Arch Microbiol (1992) 157:205-208
Archives of
Micrabinlogy
© Springer-Verlag1992
Detection of glycoproteins in Borrelia burgdorferi Vittorio Sambri 1, Claudio Stefanelli z, and Roberto Cevenini 1 1 Institute of Microbiology, University of Bologna, S. Orsola Hospital-9, via Massarenti, and 2 Department of Biochemistry, University of Bologna-48, via Irnerio, 1-40138 Bologna, Italy Received February 12, 1991/Accepted October 24~ 1991
Abstract. The presence of carbohydrates on proteins of Borrelia burgdorferi, the causative agent of L y m e disease, was investigated by using a digoxigenin labeling method together with Schiff staining and N-glycosidase F assay. The two m a j o r outer surface exposed proteins of 31 k D a and 34 k D a showed to be glycosylated and gel filtration high pressure liquid c h r o m a t o g r a p h y ( H P L C ) of proteins of B. burgdorferi metabolically labeled with 14C-N-acetylglucosamine revealed the incorporation of the carbohydrate into the glycosyl residue of these proteins.
Key words: Borrelia burgdorferi - Glycoproteins Digoxigenin labeling - Monoclonal antibodies - NGlycosidase F - Gel filtration H P L C
tectins against B. burgdorferi antigens. The aim of this study was to evaluate by chemical methods the presence of carbohydrate moieties on B. burgdorferi proteins.
Materials and methods
Growth of bacteria B. burgdorferistrain IRS (Barbour et al. 1983b), was grown in BSK II medium as previously described (Sambri and Lovett 1990). Bacteria were cultured in sterile glass bottles until the number of viable borreliae reached 3 x 108/ml, then the bacterial suspension was spun down by centrifugation, washed three times in 0.01 M ' sodium phosphate buffer, containing 0.15 M NaC1, pH 7.2 (PBS) (Sambri et al. 1991a) and frozen at - 7 0 °C until used. Detection of carbohydrates
Borrelia burgdorferi, a helical shaped bacterium transmitted to h u m a n s by the bite o f a r t h r o p o d vectors, has been recognized as the causative agent of L y m e Disease (Burgdorfer et al. 1982). Endoflagella as well as surface antigens are recognized by host antibody response during the progression of the disease (Coleman and Benach 1987; Sambri et al. 1991b). Several informations are available a b o u t the nature of the two m a j o r outer surface exposed proteins, O s p A (31 kDa) and OspB (34 kDa): Barbour identified the gene coding for these proteins on a plasmid (Barbour 1988), Wallich (Wallich et al. 1989) described the sequence o f the gene encoding the outer surface protein A of a E u r o p e a n strain of B. burgdorferi and Brandt (Brandt et al. 1990) reported on the characterization of O s p A and OspB as lipoproteins. The possibility that some of the surface antigens were carbohydrates has been,investigated by Coleman and Benach (Coleman and Benach 1988) by affinity blotting with
For the detection of carbohydrate the Glycan detection Kit (Boehringer Mannheim Biochemica, Mannheim, FRG), was used. The principle of the method is the following: adjacent hydroxyl groups in sugars are oxidized to aldehyde by mild periodate treatment, then the spacer-linked steroid hapten digoxigenin is covalently bound to the preformed aldehyde via a hydrazide group. Glycoconjugates labeled with digoxigenin are then detected in an enzyme immunoassay using an antibody-alkaline phosphatase conjugate. Borretia cells, after oxidation with periodate and labeling with digoxigenin, following Manufacturer's instructions, were solubilized in sample buffer and separated by sodium dodecyl sulphate gel electrophoresis (SDS-PAGE) as previously described (Cevenini et al. 1987), transferred to a nitrocellulose membrane (see below), and then tested with an anti-digoxigenin antibody conjugated with alkaline phosphatase.
Schiff staining Polyacrilamyde gels were also stained with the SS procedure for carbohydrates, following the method described by Glossmann (Glossmann and Neville 1971).
