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Identification of surface proteins on bovine leukocytes by a biotin-avidin protein blotting technique
Journal oflmmunologicalMethods, 85 (1985) 195-202
195
Elsevier JIM03721
Identification of Surface Proteins on Bovine Leukocytes by a Biotin-Avidin Protein Blotting Technique Walter L. Hurley *, Eve Finkelstein and Brent D. Holst Department of Animal Sciences, University of Illinois, Urbana, IL 61801. U.S.A.
(Received 18 July 1985, accepted 27 August 1985)
A method has been developed which covalently attaches biotin to proteins on the outer surface of leukocytes. These proteins are separated by polyacrylamide gel electrophoresis and transferred to nitrocellulose by protein blotting. Labeled proteins are detected using an avidin-peroxidase conjugate and color indicator. The method has been used to identify surface proteins specific to either polymorphonuclear leukocytes or mononuclear leukocytes prepared from bovine peripheral blood. The method provides a highly sensitive, non-radioactive means of examining alterations of surface proteins on leukocytes under differing functional and physiological conditions. Cell viability is not altered by this labeling method and labeled cells can be used to examine functions of surface proteins. Key words: biotin -avidin - cell surface proteins - protein blotting
Introduction L e u k o c y t e function is extensively m e d i a t e d b y p r o t e i n c o m p o n e n t s on the cell's surface. These p r o t e i n s include receptors for i m m u n o g l o b u l i n s , c o m p l e m e n t , enzymes, c h e m o t a c t i c factors and hormones, as well as o t h e r leukocyte antigens such as the m a j o r h i s t o c o m p a t i b i l i t y c o m p l e x proteins. Studies of leukocyte surface antigens have generally consisted of defining i n d i v i d u a l surface p r o t e i n s b y imm u n o c h e m i c a l techniques such as i m m u n o a f f i n i t y c h r o m a t o g r a p h y (Strosberg, 1984), antigen detection p r o t e i n blots ( R e n a r t et al., 1979; T o w b i n et al., 1979; Burnette, 1981), quantitative d o t - i m m u n o b i n d i n g assay (Jahn et al., 1984) a n d i m m u n o h i s tochemical localization (Pool et al., 1983). F u r t h e r , i m m u n o a f f i n i t y c h r o m a t o g r a p h y has been used to p u r i f y the rat l e u k o c y t e - c o m m o n antigen, from which a p a r t i a l a m i n o acid sequence was used in the synthesis of a c o r r e s p o n d i n g oligonucleotide p r o b e to identify a c D N A for this surface antigen ( T h o m a s et al., 1985). Such m e t h o d s p r o v i d e a basis for s t u d y of the m o l e c u l a r b i o l o g y of surface proteins.
* To whom correspondence should be addressed. 0022-1759/85/$03.30 © 1985 Elsevier Science Publishers B.V. (Biomedical Division)
196
While these methods specifically identify individual surface antigens, approaches which identify the spectrum of leukocyte surface proteins have not been extensively developed. Such a method of labeling all surface antigens would provide a means of examining the relationships between numerous surface proteins and could be combined with immunological approaches to define the interaction between individual antigens and other surface proteins. Here we describe a non-radioactive method for labeling and identifying leukocyte surface proteins which utilizes the high binding affinity of the biotin-avidin complex (dissociation constant, K d - 1 0 15 M). This is used to characterize patterns of surface proteins from different cell types by gel electrophoresis. The method also could be used in attempts to purify certain surface proteins. Cells labeled by this method remain viable and could be used for functional studies involving the surface proteins.
