Vol. 179, No. 3, 1991 September 30, 1991
BIOCHEMICAL
SYNTHESIS
AND BIOPHYSICAL RESEARCH COMMUNICATIONS Pages 1464-l 469
AND CHARACTERIZATION OF A PERTUSSIS TOXIN-BIOTIN CONJUGATE
Louis D. Heerze1* , Clifford G. Clarkl, Ying Chenl, Richard H. Smith2, and Glen D. Armstrong1* 1Departmentof Medical Microbiology and Infectious Diseases,University of Alberta, Edmonton, Alberta, CanadaT6G 2H7 2ChembiomedLtd., Edmonton, Alberta, Canada Received
August
9,
1991
SUMMARY: We prepareda Pertussistoxin-biotin conjugateandfound its biological propertiesto be similar to thoseof native Pertussistoxin with respectto the hemagglutination,Chinesehamster ovary cell, and lymphocyte proliferation assays.Direct binding to Chinesehamster ovary and Jurkat cells was observedusing fluorescencemicroscopy. Pertussistoxin-biotin was alsofound to possesssimilar glycoconjugate binding specificitiesasthoseof 1251-labeledPertussistoxin . 0 1991
Academx
Press,
Inc.
Pertussistoxin (PT) is one of the important virulence factors produced by Bordetella pertussis, the etiological agent of whooping cough (1). PI, like other A-B type toxins containsan enzymatically active A subunit that hasADP-ribosyltransferase activity which is responsiblefor modifying guanine nucleotide-regulatory proteins (G proteins) in eukaryotic cells (2,3). The complex B oligomer of PI is responsiblefor the lectin-like binding activity of the toxin to receptors on goose erythrocytes, Chinesehamsterovary (CHO) cells, T lymphocytes, and other host cell membranes(4- 10). The B oligomer may alsocontribute to the attachmentof Bordetefia pertussis organismsto epithelial cells lining the upper respiratory tract of humans(4); the only known host of Bordetella pertussis . It is therefore important to identify and characterize receptors for PT in order to fully understandits activity in the diseaseprocess. Researchefforts aimedat characterizingthe lectin-like binding activity of the B oligomer of PT have utilized either antibodies directed against PT (11,12) or the use of radiolabeled toxin (5,13-15). Disadvantages of using antibodies center around the necessity of having to obtain specific antibodiesthat recognize epitopesoutsidePT receptor binding domains.Although, Iodine125 labeled PT hasproved to be a useful probe for characterizing the receptor binding activity of * To whom correspondenceshouldbe addressed. m PT Pertussistoxin, ADP adenosinediphosphate, CHO Chinese hamster ovary, HA hemagglutination, PBS phosphatebuffered saline,BSA bovine serumalbumin, PBST phosphatebuffered salinecontaining Tween 20, ABTS 2, 2’-azino-bis (3-ethylbenzthiazoline- 6sulfonic acid), FBS fetal bovine serum,FlTC fluoresceinisothiocyanate. 0006-291X/91 $1.50 Copyright 0 1991 by Academic Press, Inc. All rights of reproduction in any form reserved.
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[1251]-PT only retains its activity for short periods. Therefore,
we sought an
alternative to iodinated PT for use in receptor studies. We chose to label PT with biotin since biotin has been successfully used in ELISA systems as well as immunochemical staining techniques. The added advantage of utilizing a biotin system is in the high affinity interaction with avidin. Avidin can be readily obtained conjugated to enzymes such as peroxidase which can be utilized in ELISA experiments with PT-biotin, or they may be conjugated to fluorescent probes that can be used to determine PT-biotin binding to different cell types. Highly specific anti-biotin antibody enzyme conjugates are also available to characterize PTbiotin’s lectin-like binding activity. In the present study PT-biotin was found to possess similar biological properties to those of native PT as was determined in the hemagglutination
(HA),
CHO cell and lymphocyte
proliferation assays. Binding inhibition experiments indicated that the carbohydrate binding specificities of PT-biotin were similar to those of [ 1251]-PT. Direct binding to CHO and Jurkat cells was also observed using fluorescence microscopy.