Enzymatic deglycosylation Offprint requests to. V. Sambri Abbreviations: N-glycosidase
F : peptide=N-glycosidase F (EC 3.5.1.52); SDS-PAGE: sodium dodecyl sulphate polyacrylamide gel electrophoresis; WB: Western blotting; HPLC: high pressure liquid chromatography; SDS: sodium dodecyl sulphate; mAb: monoclonal antibody; MIAF: mouse immune ascitic fluid; SS: Schiff staining; Osp: Outer surface protein
Borreliae collected by centrifugation (1.0 mg proteins/ml), were boiled for 3 min in 0.2% SDS. After cooling borreliae were incubated with N-glycosidase F (Boehringer Mannheim) used at 0.4 U/10 pg protein, for 15 h at 37 °C, in 20 mM sodium phosphate buffer containing EDTA (Sigma Chemicals St. Louis, Mo., USA) 50 raM, Triton X-100 1.0% (BDH, Poole, UK). pH 7.4. The cells were then analyzed for the presence of carbohydrate.
206
Sodium dodecyl sulphate polyacrylamide gel electrophoresis (SDS-PAGE)
adjuvant female Balb/C mice following the method described by Cevenini. (Cevenini et al. 1991).
The SDS-PAGE was performed following the method of Laemmli (Laemmli 1970) as previously described (Cevenini et al. 1987).
Surface immunofluorescence assay ( SIFA )
Western blot (WB)
The immunofluorescence assay directed to the surface exposed epitopes was performed with living borreliae, as previously reported (Sambri et al. 1991b).
After separation on SDS-PAGE, proteins were electrophoretically transferred onto a nitrocellulose sheet by using the method of Towbin (Towbin et al. 1979), as previously described (Cevenini et al. 1987) with minor modifications: after transfer the nitrocellulose sheet was incubated with gentle agitation in the blocking solution of the Glycan detection Kit for 30 min at room temperature and then washed three times with 0.05 M Tris-buffer, containing 0.15 M NaC1, pH 6.5 (TBS). The paper was then incubated with anti digoxigenin alkaline phosphatase conjugated antibody, washed again in TBS and developed with nitroblue tetrazolium chloride - X-phosphate solution in 0.01 M Tris-HC1, containing 0.05 M MgC12 and 0.1 M NaC1 0.1 M, pH 9.5.
Radiolabeling of bacteria 14C-labeled N-acetylglucosamine (56.8mCi/mmol, Amersham, Amersham, UK) was added to growing cultures of B. burgdorferi to a final concentration of 10 I~Ci/ml. Intrinsically radiolabeled mierorganisms were then prepared for gel filtration hplc analysis as described below.
High pressure liquid chromatography (HPLC) Aliquots of 0.5 ml of cell suspension (3 x 108 cells/ml) were collected, washed and finally boiled 3 rain in 0.2% SDS. After cooling and centrifugation at 10,000 g for 5 min, 0.1 ml were analyzed by gel filtration HPLC. A liquid chromatograph from Waters (Milford, Mass., USA) was used and gel filtration was performed on a Bio Sil TSK 250 column of 600 x 75 mm eluted at room temperature with 50 mM Tris-HC1 pH 7.0, 0.3 M NaC1 and 0.05% SDS at a flow rate of 0.5 ml/min. Fractions of 0.5 ml were collected and assayed for radioactivity by liquid scintillation counting.