Materials and Methods
Leukocyte collection and separation Heparinized peripheral blood was collected from several cows by venipuncture. Blood was centrifuged and the plasma was removed. Red cells (1 vol.) were lysed by adding 2 vols. of cold distilled water, and mixing gently for 50 s on ice, immediately followed by the addition of 1 vol. of cold 15 mM potassium phosphate, 450 mM sodium chloride, pH 6.8. Leukocytes were centrifuged at 400 x g for 10 min at 4°C, and resuspended in phosphate-buffered saline (PBS, 10 mM potassium phosphate, 150 mM sodium chloride, pH 7.4). Remaining erythrocytes were lysed as before, if necessary. Leukocytes were washed once with PBS and resuspended to a final concentration of 50 x 106/ml with PBS. For separation of polymorphonuclear leukocytes and mononuclear leukocyte fractions, total leukocyte preparations were centrifuged on 5 ml of Histopaque 1077 (Sigma Chemical Co., St. Louis, MO) at 400 x g for 45 min at 22°C. The interface layer (mononuclear leukocytes) and pellet (polymorphonuclear leukocytes) were washed twice with PBS and resuspended to 50 x 106/ml with PBS. Viability of preparations used in biotin labeling were 85-95% live cells as determined by exclusion of trypan blue. Differential cell counts were made on total leukocyte preparations and on Histopaque separated fractions using Wright's differential stain (Camco Quick Stain, American Scientific Products, McGaw Park, IL).
Biotin labeling leukocytes About 5 X 10 6 cells were diluted to 0.5 ml with PBS. Generally, 100 fig of biotin labeling reagent (Sulfo-NHS-Biotin, Pierce Chemical Co., Rockford, IL, 10 m g / m l in dimethylsulfoxide) was added to the cells and incubated 10 min at 22°C with shaking. One hundred/~g of biotin reagent provided optimal labeling while minimizing background color development in the detection procedure. Satisfactory labeling was obtained with 50 /~g biotin reagent, but 10 fig reagent per reaction gave insufficient labeling. Labeled cells were centrifuged at 400 x g for 2 min in a
197 Beckman Microfuge 11 and washed once with PBS. Cells then were disrupted by brief sonication (10 s, Tekmar Sonic Disruptor, Tekmar Co., Cincinnati, OH) and washed twice with PBS. This step was found to reduce the background color development during detection. The final pellet was resuspended in 100 ffl distilled water, 20 ffl of sample buffer (25% 2-mercaptoethanol, 14.5% sodium dodecylsulfate, SDS, and 0.28 M Tris, pH 6.8) and 20 ffl dye solution (70% glycerol, with 0.12% bromphenol blue), in preparation for gel electrophoresis. Cell viability was determined as above on intact biotin-labeled cells that had been washed twice with PBS. Viability of biotin-labeled cells was not changed when compared to non-biotin-labeled cells.
Gel electrophoresis, protein blotting and detection Samples were boiled 5-10 min and generally 10 ffl were loaded per gel lane (equivalent to 3.5 x 105 cells). Samples were electrophoresed on 12.5% or 15% SDS-polyacrylamide slab gels (8 x 10 cm, 0.8 mm thick, Idea Scientific, Corvallis, OR), as described by Laemmli (1970). Proteins were transferred to nitrocellulose (BA-85, Schleicher and Schuell, Keene, NH) by electroblotting (Transblot, BioRad, Richmond, CA) at 0.06 A for 12-14 h at 22°C or at 0.36 A for 3 h at 4°C, in 25 mM Tris, pH 8.3, 192 mM glycine, and 20% methanol (v/v). Nitrocellulose blots were blocked in 2% BSA (bovine serum albumin), 0.1% Triton X-100 for 30 min at 37°C. Blots were incubated for 30 min at 37°C in avidin-horseradish peroxidase (HRP) conjugate (Miles-Yeda, Rehovot, Israel) at a concentration of 3.5 ffg/ml PBS; usually 2 ml were used per 8 x 10 cm blot. Blots then were washed 3 times with 0.1% BSA, 0.05% Tween 20 in PBS, for 5 min per wash, at 37°C. Bound avidin-peroxidase was detected as follows: for each blot, 5 ml of a freshly prepared solution containing 2.5 mg diaminobenzidine tetrahydrochloride (DAB; Sigma, St. Louis, MO), 10 mM Tris, pH 7.5 and 0.84 mM cobalt chloride was incubated for 10 min on ice in the dark. Hydrogen peroxide, 7.5/~1 of a 30% solution, was added to the DAB solution, mixed and immediately applied to the blot in a flat-bottom tray. The D A B / h y d r o gen peroxide solution was washed back and forth over the blot until the desired exposure was obtained (usually 0.5-1 min). The developed blot was rinsed well in tap water and dried for 5-10 min at 80°C. A useful method of preserving the blots was to place the blot face down on an acetate-sheet transparency pre-cut to a size larger than the blot, then carefully sealing the blot to the transparency with transparent adhesive plastic. This preserves the fragile nitrocellulose in a durable, yet clearly visible form.