EXPERIMENTAL
PROCEDURES
Materials; All of the reagents were obtained from Sigma except for: PT and human ceruloplasmin which were a gift from the Connaught Centte for Biotechnology Research, Sulfo-NHS-biotin was obtained from Pierce, and a-Chymotrypsin was from Calbiochem-Behring. Goose blood was from Gibmar Laboratories and Jurkat cells were kindly provided by Dr. V. Paetkau, Dept. of Biochemistry, University of Alberta. LEC 1 CHO cells (ATCC CRL 1735) were from ATCC. Preparation sf PT-biotic PT (100 uL,190 pg) in 50 mM phosphate buffer, pH 7.5. containing 50% glycerol was sonicated prior to use, and then diluted with 100 pL of 0.1 M borate buffer, pH 8.5. Sulfo-NHS-biotin (20 pL, 2 mg./mL in H20) was added to the PT solution and the reaction was allowed to proceed for 30 min at room temperature. Subsequently, aliquots (50 pL) of the PTbiotin were incubated with 100 l.tL volumes of washed fetuin-agarose for 60 min at room temperature. Mixtures were then transferred to glass wool-plugged Pasteur pipets, washed with PBS (20 mL), and eluted with 200 pL volumes of 50 mM diethanolamine buffer, pH 11.5, as previously described (16). The various biological activities of the PT-biotin preparation were then tested as described below. Hemdutination and CHO Cell AssavG Chymotrypsin-treated goose erythrocytes and Chinese hamster ovary (CHO) cells were used in their respective assays (13,17) to compare the endpoint dilutions of the PT-biotin preparations with that of underivatized PT . Lvmohocvte Proliferation Assavs: Routine mitogenesis assays were performed as described previously (18) by measuring the incorporation of r3H]-thymidine into peripheral blood lymphocytes. Binding Inhibition Assa-w Microtiter wells were coated with 100 l.tL of fetuin (50 pg/mL) in 50 mM sodium phosphate buffer (pH 6.8) containing 5 mM MgC12 and 15 mM NaN3 for 16 hr at 4°C. The solution was removed by aspiration and replaced with 100 pL of 1% BSA in PBS containing 0.05% Tween 20 (PBST). After incubation for 4 hr at room temperature, the microtiter wells were washed four times with 300 l.tL of PBST . Glycoprotein inhibitors ranging from 10w4to 10m8M or PT (0.2-7pM) in PBS (40 uL) were added to each well and PT-biotin (10 pL, diluted l/20 in PBST) was then added to the microtiter wells. After incubating for 1 hr the binding reaction was stopped by aspirating the solutions and the plate was washed with PBST (4X3OOpL). Horseradish peroxidase conjugated anti-biotin antiboby (lOOpL, l/800 dilution in 1% BSA in PBST) was then added to the wells. The plates were incubated at ambient temperature for 2 hr. Avidin-peroxidase (5OpL of a l/3000 dilution in PBST for 1 hr) could also be used. After washing the wells as described above, the substrate solution (1 mM ABTS in 5 mM citrate buffer, pH 4.2, containing 0.1% hydrogen peroxide, v/v) was added and the plates were incubated for 30 min. Color development was recorded at 405 nm using a Titertek Multiskan MC plate reader. Maximum binding was determined in the absence of glycoprotein inhibitor and background binding was measured in wells coated with BSA only. Binding assays for each inhibitor concentration were 1465
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done in at least duplicate and the average value varied less than 15%. The concentration of glycoprotein required for 50% inhibition (IC,,) wasdeterminedby plotting the amount of binding observed in the presenceof glycoprotein inhibitor asa percent of the maximum binding achieved without . inhibitor. . . . lotin BlndmP to Ju kat Cells: Jurkat cells grown in RPM1 1640 (Gibco) media containing 10% fetal bovine serum[FBS) were washedfour timeswith PBS, counted, and suspendedto give 1 x lo7 cells/ml in PBS. PT-biotin (0.2 pg in 20 uL PBS) was addedto 100 pL (1 x 106) Jurkat cells and incubatedon ice for 90 min with occasionalshaking.PBS (20 pL) wasaddedto the same number of cells to provide a negative control. After incubation, the cell suspensionswere washed twice with 3 mL of cold PBS. Avidin-FlTC (50 pL, l/100 in PBS) was then added and the cells were allowed to incubate for 30 min on ice. Cell suspensions were again washedtwice with cold PBS, and wet mounts were prepared for fluorescence microscopy using a Leitz Laborlux K fluorescencemicroscope. PT-biotin Bindim to CHO Cells; Wild type and LEC 1 CHO cells were grown on 10 well Linbro (Flow) glassmicroscope slides in Ham’s F-12 medium (Gibco) supplementedwith 10% FBS. Monolayers adherent to the glassslideswere washedgently with approximately 10 mL of PBS, PT-biotin (0.2 ug in 10 uL PBS) wasadded,and the mixtures were allowed to incubate on ice for 90 min. Controls were prepared using either 20 pL of PBS without PT-biotin or 0.2 pg of underivatized PT. Control experimentswere alsodoneusing a CHO cell LEC 1 mutant. The CHO cell monolayers were washedagain with PBS and incubated with avidin-FITC (20 pL, l/100 in PBS) on ice for an additional 30 min. After a final washwith 15 mL of cold PBS, the CHO cells were preparedfor fluorescencemicroscopyasdescribedabove. RESULTS