Results
Borrelia burgdorferi p r e p a r a t i o n s s h o w e d o n l y two intensely reactive b a n d s with a n a p p a r e n t m o l e c u l a r m a s s o f 31 k D a a n d 34 k D a respectively, w h e n a n a l y z e d using the G l y c a n d e t e c t i o n Kit. S o m e faint b a n d s with h i g h e r m o l e c u l a r weight were also detected. T w o b a n d s with the s a m e m o l e c u l a r m a s s were d e t e c t e d b y the SS o f the gels ( d a t a n o t shown). I n o r d e r to verify the presence o f c a r b o h y d r a t e o n t o the 31 k D a a n d 3 4 k D a p r o t e i n s , b o r r e l i a cells were t r e a t e d w i t h N - g l y c o s i d a s e F. A f t e r e n z y m a t i c t r e a t m e n t the c a r b o h y d r a t e r e a c t i o n was negative, suggesting the presence o f N - l i n k e d glycans o n the 31 k D a a n d 34 k D a p r o t e i n s (Fig. 1). A p a n e l o f 11 m A b s r e a c t i n g w i t h the 31 k D a a n d 34 k D a b a n d s , b y W B , were also tested b y S I F A with living bacteria. A l l the m A b s s t u d i e d were positive, s h o w i n g the surface e x p o s u r e o f the epitopes. In a d d i t i o n , the m A b s were a n a l y z e d for their specificity for c a r b o h y d r a t e e p i t o p e s p r e s e n t on o u t e r surface p r o t e i n s o f B. burgdorferi. I n this e x p e r i m e n t 6 m o n o c l o n a l antib o d i e s r e c o g n i z i n g O s p A a n d 5 r e c o g n i z i n g OspB, were tested b y i m m u n o f l u o r e s c e n c e w i t h B. burgdorferi cells subjected to m i l d t r e a t m e n t w i t h s o d i u m p e r i o d a t e . O u t
Production of monoclonal antibodies Mouse monoclonal antibodies (mAbs) against B. burgdorferi strain IRS (Barbour et al. 1983b) were obtained using the method of K6hler (K6hler and Milstein 1975) as previously described (Cevenini et al. 1987): spleen lymphocytes from intraperitoneally immunized female Balb/C mouse were fused with NSO/U routine myeloma cells. Hybridoma antibody production was detected by testing cell culture supernatants with an enzyme linked immunosorbent assay performed with sonicated borrelia cells. Specific monoclonal antibodies for a 31 kDa (Barbour et al. 1983c) and for the 34 kDa (Barbour et al. 1984) proteins were identified by using WB.
Detection of monoclonal antibodies specific for carbohydrate epitopes Monoclonal antibodies directed against carbohydrate epitopes belonging to the outer surface proteins of B. burgdorferi was determined using mild periodate oxidation, following the method of Woodward (Woodward et al. 1985).
Production of immune serum Guinea pigs were inoculated intramuscularly with 1 ml of heat inactivated B. burgdorferi whole cells (1 gg/gl proteins) emulsified (1 : 4 ratio) with Freund's complete adjuvant. Animals were boosted on day 14 and 28 and then bled by cardiac puncture.
Production of mouse immune ascitic fluid (MIAF) MIAF was obtained by immunizing intraperitoneally with heat inactivated and sonicated bacterial bodies and complete Freund's
F i g . 1. Detection of carbohydrates in proteins of B. burgdorferi. Lanes 1 and 2 contain the control glycosylated protein transferrin treated and untreated with N-glycosidase F, respectively. Lanes 3
and 4 contain borreliae preparations treated and untreated with the N-glycosidase F, respectively. Positions of molecular mass markers are indicated on the right
207
of the 11 monoclonal antobodies evaluated, two, 1El and 8C3, showed to react with carbohydrate epitopes present on OspA and OspB, respectively, since their immunoreactivity was abolished by prior treatment of borreliae with periodate. The remaining 9 monoclonals still recognized the antigen after the treatment. When B. burgdorferi preparations deglycosylated by enzymatic treatment were tested with all the above mAbs and with guinea pig B. burgdorferi hyperimmune serum, only mAb 1El and 8C3 gave negative results, by WB assay (Fig. 2). To obtain further evidence of the presence of proteinbound glycosyl residue(s), bacteria were grown in medium containing 14C N-acetylglucosamine. When cell extracts were analyzed by gel filtration (HPLC) (Fig. 3) a peak of radioactivity was found in a zone of the chromatogram corresponding to a M, of about 30 kDa. The SDS-PAGE and Coomassie-blue staining of the chromatographic fraction corresponding to the highest radioactivity peak (Fig. 3) revealed the presence of two bands of 31 kDa and 34 kDa, respectively, which were recognized in WB by mAbs 1El and 8C3, specific for carbohydrates epitopes of OspA and OspB, respectively (Fig. 4). Another part of radioactivity was found in the chromatogram in a position corresponding to proteins in the range of 69-90 kDa (Fig. 3). This peak of radioactivity was lowered if 2 mM dithiothreitol was present in the elution buffer. Part of the injected radioactivity was incorporated in low molecular mass compounds with a M r < 10 kDa.