Results
Biotin-labeling procedure and avidin-HRP detection A typical electrophoresis pattern of surface proteins labeled with biotin and detected by avidin-HRP is shown in Fig. 1, lanes 1 and 2 for total bovine peripheral blood leukocytes. Lane 3 in Fig. 1 shows the level of non-specific color development when unlabeled leukocytes are blotted and treated with avidin-HRP. In experiments
198
1
2
3
4 Mw -68 -43
-25.7
- 18.4
- 14.3
Fig. 1. Electrophoresis of proteins from biotin-labeled, unlabeled and trypsinized cells. Lanes 1 and 2 are from biotin-labeled cells, lane 3 from unlabeled cells, and lane 4 from cells that were trypsin digested after biotin labeling. Samples in all lanes are total peripheral blood leukocytes. Electrophoresis was on a 15% gel. Molecular weights (Mw) are shown × 10 -3. Details of procedures were as described in Materials and Methods, and in Results. where identical blots of labeled and unlabeled proteins were incubated with avidin only, H R P only, avidin-HRP conjugate, or neither avidin or H R P , only when a v i d i n - H R P was used were protein bands observed (data not shown). Assessment of sonication after the biotin-labeling step revealed that such sonication reduced the background color development on the blot (data not shown). This p r o b a b l y was due to the reduction in total protein in the sample after sonication, where about 14/~g total protein of nonsonicated samples were loaded per gel lane c o m p a r e d to 4 / z g for sonicated samples. Biotin labeling does not substantially alter migration on electrophoresis under the denaturing conditions used in this procedure. The m e t h o d is highly sensitive: as little as 1 ng of a purified, biotin-labeled protein can be detected (data not shown). Viability of biotin-labeled cells, as determined in several experiments, was equal to that of unlabeled cells.
Labeling surface proteins A key c o m p o n e n t of this procedure is the degree to which the biotin labeling is specific for external cell-surface proteins vs. internal proteins. Fig. 1, lane 4 shows the effect of digestion of previously biotin-labeled cells with trypsin (0.5 m g / m l , 15 min, 37°C) where the detection of labeled proteins was reduced to background
199
1
2
3
4
5
6 IIw
,ram,
-43
-14
-1113
Fig. 2. Electrophoresis of biotin labeled proteins in disrupted and intact leukocytes. Odd numbered lanes contain proteins from cells disrupted (sonicated) prior to labeling and even numbered lanes contain proteins from cells labeled intact. Each pair of lanes (1 and 2, 3 and 4, 5 and 6) are from individual cows. The arrows indicate proteins specifically labeled in the disrupted cells. Electrophoresis was on a 12.5% gel. Molecular weights (Mw) are shown x l 0 -3. Procedures were as described in Materials and Methods.
levels. Specific labeling of external proteins also is demonstrated in Fig. 2. Total peripheral blood leukocytes were sonicated before labeling and compared with intact biotin-labeled cells. Several major proteins were labeled in the disrupted cells which were minor components in intact cells. These bands are presumed to be major internal proteins which are accessible to the biotin reagent upon disruption of the cells.
Identifying leukocyte subtype-specific proteins The biotin-labeling method was used to identify leukocyte surface proteins specific for cell types. Polymorphonuclear leukocyte and mononuclear leukocyte fractions were separated on Ficoll-Hypaque. Ceils from these fractions were biotinlabeled and typical protein profiles are shown in Fig. 3. Several protein bands were found to be specific for polymorphonuclear leukocytes, including bands of molecular weights 81,000, 20,000, and 13,500. Although several proteins specific for mononuclear leukocytes have been observed, these often are not as distinct as the
200
MW
-68 -43
-26.7
P-18.4
P-
Fig. 3 Electrophoresis of biotin labeled proteins from different leukocyte cell types. Odd numbered lanes are from polymorphonuclear leukocyte fractions and even numbered lanes from mononuclear leukocyte fractions. Each pair of lanes (1 and 2, 3 and 4) are from individual cows. P indicates polymorphonuclear specific proteins and M identifies mononuclear specific protein. Electrophoresis was on a 15% gel. Molecular weights (Mw) are shown X10-‘. Procedures were as described in Materials and Methods.