AND DISCUSSION
When PT-biotin waspreparedasdescribedabove usingfetuin-agaroseaffinity columnsto purify the product the final concentration of the PT-biotin preparationsvaried from 1 to 10l@rnL as estimated by the HA assay. On average the recovery of PT-biotin was approximately 20% which was consistent with recoveries of [ 1251]-PTobtained on fetuin-agarose (13). However, unlike [1251]-PT, PT-biotin could be storedat 4°C for several months without any detectableloss of HA activity. The biological propertiesof the PT-biotin were further characterizedin the CHO cell assay. Approximately 0.5 ng/mL (minimum concentration) of both native PT and PT-biotin causedthe characteristicclustering of CHO cells (17) in two independenttests.This value correlated with the minimum concentration of PT previously found to causeCHO cell clustering (17). Moreover, this
n PT H
F 0
PT-biotin
0 1
0.33
0.66 Concentration
&g/mL)
m. Dosedependent lymphcqte proliferationby PTor PT-biotin.Mitogenesisassays were performedas describedpreviously (18) usingperipheralblood lymphocytes.Three different concentrations of eitherPTor PT-biotin(estimated by HA assay)wereutilized to comparetheir mitogenicproperties.Eachbar is a meanof six determinations + the standarddeviationof the mean. 1466
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result indicated that the enzymatic activity associated with the A-subunit of PT responsible for the characteristic CHO cell clustering (19) was not altered during the biotinylation reaction. Similar dose-dependent mitogenic activities were observed for underivatized PT and PT-biotin (Figure 1)
c
B
c
Figure. Fluorescent staining of Jurkat (A), Chinese hamster ovary cells (B) and Chinese hamster ovary LEC 1 mutant cells (C) using avidin-FITC. Panels Al, A3, Bl, B3, Cl and C3 are visible light micrographs of the corresponding fluorescence micrographs shown in panels A2, A4, B2, B4, C2 and C4. The wells in panels Al, A2, Bl, B2, Cl and C2 are negative controls (no PTbiotin) for those shown in panels A3, A4, B3, B4, C3 and C4. Magnification 400X.
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indicating that the biotinylation
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reaction had not affected the mitogenic activity found in the B
oligomer. Therefore, the concentration of biotinylated PT as determined by the goose HA assay was confirmed in both the CHO cell and mitogenicity assays. In order investigate the utility of PT-biotin further, we directly visualized PT-biotin binding to intact CHO and Jurkat cells using fluorescence microscopy
(Figure 2). Control experiments
utilizing a CHO cell LEC 1 mutant which expresses incomplete N-linked oligosaccharide structures on cell-surface glycoproteins were unable to bind PT-biotin. This result was consistent with earlier findings (9,lO) that CHO cell LEC mutants which lack terminal sialylactosamine oligosaccharide sequences required for high affinity PT binding, were less sensitive to the effects of PT. PT-biotin also bound to Jurkat cells, a cell line which has been useful for investigating lymphocyte receptors for PT (16). Binding inhibition ELISA experiments (Table 1) using glycoconjugates
terminating
in
sialylactosamine demonstrated that PI-biotin binds with similar specificity as [ 1251]-PT. The ability of unlabeled PT to compete effectively for PT-biotin binding suggests that both derivatized PT and underivatized PT recognize the same oligosaccharide sequences found on glycoconjugates. In summary,
PT-biotin
has been shown
to be a useful, non-radioactive
probe for
characterizing the lectin-like binding activity of PT. PT-biotin retains similar biological activities (in HA and lymphocyte proliferation assays) associated with the B oligomer of unmodified PT as well as possessing similar binding specificities as [ 12$labeled PT. PT-biotin has also been shown to be a useful derivative for examining PT binding to different cells and may be used in combination with fluorescent or heavy metal probes coupled to avidin to investigate PT binding by fluorescence or electron microscopy.