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Archives of
Micrabinlogy
© Springer-Verlag1992
Detection of glycoproteins in Borrelia burgdorferi Vittorio Sambri 1, Claudio Stefanelli z, and Roberto Cevenini 1 1 Institute of Microbiology, University of Bologna, S. Orsola Hospital-9, via Massarenti, and 2 Department of Biochemistry, University of Bologna-48, via Irnerio, 1-40138 Bologna, Italy Received February 12, 1991/Accepted October 24~ 1991
Abstract. The presence of carbohydrates on proteins of Borrelia burgdorferi, the causative agent of L y m e disease, was investigated by using a digoxigenin labeling method together with Schiff staining and N-glycosidase F assay. The two m a j o r outer surface exposed proteins of 31 k D a and 34 k D a showed to be glycosylated and gel filtration high pressure liquid c h r o m a t o g r a p h y ( H P L C ) of proteins of B. burgdorferi metabolically labeled with 14C-N-acetylglucosamine revealed the incorporation of the carbohydrate into the glycosyl residue of these proteins.
Key words: Borrelia burgdorferi - Glycoproteins Digoxigenin labeling - Monoclonal antibodies - NGlycosidase F - Gel filtration H P L C
tectins against B. burgdorferi antigens. The aim of this study was to evaluate by chemical methods the presence of carbohydrate moieties on B. burgdorferi proteins.
Materials and methods
Growth of bacteria B. burgdorferistrain IRS (Barbour et al. 1983b), was grown in BSK II medium as previously described (Sambri and Lovett 1990). Bacteria were cultured in sterile glass bottles until the number of viable borreliae reached 3 x 108/ml, then the bacterial suspension was spun down by centrifugation, washed three times in 0.01 M ' sodium phosphate buffer, containing 0.15 M NaC1, pH 7.2 (PBS) (Sambri et al. 1991a) and frozen at - 7 0 °C until used. Detection of carbohydrates
Borrelia burgdorferi, a helical shaped bacterium transmitted to h u m a n s by the bite o f a r t h r o p o d vectors, has been recognized as the causative agent of L y m e Disease (Burgdorfer et al. 1982). Endoflagella as well as surface antigens are recognized by host antibody response during the progression of the disease (Coleman and Benach 1987; Sambri et al. 1991b). Several informations are available a b o u t the nature of the two m a j o r outer surface exposed proteins, O s p A (31 kDa) and OspB (34 kDa): Barbour identified the gene coding for these proteins on a plasmid (Barbour 1988), Wallich (Wallich et al. 1989) described the sequence o f the gene encoding the outer surface protein A of a E u r o p e a n strain of B. burgdorferi and Brandt (Brandt et al. 1990) reported on the characterization of O s p A and OspB as lipoproteins. The possibility that some of the surface antigens were carbohydrates has been,investigated by Coleman and Benach (Coleman and Benach 1988) by affinity blotting with
For the detection of carbohydrate the Glycan detection Kit (Boehringer Mannheim Biochemica, Mannheim, FRG), was used. The principle of the method is the following: adjacent hydroxyl groups in sugars are oxidized to aldehyde by mild periodate treatment, then the spacer-linked steroid hapten digoxigenin is covalently bound to the preformed aldehyde via a hydrazide group. Glycoconjugates labeled with digoxigenin are then detected in an enzyme immunoassay using an antibody-alkaline phosphatase conjugate. Borretia cells, after oxidation with periodate and labeling with digoxigenin, following Manufacturer's instructions, were solubilized in sample buffer and separated by sodium dodecyl sulphate gel electrophoresis (SDS-PAGE) as previously described (Cevenini et al. 1987), transferred to a nitrocellulose membrane (see below), and then tested with an anti-digoxigenin antibody conjugated with alkaline phosphatase.
Schiff staining Polyacrilamyde gels were also stained with the SS procedure for carbohydrates, following the method described by Glossmann (Glossmann and Neville 1971).