polymorphonuclear specific bands because of the contamination of the polymorphonuclear leukocyte fractions with mononuclear leukocytes, and because of the more diverse population of cell types in the mononuclear leukocytes. Typically, separations on the Histopaque 1077 yielded 77% polymorphonuclear and 23% mononuclear cells in the polymorphonuclear fraction (pellet), and 8% polymorphonuclear and 92% mononuclear cells in the mononuclear fraction (interface). One protein identified as enriched in mononuclear leukocytes is identified in Fig. 3 (molecular weight about 63,000).
Discussion A method has been developed to specifically label surface proteins of leukocytes with a non-radioactive probe, which can be detected with avidin-HRP. The labeling reagent used in this procedure is a N-hydroxysuccinimide ester derivative of biotin which binds covalently to lysine residues of proteins (Bayer and Wilchek, 1980). The method takes advantage of the reactivity of this derivative for lysine residues in the
201 labeling step, and the high binding affinity of biotin and avidin (Kd - 10 1~ M) (Green, 1975) in the detection step. To study surface proteins, the labeling of internal proteins must be minimal. Bovine peripheral blood leukocytes were used to compare the labeling of proteins from intact and disrupted cells. Several major bands were found primarily in disrupted cells and were only minor components of the intact cell proteins detected. These are presumed to be internal proteins and are probably membrane associated as the procedure would eliminate most soluble proteins in the post-labeling washes. In addition, trypsin treatment of biotin-labeled cells resulted in a protein pattern comparable to that observed in unlabeled cells. It is expected that trypsin primarily digests external proteins under these conditions. The results of trypsin treatment also confirm that the components visualized on blots are proteinaceous in nature. The comparison of labeled proteins from polymorphonuclear leukocytes and mononuclear leukocytes showed several distinct protein bands specific for cell types. The polymorphonuclear leukocyte fraction was primarily composed of polymorphonuclear neutrophils, some eosinophils and about 23% mononuclear cells, while the mononuclear fraction contained few polymorphonuclear leukocytes. The polymorphonuclear leukocyte specific proteins indicated in Fig. 3 are probably surface antigens of neutrophils, as these are the major cell type in this fraction. A number of proteins are common between the cell types. Identification of these proteins using this method can be integrated with immunological identification of specific surface antigens. For example, a set of monoclonal antibodies has been developed which are specific for bovine polymorphonuclear neutrophils (Nickerson et al., 1983). Combining the methodologies of immunoblotting (Towbin et al., 1979) with the surface labeling procedure described here will provide a more complete characterization of bovine neutrophil surface proteins. An interesting feature of this procedure is the maintenance of viability of biotin-labeled cells. Labeled cells thus can be used to study various cell functions, to examine the interaction of surface receptors with ligands, and to measure the turnover of surface proteins.
Acknowledgements This research was supported by Hatch project 35-0370 of the Illinois Agricultural Experiment Station, USDA Regional project NEl12-354, and a University of Illinois Undergraduate Research Award.
References Bayer, E.A. and M. Wilchek, 1980, Methods Biochem.Anal. 26, 1. Burnette, W.N., 1981, Anal. Biochem. 112, 195. Green, N.M., 1975, Adv. Protein Chem. 29, 85. Jahn, R., W. Schieblerand P. Greengard, 1984, Proc. Natl. Acad. Sci. U.S.A. 81, 1684.