Based on the high affinity interaction of biotin for avidin, PT-biotin can
also be used in combination with avidin-agarose as a potential immunoprecipitation obtaining PT-receptor complexes.Work
technique for
in these areas are presently in progress.
Table 1 Concentrations of Glycoproteins or PI Resulting in 50% Inhibition of PT-biotin
and [ 12%]-F’T Binding
Glycoprotein Inhibitor
IC5O(cLM) FT-bioti
ICejO
fibrinogen ceruloplasmin fetuin transferrin laminin unlabeled-ET
0.36 310.04 l.lf0.7 1.0fO.l 28+12 0.9 f 0.2 2.24*
0.29 it 0.09 1.17* 0.39 f 0.34 43+3 N.D. 3.79 f2.39
* One determination only. ** Previously published results (1.5). N.D. not determined.
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ACKNOWLEDGMENTS This work was supported by a grant (MT 10220) from the Medical Research Council of Canada. C.G.C was supported by a studentship from the Alberta Heritage Foundation for Medical Research. REFERENCES 1. Munoz, 3.J. (1985) in Pertussis Toxin (R. Sekura, J.Moss, and M. Vaughan, Eds.) pp 1-18. Academic Press, Orlando, FL. 2. Reisine, T. (1990) Biochem. Pharmacol. 39, 1499-1504. 3. Ribiero-Neto, F.A.P., and Rodbell, M. (1989) Proc. Natl. Acad. Sci. USA 86, 2577-2581. 4. Tuomanen, E., Towbin, H., Rosenfelder, G., Braun, D., Larson, G., Hansson, G.C., and Hill, R. (1988) J. Exp. Med. 168, 267-277. 5. Armstrong, G.D., Howard, L.A., and Peppler, M.S. (1988) J. Biol. Chem. 263, 8677-8684. 6. Sekura, R., and Zhang, Y. (1985) in Pertussis Toxin (R. Sekura, J.Moss, and M. Vaughan, Eds.) pp 45-64. Academic Press, Orlando, FL. 7. Tamura, M., Nogimori, K., Yajima, M., Ase, K., and Ui, M. (1983) J. Biol. Chem. 258, 6756-6761. 8. Strnad, CF., Lin, W.-Q., and Carchman, R.A. (1989) Immunology 66, 539-545. 9. Witvleit, M.H., Bums, D.L., Brennan, M.J., Poolman, J.T., and Manclark, C.R. (1989) Infect. Immun. 57,3324-3330. 10. Brennan, M.J., David, J.L., Kenimer, J.G., and Manclark, CR. (1988) J.Biol. Chem. 263, 4895-4899. 11. Kenimer, J.G., Kim, K.J., Probst, P.G., Manclark, C.R., Burstyn, D.G., and Cowell, J.L. (1989) Hybridoma 8,37-5 1. 12. Presentini, R., Perin, F., Ancilli, G., Nucci, D., Bartoloni, A., Rappuoli, R., and Antoni, G. (1989) Mol. Immun. 26,95-100. 13. Armstrong, G.D., and Peppler, M.S. (1987) Infect. Immun. 55, 1294-1299. 14. Tyrrell, G.J., Peppler, M.S., Bonnah, R.A., Clark, C.G., Chong, P., and Armstrong, G.D. (1989) Infect. Immun. 57, 1854-1857. 15. Heerze, L.D., and Armstrong, G.D. (1990) Biochem. Biophys. Res. Commun. 172, 12241229. 16. Clark, C.G., and Armstrong, G.D. (1990) Infect. Immun. 58, 3840-3846. 17. Hewlett, E.1.. Sauer, K.T., Myers, G.A., Cowell, J.L., and Guerrant, R.L. (1983) Infect. Immun. 40,1198-1203. 18. Salonen, R., Ilonen, J., and Salmi, A.A. (1989) Exp. Immunol. 75, 376-380. 19. Burns, D.L., Kenimer, J.G., and Manclark, C.R. (1987) Infect. Immun. 55, 24-28,
1469
BIOCHEMICAL
SYNTHESIS
AND BIOPHYSICAL RESEARCH COMMUNICATIONS Pages 1464-l 469
AND CHARACTERIZATION OF A PERTUSSIS TOXIN-BIOTIN CONJUGATE
Louis D. Heerze1* , Clifford G. Clarkl, Ying Chenl, Richard H. Smith2, and Glen D. Armstrong1* 1Departmentof Medical Microbiology and Infectious Diseases,University of Alberta, Edmonton, Alberta, CanadaT6G 2H7 2ChembiomedLtd., Edmonton, Alberta, Canada Received
August
9,
1991
SUMMARY: We prepareda Pertussistoxin-biotin conjugateandfound its biological propertiesto be similar to thoseof native Pertussistoxin with respectto the hemagglutination,Chinesehamster ovary cell, and lymphocyte proliferation assays.Direct binding to Chinesehamster ovary and Jurkat cells was observedusing fluorescencemicroscopy. Pertussistoxin-biotin was alsofound to possesssimilar glycoconjugate binding specificitiesasthoseof 1251-labeledPertussistoxin . 0 1991
Academx
Press,
Inc.