Enzymatic deglycosylation Offprint requests to. V. Sambri Abbreviations: N-glycosidase
F : peptide=N-glycosidase F (EC 3.5.1.52); SDS-PAGE: sodium dodecyl sulphate polyacrylamide gel electrophoresis; WB: Western blotting; HPLC: high pressure liquid chromatography; SDS: sodium dodecyl sulphate; mAb: monoclonal antibody; MIAF: mouse immune ascitic fluid; SS: Schiff staining; Osp: Outer surface protein
Borreliae collected by centrifugation (1.0 mg proteins/ml), were boiled for 3 min in 0.2% SDS. After cooling borreliae were incubated with N-glycosidase F (Boehringer Mannheim) used at 0.4 U/10 pg protein, for 15 h at 37 °C, in 20 mM sodium phosphate buffer containing EDTA (Sigma Chemicals St. Louis, Mo., USA) 50 raM, Triton X-100 1.0% (BDH, Poole, UK). pH 7.4. The cells were then analyzed for the presence of carbohydrate.
206
Sodium dodecyl sulphate polyacrylamide gel electrophoresis (SDS-PAGE)
adjuvant female Balb/C mice following the method described by Cevenini. (Cevenini et al. 1991).
The SDS-PAGE was performed following the method of Laemmli (Laemmli 1970) as previously described (Cevenini et al. 1987).
Surface immunofluorescence assay ( SIFA )
Western blot (WB)
The immunofluorescence assay directed to the surface exposed epitopes was performed with living borreliae, as previously reported (Sambri et al. 1991b).
After separation on SDS-PAGE, proteins were electrophoretically transferred onto a nitrocellulose sheet by using the method of Towbin (Towbin et al. 1979), as previously described (Cevenini et al. 1987) with minor modifications: after transfer the nitrocellulose sheet was incubated with gentle agitation in the blocking solution of the Glycan detection Kit for 30 min at room temperature and then washed three times with 0.05 M Tris-buffer, containing 0.15 M NaC1, pH 6.5 (TBS). The paper was then incubated with anti digoxigenin alkaline phosphatase conjugated antibody, washed again in TBS and developed with nitroblue tetrazolium chloride - X-phosphate solution in 0.01 M Tris-HC1, containing 0.05 M MgC12 and 0.1 M NaC1 0.1 M, pH 9.5.
Radiolabeling of bacteria 14C-labeled N-acetylglucosamine (56.8mCi/mmol, Amersham, Amersham, UK) was added to growing cultures of B. burgdorferi to a final concentration of 10 I~Ci/ml. Intrinsically radiolabeled mierorganisms were then prepared for gel filtration hplc analysis as described below.
High pressure liquid chromatography (HPLC) Aliquots of 0.5 ml of cell suspension (3 x 108 cells/ml) were collected, washed and finally boiled 3 rain in 0.2% SDS. After cooling and centrifugation at 10,000 g for 5 min, 0.1 ml were analyzed by gel filtration HPLC. A liquid chromatograph from Waters (Milford, Mass., USA) was used and gel filtration was performed on a Bio Sil TSK 250 column of 600 x 75 mm eluted at room temperature with 50 mM Tris-HC1 pH 7.0, 0.3 M NaC1 and 0.05% SDS at a flow rate of 0.5 ml/min. Fractions of 0.5 ml were collected and assayed for radioactivity by liquid scintillation counting.