202 Laemmli, U.K., 1970, Nature (London) 227, 680. Nickerson, S.C., R.P. Shapiro, A.J. Guidry, S. Srikumaran and R.A. Goldsby, 1983, J. Dairy Sci. 66, 1547 Pool, Chr. W., R.M. Buijs, D.F. Swaab, G.J. Boer and F.W. Van Leeuwen, 1983, in: Immunohistochem istry, ed. A.C. Cuello (Wiley, New York) p. 1. Renart, J., J. Reiser and G.R. Stark, 1979, Proc. Natl. Acad. Sci. U.S.A. 76, 3116. Strosberg, A.D., 1984, in: Receptor Biochemistry and Methodology, Vol. 2, eds. J.C. Venter and L.C Harrison (Alan R. Liss, New York) p. 1 Thomas, M.L., A.N. Barclay, J. Gagnon and A.F. Williams, 1985, Cell 41, 83. Towbin, H., T. Staehelin and J. Gordon, 1979, Proc. Natl. Acad. Sci. U.S.A. 76, 4350.
195
Elsevier JIM03721
Identification of Surface Proteins on Bovine Leukocytes by a Biotin-Avidin Protein Blotting Technique Walter L. Hurley *, Eve Finkelstein and Brent D. Holst Department of Animal Sciences, University of Illinois, Urbana, IL 61801. U.S.A.
(Received 18 July 1985, accepted 27 August 1985)
A method has been developed which covalently attaches biotin to proteins on the outer surface of leukocytes. These proteins are separated by polyacrylamide gel electrophoresis and transferred to nitrocellulose by protein blotting. Labeled proteins are detected using an avidin-peroxidase conjugate and color indicator. The method has been used to identify surface proteins specific to either polymorphonuclear leukocytes or mononuclear leukocytes prepared from bovine peripheral blood. The method provides a highly sensitive, non-radioactive means of examining alterations of surface proteins on leukocytes under differing functional and physiological conditions. Cell viability is not altered by this labeling method and labeled cells can be used to examine functions of surface proteins. Key words: biotin -avidin - cell surface proteins - protein blotting
Introduction L e u k o c y t e function is extensively m e d i a t e d b y p r o t e i n c o m p o n e n t s on the cell's surface. These p r o t e i n s include receptors for i m m u n o g l o b u l i n s , c o m p l e m e n t , enzymes, c h e m o t a c t i c factors and hormones, as well as o t h e r leukocyte antigens such as the m a j o r h i s t o c o m p a t i b i l i t y c o m p l e x proteins. Studies of leukocyte surface antigens have generally consisted of defining i n d i v i d u a l surface p r o t e i n s b y imm u n o c h e m i c a l techniques such as i m m u n o a f f i n i t y c h r o m a t o g r a p h y (Strosberg, 1984), antigen detection p r o t e i n blots ( R e n a r t et al., 1979; T o w b i n et al., 1979; Burnette, 1981), quantitative d o t - i m m u n o b i n d i n g assay (Jahn et al., 1984) a n d i m m u n o h i s tochemical localization (Pool et al., 1983). F u r t h e r , i m m u n o a f f i n i t y c h r o m a t o g r a p h y has been used to p u r i f y the rat l e u k o c y t e - c o m m o n antigen, from which a p a r t i a l a m i n o acid sequence was used in the synthesis of a c o r r e s p o n d i n g oligonucleotide p r o b e to identify a c D N A for this surface antigen ( T h o m a s et al., 1985). Such m e t h o d s p r o v i d e a basis for s t u d y of the m o l e c u l a r b i o l o g y of surface proteins.
* To whom correspondence should be addressed. 0022-1759/85/$03.30 © 1985 Elsevier Science Publishers B.V. (Biomedical Division)
196
While these methods specifically identify individual surface antigens, approaches which identify the spectrum of leukocyte surface proteins have not been extensively developed. Such a method of labeling all surface antigens would provide a means of examining the relationships between numerous surface proteins and could be combined with immunological approaches to define the interaction between individual antigens and other surface proteins. Here we describe a non-radioactive method for labeling and identifying leukocyte surface proteins which utilizes the high binding affinity of the biotin-avidin complex (dissociation constant, K d - 1 0 15 M). This is used to characterize patterns of surface proteins from different cell types by gel electrophoresis. The method also could be used in attempts to purify certain surface proteins. Cells labeled by this method remain viable and could be used for functional studies involving the surface proteins.