Pertussistoxin (PT) is one of the important virulence factors produced by Bordetella pertussis, the etiological agent of whooping cough (1). PI, like other A-B type toxins containsan enzymatically active A subunit that hasADP-ribosyltransferase activity which is responsiblefor modifying guanine nucleotide-regulatory proteins (G proteins) in eukaryotic cells (2,3). The complex B oligomer of PI is responsiblefor the lectin-like binding activity of the toxin to receptors on goose erythrocytes, Chinesehamsterovary (CHO) cells, T lymphocytes, and other host cell membranes(4- 10). The B oligomer may alsocontribute to the attachmentof Bordetefia pertussis organismsto epithelial cells lining the upper respiratory tract of humans(4); the only known host of Bordetella pertussis . It is therefore important to identify and characterize receptors for PT in order to fully understandits activity in the diseaseprocess. Researchefforts aimedat characterizingthe lectin-like binding activity of the B oligomer of PT have utilized either antibodies directed against PT (11,12) or the use of radiolabeled toxin (5,13-15). Disadvantages of using antibodies center around the necessity of having to obtain specific antibodiesthat recognize epitopesoutsidePT receptor binding domains.Although, Iodine125 labeled PT hasproved to be a useful probe for characterizing the receptor binding activity of * To whom correspondenceshouldbe addressed. m PT Pertussistoxin, ADP adenosinediphosphate, CHO Chinese hamster ovary, HA hemagglutination, PBS phosphatebuffered saline,BSA bovine serumalbumin, PBST phosphatebuffered salinecontaining Tween 20, ABTS 2, 2’-azino-bis (3-ethylbenzthiazoline- 6sulfonic acid), FBS fetal bovine serum,FlTC fluoresceinisothiocyanate. 0006-291X/91 $1.50 Copyright 0 1991 by Academic Press, Inc. All rights of reproduction in any form reserved.
1464
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[1251]-PT only retains its activity for short periods. Therefore,
we sought an
alternative to iodinated PT for use in receptor studies. We chose to label PT with biotin since biotin has been successfully used in ELISA systems as well as immunochemical staining techniques. The added advantage of utilizing a biotin system is in the high affinity interaction with avidin. Avidin can be readily obtained conjugated to enzymes such as peroxidase which can be utilized in ELISA experiments with PT-biotin, or they may be conjugated to fluorescent probes that can be used to determine PT-biotin binding to different cell types. Highly specific anti-biotin antibody enzyme conjugates are also available to characterize PTbiotin’s lectin-like binding activity. In the present study PT-biotin was found to possess similar biological properties to those of native PT as was determined in the hemagglutination
(HA),
CHO cell and lymphocyte
proliferation assays. Binding inhibition experiments indicated that the carbohydrate binding specificities of PT-biotin were similar to those of [ 1251]-PT. Direct binding to CHO and Jurkat cells was also observed using fluorescence microscopy.