Results
Borrelia burgdorferi p r e p a r a t i o n s s h o w e d o n l y two intensely reactive b a n d s with a n a p p a r e n t m o l e c u l a r m a s s o f 31 k D a a n d 34 k D a respectively, w h e n a n a l y z e d using the G l y c a n d e t e c t i o n Kit. S o m e faint b a n d s with h i g h e r m o l e c u l a r weight were also detected. T w o b a n d s with the s a m e m o l e c u l a r m a s s were d e t e c t e d b y the SS o f the gels ( d a t a n o t shown). I n o r d e r to verify the presence o f c a r b o h y d r a t e o n t o the 31 k D a a n d 3 4 k D a p r o t e i n s , b o r r e l i a cells were t r e a t e d w i t h N - g l y c o s i d a s e F. A f t e r e n z y m a t i c t r e a t m e n t the c a r b o h y d r a t e r e a c t i o n was negative, suggesting the presence o f N - l i n k e d glycans o n the 31 k D a a n d 34 k D a p r o t e i n s (Fig. 1). A p a n e l o f 11 m A b s r e a c t i n g w i t h the 31 k D a a n d 34 k D a b a n d s , b y W B , were also tested b y S I F A with living bacteria. A l l the m A b s s t u d i e d were positive, s h o w i n g the surface e x p o s u r e o f the epitopes. In a d d i t i o n , the m A b s were a n a l y z e d for their specificity for c a r b o h y d r a t e e p i t o p e s p r e s e n t on o u t e r surface p r o t e i n s o f B. burgdorferi. I n this e x p e r i m e n t 6 m o n o c l o n a l antib o d i e s r e c o g n i z i n g O s p A a n d 5 r e c o g n i z i n g OspB, were tested b y i m m u n o f l u o r e s c e n c e w i t h B. burgdorferi cells subjected to m i l d t r e a t m e n t w i t h s o d i u m p e r i o d a t e . O u t
Production of monoclonal antibodies Mouse monoclonal antibodies (mAbs) against B. burgdorferi strain IRS (Barbour et al. 1983b) were obtained using the method of K6hler (K6hler and Milstein 1975) as previously described (Cevenini et al. 1987): spleen lymphocytes from intraperitoneally immunized female Balb/C mouse were fused with NSO/U routine myeloma cells. Hybridoma antibody production was detected by testing cell culture supernatants with an enzyme linked immunosorbent assay performed with sonicated borrelia cells. Specific monoclonal antibodies for a 31 kDa (Barbour et al. 1983c) and for the 34 kDa (Barbour et al. 1984) proteins were identified by using WB.
Detection of monoclonal antibodies specific for carbohydrate epitopes Monoclonal antibodies directed against carbohydrate epitopes belonging to the outer surface proteins of B. burgdorferi was determined using mild periodate oxidation, following the method of Woodward (Woodward et al. 1985).
Production of immune serum Guinea pigs were inoculated intramuscularly with 1 ml of heat inactivated B. burgdorferi whole cells (1 gg/gl proteins) emulsified (1 : 4 ratio) with Freund's complete adjuvant. Animals were boosted on day 14 and 28 and then bled by cardiac puncture.
Production of mouse immune ascitic fluid (MIAF) MIAF was obtained by immunizing intraperitoneally with heat inactivated and sonicated bacterial bodies and complete Freund's
F i g . 1. Detection of carbohydrates in proteins of B. burgdorferi. Lanes 1 and 2 contain the control glycosylated protein transferrin treated and untreated with N-glycosidase F, respectively. Lanes 3
and 4 contain borreliae preparations treated and untreated with the N-glycosidase F, respectively. Positions of molecular mass markers are indicated on the right
207
of the 11 monoclonal antobodies evaluated, two, 1El and 8C3, showed to react with carbohydrate epitopes present on OspA and OspB, respectively, since their immunoreactivity was abolished by prior treatment of borreliae with periodate. The remaining 9 monoclonals still recognized the antigen after the treatment. When B. burgdorferi preparations deglycosylated by enzymatic treatment were tested with all the above mAbs and with guinea pig B. burgdorferi hyperimmune serum, only mAb 1El and 8C3 gave negative results, by WB assay (Fig. 2). To obtain further evidence of the presence of proteinbound glycosyl residue(s), bacteria were grown in medium containing 14C N-acetylglucosamine. When cell extracts were analyzed by gel filtration (HPLC) (Fig. 3) a peak of radioactivity was found in a zone of the chromatogram corresponding to a M, of about 30 kDa. The SDS-PAGE and Coomassie-blue staining of the chromatographic fraction corresponding to the highest radioactivity peak (Fig. 3) revealed the presence of two bands of 31 kDa and 34 kDa, respectively, which were recognized in WB by mAbs 1El and 8C3, specific for carbohydrates epitopes of OspA and OspB, respectively (Fig. 4). Another part of radioactivity was found in the chromatogram in a position corresponding to proteins in the range of 69-90 kDa (Fig. 3). This peak of radioactivity was lowered if 2 mM dithiothreitol was present in the elution buffer. Part of the injected radioactivity was incorporated in low molecular mass compounds with a M r < 10 kDa.
10
158
66
44
26
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