Materials and Methods
Leukocyte collection and separation Heparinized peripheral blood was collected from several cows by venipuncture. Blood was centrifuged and the plasma was removed. Red cells (1 vol.) were lysed by adding 2 vols. of cold distilled water, and mixing gently for 50 s on ice, immediately followed by the addition of 1 vol. of cold 15 mM potassium phosphate, 450 mM sodium chloride, pH 6.8. Leukocytes were centrifuged at 400 x g for 10 min at 4°C, and resuspended in phosphate-buffered saline (PBS, 10 mM potassium phosphate, 150 mM sodium chloride, pH 7.4). Remaining erythrocytes were lysed as before, if necessary. Leukocytes were washed once with PBS and resuspended to a final concentration of 50 x 106/ml with PBS. For separation of polymorphonuclear leukocytes and mononuclear leukocyte fractions, total leukocyte preparations were centrifuged on 5 ml of Histopaque 1077 (Sigma Chemical Co., St. Louis, MO) at 400 x g for 45 min at 22°C. The interface layer (mononuclear leukocytes) and pellet (polymorphonuclear leukocytes) were washed twice with PBS and resuspended to 50 x 106/ml with PBS. Viability of preparations used in biotin labeling were 85-95% live cells as determined by exclusion of trypan blue. Differential cell counts were made on total leukocyte preparations and on Histopaque separated fractions using Wright's differential stain (Camco Quick Stain, American Scientific Products, McGaw Park, IL).
Biotin labeling leukocytes About 5 X 10 6 cells were diluted to 0.5 ml with PBS. Generally, 100 fig of biotin labeling reagent (Sulfo-NHS-Biotin, Pierce Chemical Co., Rockford, IL, 10 m g / m l in dimethylsulfoxide) was added to the cells and incubated 10 min at 22°C with shaking. One hundred/~g of biotin reagent provided optimal labeling while minimizing background color development in the detection procedure. Satisfactory labeling was obtained with 50 /~g biotin reagent, but 10 fig reagent per reaction gave insufficient labeling. Labeled cells were centrifuged at 400 x g for 2 min in a
197 Beckman Microfuge 11 and washed once with PBS. Cells then were disrupted by brief sonication (10 s, Tekmar Sonic Disruptor, Tekmar Co., Cincinnati, OH) and washed twice with PBS. This step was found to reduce the background color development during detection. The final pellet was resuspended in 100 ffl distilled water, 20 ffl of sample buffer (25% 2-mercaptoethanol, 14.5% sodium dodecylsulfate, SDS, and 0.28 M Tris, pH 6.8) and 20 ffl dye solution (70% glycerol, with 0.12% bromphenol blue), in preparation for gel electrophoresis. Cell viability was determined as above on intact biotin-labeled cells that had been washed twice with PBS. Viability of biotin-labeled cells was not changed when compared to non-biotin-labeled cells.
Gel electrophoresis, protein blotting and detection Samples were boiled 5-10 min and generally 10 ffl were loaded per gel lane (equivalent to 3.5 x 105 cells). Samples were electrophoresed on 12.5% or 15% SDS-polyacrylamide slab gels (8 x 10 cm, 0.8 mm thick, Idea Scientific, Corvallis, OR), as described by Laemmli (1970). Proteins were transferred to nitrocellulose (BA-85, Schleicher and Schuell, Keene, NH) by electroblotting (Transblot, BioRad, Richmond, CA) at 0.06 A for 12-14 h at 22°C or at 0.36 A for 3 h at 4°C, in 25 mM Tris, pH 8.3, 192 mM glycine, and 20% methanol (v/v). Nitrocellulose blots were blocked in 2% BSA (bovine serum albumin), 0.1% Triton X-100 for 30 min at 37°C. Blots were incubated for 30 min at 37°C in avidin-horseradish peroxidase (HRP) conjugate (Miles-Yeda, Rehovot, Israel) at a concentration of 3.5 ffg/ml PBS; usually 2 ml were used per 8 x 10 cm blot. Blots then were washed 3 times with 0.1% BSA, 0.05% Tween 20 in PBS, for 5 min per wash, at 37°C. Bound avidin-peroxidase was detected as follows: for each blot, 5 ml of a freshly prepared solution containing 2.5 mg diaminobenzidine tetrahydrochloride (DAB; Sigma, St. Louis, MO), 10 mM Tris, pH 7.5 and 0.84 mM cobalt chloride was incubated for 10 min on ice in the dark. Hydrogen peroxide, 7.5/~1 of a 30% solution, was added to the DAB solution, mixed and immediately applied to the blot in a flat-bottom tray. The D A B / h y d r o gen peroxide solution was washed back and forth over the blot until the desired exposure was obtained (usually 0.5-1 min). The developed blot was rinsed well in tap water and dried for 5-10 min at 80°C. A useful method of preserving the blots was to place the blot face down on an acetate-sheet transparency pre-cut to a size larger than the blot, then carefully sealing the blot to the transparency with transparent adhesive plastic. This preserves the fragile nitrocellulose in a durable, yet clearly visible form.