EXPERIMENTAL
PROCEDURES
Materials; All of the reagents were obtained from Sigma except for: PT and human ceruloplasmin which were a gift from the Connaught Centte for Biotechnology Research, Sulfo-NHS-biotin was obtained from Pierce, and a-Chymotrypsin was from Calbiochem-Behring. Goose blood was from Gibmar Laboratories and Jurkat cells were kindly provided by Dr. V. Paetkau, Dept. of Biochemistry, University of Alberta. LEC 1 CHO cells (ATCC CRL 1735) were from ATCC. Preparation sf PT-biotic PT (100 uL,190 pg) in 50 mM phosphate buffer, pH 7.5. containing 50% glycerol was sonicated prior to use, and then diluted with 100 pL of 0.1 M borate buffer, pH 8.5. Sulfo-NHS-biotin (20 pL, 2 mg./mL in H20) was added to the PT solution and the reaction was allowed to proceed for 30 min at room temperature. Subsequently, aliquots (50 pL) of the PTbiotin were incubated with 100 l.tL volumes of washed fetuin-agarose for 60 min at room temperature. Mixtures were then transferred to glass wool-plugged Pasteur pipets, washed with PBS (20 mL), and eluted with 200 pL volumes of 50 mM diethanolamine buffer, pH 11.5, as previously described (16). The various biological activities of the PT-biotin preparation were then tested as described below. Hemdutination and CHO Cell AssavG Chymotrypsin-treated goose erythrocytes and Chinese hamster ovary (CHO) cells were used in their respective assays (13,17) to compare the endpoint dilutions of the PT-biotin preparations with that of underivatized PT . Lvmohocvte Proliferation Assavs: Routine mitogenesis assays were performed as described previously (18) by measuring the incorporation of r3H]-thymidine into peripheral blood lymphocytes. Binding Inhibition Assa-w Microtiter wells were coated with 100 l.tL of fetuin (50 pg/mL) in 50 mM sodium phosphate buffer (pH 6.8) containing 5 mM MgC12 and 15 mM NaN3 for 16 hr at 4°C. The solution was removed by aspiration and replaced with 100 pL of 1% BSA in PBS containing 0.05% Tween 20 (PBST). After incubation for 4 hr at room temperature, the microtiter wells were washed four times with 300 l.tL of PBST . Glycoprotein inhibitors ranging from 10w4to 10m8M or PT (0.2-7pM) in PBS (40 uL) were added to each well and PT-biotin (10 pL, diluted l/20 in PBST) was then added to the microtiter wells. After incubating for 1 hr the binding reaction was stopped by aspirating the solutions and the plate was washed with PBST (4X3OOpL). Horseradish peroxidase conjugated anti-biotin antiboby (lOOpL, l/800 dilution in 1% BSA in PBST) was then added to the wells. The plates were incubated at ambient temperature for 2 hr. Avidin-peroxidase (5OpL of a l/3000 dilution in PBST for 1 hr) could also be used. After washing the wells as described above, the substrate solution (1 mM ABTS in 5 mM citrate buffer, pH 4.2, containing 0.1% hydrogen peroxide, v/v) was added and the plates were incubated for 30 min. Color development was recorded at 405 nm using a Titertek Multiskan MC plate reader. Maximum binding was determined in the absence of glycoprotein inhibitor and background binding was measured in wells coated with BSA only. Binding assays for each inhibitor concentration were 1465
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done in at least duplicate and the average value varied less than 15%. The concentration of glycoprotein required for 50% inhibition (IC,,) wasdeterminedby plotting the amount of binding observed in the presenceof glycoprotein inhibitor asa percent of the maximum binding achieved without . inhibitor. . . . lotin BlndmP to Ju kat Cells: Jurkat cells grown in RPM1 1640 (Gibco) media containing 10% fetal bovine serum[FBS) were washedfour timeswith PBS, counted, and suspendedto give 1 x lo7 cells/ml in PBS. PT-biotin (0.2 pg in 20 uL PBS) was addedto 100 pL (1 x 106) Jurkat cells and incubatedon ice for 90 min with occasionalshaking.PBS (20 pL) wasaddedto the same number of cells to provide a negative control. After incubation, the cell suspensionswere washed twice with 3 mL of cold PBS. Avidin-FlTC (50 pL, l/100 in PBS) was then added and the cells were allowed to incubate for 30 min on ice. Cell suspensions were again washedtwice with cold PBS, and wet mounts were prepared for fluorescence microscopy using a Leitz Laborlux K fluorescencemicroscope. PT-biotin Bindim to CHO Cells; Wild type and LEC 1 CHO cells were grown on 10 well Linbro (Flow) glassmicroscope slides in Ham’s F-12 medium (Gibco) supplementedwith 10% FBS. Monolayers adherent to the glassslideswere washedgently with approximately 10 mL of PBS, PT-biotin (0.2 ug in 10 uL PBS) wasadded,and the mixtures were allowed to incubate on ice for 90 min. Controls were prepared using either 20 pL of PBS without PT-biotin or 0.2 pg of underivatized PT. Control experimentswere alsodoneusing a CHO cell LEC 1 mutant. The CHO cell monolayers were washedagain with PBS and incubated with avidin-FITC (20 pL, l/100 in PBS) on ice for an additional 30 min. After a final washwith 15 mL of cold PBS, the CHO cells were preparedfor fluorescencemicroscopyasdescribedabove. RESULTS
