl sulfoxide (1 mg/ml) was added to 1 ml of the isolated antibody (1 mg/ml). After a 4-h incubation at room temperature, the mixture was dialyzed against PBS overnight at 4°C.
Antibody binding studies using glycoproteins and sugar peptides. In the case of binding studies using glycoproteins, a 96-well Nunc microtiter plate was coated overnight at 4°C with 50 ng of each glycoprotein in 50 ~1. For binding studies using sugar peptides, to provide for the greater reactivity of the sugar chains with the microtiter plate, the proteins were digested with Pronase for 24 h at 37°C and the sugar portions were isolated by gel filtration on a Bio-Gel P4 column. Transferrin was first treated with neuraminidase prior to digestion. These sugar peptides were coated on MM Inc. plates with 1 pg peptides/ml overnight at 4°C and the reactivity with two monoclonal antibodies was checked as described before. Enzyme treatment. All enzymatic digestions were carried out overnight at 37°C in a 96-well plate in an incubator. Asialoglycophorin-coated plates was sequentially digested with, P-galactosidase, /3-N-acetylhexosaminidase, Lu-mannosidase,and P-mannosidase. P-Galac-
ANTIBODIES
TO
AN
N-LINKED
151
OLIGOSACCHARIDE
0
2. .e .> t; I [1:
0 8
500 -
al .e 2