Results
Biotin-labeling procedure and avidin-HRP detection A typical electrophoresis pattern of surface proteins labeled with biotin and detected by avidin-HRP is shown in Fig. 1, lanes 1 and 2 for total bovine peripheral blood leukocytes. Lane 3 in Fig. 1 shows the level of non-specific color development when unlabeled leukocytes are blotted and treated with avidin-HRP. In experiments
198
1
2
3
4 Mw -68 -43
-25.7
- 18.4
- 14.3
Fig. 1. Electrophoresis of proteins from biotin-labeled, unlabeled and trypsinized cells. Lanes 1 and 2 are from biotin-labeled cells, lane 3 from unlabeled cells, and lane 4 from cells that were trypsin digested after biotin labeling. Samples in all lanes are total peripheral blood leukocytes. Electrophoresis was on a 15% gel. Molecular weights (Mw) are shown × 10 -3. Details of procedures were as described in Materials and Methods, and in Results. where identical blots of labeled and unlabeled proteins were incubated with avidin only, H R P only, avidin-HRP conjugate, or neither avidin or H R P , only when a v i d i n - H R P was used were protein bands observed (data not shown). Assessment of sonication after the biotin-labeling step revealed that such sonication reduced the background color development on the blot (data not shown). This p r o b a b l y was due to the reduction in total protein in the sample after sonication, where about 14/~g total protein of nonsonicated samples were loaded per gel lane c o m p a r e d to 4 / z g for sonicated samples. Biotin labeling does not substantially alter migration on electrophoresis under the denaturing conditions used in this procedure. The m e t h o d is highly sensitive: as little as 1 ng of a purified, biotin-labeled protein can be detected (data not shown). Viability of biotin-labeled cells, as determined in several experiments, was equal to that of unlabeled cells.
Labeling surface proteins A key c o m p o n e n t of this procedure is the degree to which the biotin labeling is specific for external cell-surface proteins vs. internal proteins. Fig. 1, lane 4 shows the effect of digestion of previously biotin-labeled cells with trypsin (0.5 m g / m l , 15 min, 37°C) where the detection of labeled proteins was reduced to background
199
1
2
3
4
5
6 IIw
,ram,
-43
-14
-1113
Fig. 2. Electrophoresis of biotin labeled proteins in disrupted and intact leukocytes. Odd numbered lanes contain proteins from cells disrupted (sonicated) prior to labeling and even numbered lanes contain proteins from cells labeled intact. Each pair of lanes (1 and 2, 3 and 4, 5 and 6) are from individual cows. The arrows indicate proteins specifically labeled in the disrupted cells. Electrophoresis was on a 12.5% gel. Molecular weights (Mw) are shown x l 0 -3. Procedures were as described in Materials and Methods.
levels. Specific labeling of external proteins also is demonstrated in Fig. 2. Total peripheral blood leukocytes were sonicated before labeling and compared with intact biotin-labeled cells. Several major proteins were labeled in the disrupted cells which were minor components in intact cells. These bands are presumed to be major internal proteins which are accessible to the biotin reagent upon disruption of the cells.