AND DISCUSSION
When PT-biotin waspreparedasdescribedabove usingfetuin-agaroseaffinity columnsto purify the product the final concentration of the PT-biotin preparationsvaried from 1 to 10l@rnL as estimated by the HA assay. On average the recovery of PT-biotin was approximately 20% which was consistent with recoveries of [ 1251]-PTobtained on fetuin-agarose (13). However, unlike [1251]-PT, PT-biotin could be storedat 4°C for several months without any detectableloss of HA activity. The biological propertiesof the PT-biotin were further characterizedin the CHO cell assay. Approximately 0.5 ng/mL (minimum concentration) of both native PT and PT-biotin causedthe characteristicclustering of CHO cells (17) in two independenttests.This value correlated with the minimum concentration of PT previously found to causeCHO cell clustering (17). Moreover, this
n PT H
F 0
PT-biotin
0 1
0.33
0.66 Concentration
&g/mL)
m. Dosedependent lymphcqte proliferationby PTor PT-biotin.Mitogenesisassays were performedas describedpreviously (18) usingperipheralblood lymphocytes.Three different concentrations of eitherPTor PT-biotin(estimated by HA assay)wereutilized to comparetheir mitogenicproperties.Eachbar is a meanof six determinations + the standarddeviationof the mean. 1466
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result indicated that the enzymatic activity associated with the A-subunit of PT responsible for the characteristic CHO cell clustering (19) was not altered during the biotinylation reaction. Similar dose-dependent mitogenic activities were observed for underivatized PT and PT-biotin (Figure 1)
c
B
c
Figure. Fluorescent staining of Jurkat (A), Chinese hamster ovary cells (B) and Chinese hamster ovary LEC 1 mutant cells (C) using avidin-FITC. Panels Al, A3, Bl, B3, Cl and C3 are visible light micrographs of the corresponding fluorescence micrographs shown in panels A2, A4, B2, B4, C2 and C4. The wells in panels Al, A2, Bl, B2, Cl and C2 are negative controls (no PTbiotin) for those shown in panels A3, A4, B3, B4, C3 and C4. Magnification 400X.
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indicating that the biotinylation
AND
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reaction had not affected the mitogenic activity found in the B
oligomer. Therefore, the concentration of biotinylated PT as determined by the goose HA assay was confirmed in both the CHO cell and mitogenicity assays. In order investigate the utility of PT-biotin further, we directly visualized PT-biotin binding to intact CHO and Jurkat cells using fluorescence microscopy
(Figure 2). Control experiments
utilizing a CHO cell LEC 1 mutant which expresses incomplete N-linked oligosaccharide structures on cell-surface glycoproteins were unable to bind PT-biotin. This result was consistent with earlier findings (9,lO) that CHO cell LEC mutants which lack terminal sialylactosamine oligosaccharide sequences required for high affinity PT binding, were less sensitive to the effects of PT. PT-biotin also bound to Jurkat cells, a cell line which has been useful for investigating lymphocyte receptors for PT (16). Binding inhibition ELISA experiments (Table 1) using glycoconjugates
terminating
in
sialylactosamine demonstrated that PI-biotin binds with similar specificity as [ 1251]-PT. The ability of unlabeled PT to compete effectively for PT-biotin binding suggests that both derivatized PT and underivatized PT recognize the same oligosaccharide sequences found on glycoconjugates. In summary,
PT-biotin
has been shown
to be a useful, non-radioactive
probe for
characterizing the lectin-like binding activity of PT. PT-biotin retains similar biological activities (in HA and lymphocyte proliferation assays) associated with the B oligomer of unmodified PT as well as possessing similar binding specificities as [ 12$labeled PT. PT-biotin has also been shown to be a useful derivative for examining PT binding to different cells and may be used in combination with fluorescent or heavy metal probes coupled to avidin to investigate PT binding by fluorescence or electron microscopy.
Based on the high affinity interaction of biotin for avidin, PT-biotin can
also be used in combination with avidin-agarose as a potential immunoprecipitation obtaining PT-receptor complexes.Work
technique for
in these areas are presently in progress.
Table 1 Concentrations of Glycoproteins or PI Resulting in 50% Inhibition of PT-biotin
and [ 12%]-F’T Binding
Glycoprotein Inhibitor
IC5O(cLM) FT-bioti
ICejO
fibrinogen ceruloplasmin fetuin transferrin laminin unlabeled-ET
0.36 310.04 l.lf0.7 1.0fO.l 28+12 0.9 f 0.2 2.24*
0.29 it 0.09 1.17* 0.39 f 0.34 43+3 N.D. 3.79 f2.39
* One determination only. ** Previously published results (1.5). N.D. not determined.
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ACKNOWLEDGMENTS This work was supported by a grant (MT 10220) from the Medical Research Council of Canada. C.G.C was supported by a studentship from the Alberta Heritage Foundation for Medical Research. REFERENCES 1. Munoz, 3.J. (1985) in Pertussis Toxin (R. Sekura, J.Moss, and M. Vaughan, Eds.) pp 1-18. Academic Press, Orlando, FL. 2. Reisine, T. (1990) Biochem. Pharmacol. 39, 1499-1504. 3. Ribiero-Neto, F.A.P., and Rodbell, M. (1989) Proc. Natl. Acad. Sci. USA 86, 2577-2581. 4. Tuomanen, E., Towbin, H., Rosenfelder, G., Braun, D., Larson, G., Hansson, G.C., and Hill, R. (1988) J. Exp. Med. 168, 267-277. 5. Armstrong, G.D., Howard, L.A., and Peppler, M.S. (1988) J. Biol. Chem. 263, 8677-8684. 6. Sekura, R., and Zhang, Y. (1985) in Pertussis Toxin (R. Sekura, J.Moss, and M. Vaughan, Eds.) pp 45-64. Academic Press, Orlando, FL. 7. Tamura, M., Nogimori, K., Yajima, M., Ase, K., and Ui, M. (1983) J. Biol. Chem. 258, 6756-6761. 8. Strnad, CF., Lin, W.-Q., and Carchman, R.A. (1989) Immunology 66, 539-545. 9. Witvleit, M.H., Bums, D.L., Brennan, M.J., Poolman, J.T., and Manclark, C.R. (1989) Infect. Immun. 57,3324-3330. 10. Brennan, M.J., David, J.L., Kenimer, J.G., and Manclark, CR. (1988) J.Biol. Chem. 263, 4895-4899. 11. Kenimer, J.G., Kim, K.J., Probst, P.G., Manclark, C.R., Burstyn, D.G., and Cowell, J.L. (1989) Hybridoma 8,37-5 1. 12. Presentini, R., Perin, F., Ancilli, G., Nucci, D., Bartoloni, A., Rappuoli, R., and Antoni, G. (1989) Mol. Immun. 26,95-100. 13. Armstrong, G.D., and Peppler, M.S. (1987) Infect. Immun. 55, 1294-1299. 14. Tyrrell, G.J., Peppler, M.S., Bonnah, R.A., Clark, C.G., Chong, P., and Armstrong, G.D. (1989) Infect. Immun. 57, 1854-1857. 15. Heerze, L.D., and Armstrong, G.D. (1990) Biochem. Biophys. Res. Commun. 172, 12241229. 16. Clark, C.G., and Armstrong, G.D. (1990) Infect. Immun. 58, 3840-3846. 17. Hewlett, E.1.. Sauer, K.T., Myers, G.A., Cowell, J.L., and Guerrant, R.L. (1983) Infect. Immun. 40,1198-1203. 18. Salonen, R., Ilonen, J., and Salmi, A.A. (1989) Exp. Immunol. 75, 376-380. 19. Burns, D.L., Kenimer, J.G., and Manclark, C.R. (1987) Infect. Immun. 55, 24-28,
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