Identifying leukocyte subtype-specific proteins The biotin-labeling method was used to identify leukocyte surface proteins specific for cell types. Polymorphonuclear leukocyte and mononuclear leukocyte fractions were separated on Ficoll-Hypaque. Ceils from these fractions were biotinlabeled and typical protein profiles are shown in Fig. 3. Several protein bands were found to be specific for polymorphonuclear leukocytes, including bands of molecular weights 81,000, 20,000, and 13,500. Although several proteins specific for mononuclear leukocytes have been observed, these often are not as distinct as the
200
MW
-68 -43
-26.7
P-18.4
P-
Fig. 3 Electrophoresis of biotin labeled proteins from different leukocyte cell types. Odd numbered lanes are from polymorphonuclear leukocyte fractions and even numbered lanes from mononuclear leukocyte fractions. Each pair of lanes (1 and 2, 3 and 4) are from individual cows. P indicates polymorphonuclear specific proteins and M identifies mononuclear specific protein. Electrophoresis was on a 15% gel. Molecular weights (Mw) are shown X10-‘. Procedures were as described in Materials and Methods.
polymorphonuclear specific bands because of the contamination of the polymorphonuclear leukocyte fractions with mononuclear leukocytes, and because of the more diverse population of cell types in the mononuclear leukocytes. Typically, separations on the Histopaque 1077 yielded 77% polymorphonuclear and 23% mononuclear cells in the polymorphonuclear fraction (pellet), and 8% polymorphonuclear and 92% mononuclear cells in the mononuclear fraction (interface). One protein identified as enriched in mononuclear leukocytes is identified in Fig. 3 (molecular weight about 63,000).
Discussion A method has been developed to specifically label surface proteins of leukocytes with a non-radioactive probe, which can be detected with avidin-HRP. The labeling reagent used in this procedure is a N-hydroxysuccinimide ester derivative of biotin which binds covalently to lysine residues of proteins (Bayer and Wilchek, 1980). The method takes advantage of the reactivity of this derivative for lysine residues in the
201 labeling step, and the high binding affinity of biotin and avidin (Kd - 10 1~ M) (Green, 1975) in the detection step. To study surface proteins, the labeling of internal proteins must be minimal. Bovine peripheral blood leukocytes were used to compare the labeling of proteins from intact and disrupted cells. Several major bands were found primarily in disrupted cells and were only minor components of the intact cell proteins detected. These are presumed to be internal proteins and are probably membrane associated as the procedure would eliminate most soluble proteins in the post-labeling washes. In addition, trypsin treatment of biotin-labeled cells resulted in a protein pattern comparable to that observed in unlabeled cells. It is expected that trypsin primarily digests external proteins under these conditions. The results of trypsin treatment also confirm that the components visualized on blots are proteinaceous in nature. The comparison of labeled proteins from polymorphonuclear leukocytes and mononuclear leukocytes showed several distinct protein bands specific for cell types. The polymorphonuclear leukocyte fraction was primarily composed of polymorphonuclear neutrophils, some eosinophils and about 23% mononuclear cells, while the mononuclear fraction contained few polymorphonuclear leukocytes. The polymorphonuclear leukocyte specific proteins indicated in Fig. 3 are probably surface antigens of neutrophils, as these are the major cell type in this fraction. A number of proteins are common between the cell types. Identification of these proteins using this method can be integrated with immunological identification of specific surface antigens. For example, a set of monoclonal antibodies has been developed which are specific for bovine polymorphonuclear neutrophils (Nickerson et al., 1983). Combining the methodologies of immunoblotting (Towbin et al., 1979) with the surface labeling procedure described here will provide a more complete characterization of bovine neutrophil surface proteins. An interesting feature of this procedure is the maintenance of viability of biotin-labeled cells. Labeled cells thus can be used to study various cell functions, to examine the interaction of surface receptors with ligands, and to measure the turnover of surface proteins.
Acknowledgements This research was supported by Hatch project 35-0370 of the Illinois Agricultural Experiment Station, USDA Regional project NEl12-354, and a University of Illinois Undergraduate Research Award.
References Bayer, E.A. and M. Wilchek, 1980, Methods Biochem.Anal. 26, 1. Burnette, W.N., 1981, Anal. Biochem. 112, 195. Green, N.M., 1975, Adv. Protein Chem. 29, 85. Jahn, R., W. Schieblerand P. Greengard, 1984, Proc. Natl. Acad. Sci. U.S.A. 81, 1684.
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