Neurochemical Research, Vol. 17, No. 10, 1992, pp. 1011-1014
Analysis of Con A-Binding Glycoproteins in Synaptosomal Membranes Noriaki Ishioka 1 a n d S u s u m u Kllrioka I
(Accepted February 7, 1992) Concanavalin A (Con A)-binding proteins obtained from solubilized synaptosomal membranes of bovine brain were analyzed by two-dimensional electrophoresis (2DE), and were identified by peroxidase conjugated Con A (Con A-peroxidase staining), after transfer from 2DE gel to nitrocellulose paper. The Con A-binding proteins were resolved up to 40 spots, ranging in isoelectric points (pI) from 4.5 to 8.0 and molecular weight (MW) from 10 kDa to 120 kDa. Most of the Con A-binding proteins were streaked across a pH gradient and/or exhibited as multiple spots, indicating broad charge and molecular weight heterogeneity. The presence of protein groups that showed high affinities for Con A were revealed. Most interesting group (named GP51), which consisted of seven spots separated horizontally in charge heterogeneity (pi5.85-7.5) with MW 51kDa, was characterized by its binding to an immobilized protein A gel. This implies that GP51 is related to immunoglobulins and/or GP51 may be a new member of the immunoglobulin supergene family. KEY WORDS: Synaptosomes; membrane glycoproteins; Con A-binding; protein A-binding; two-dimensional electrophoresis; immunoglobulin supergene family.
INTRODUCTION
purpose, we analyze total Con A-binding proteins of the membranes and especially Con A-binding proteins which have ability to associate with protein-A molecule.
The membrane glycoproteins in nervous system such as N-CAM (1,2), 1,1 protein (2-4), MAG (2,5), and fasciclin II (6,7), are neural cell adhesion molecules, and belong to the immunoglobulin supergene family (8,9). It is assumed that tlhese membrane glycoproteins take part in neuronal cell-to-cell interaction as in the case of recognition in immune system. The others such as fasciclin I (7,10), 5B4 antigen (11), gp27-30 (12) are in nerve growth cone. These glycoproteins also play important roles on neural cells. In this study, we focused to seek for some undiscovered immunoglobulin supergene family proteins involved in synaptosomal membranes. To attain this
EXPERIMENTAL PROCEDURE Materials. All of the reagents were generally of analytical grade. Ampholines (pH 3.5-10 and pH 3.5-5.0) were obtained from LKB (Bromma, Sweden). 3-[(3-Cholamidopropyl)dimethylammonio]-lpropane-sulfonate (CHAPS) for sohbilization of membranes was purchased from Wako Pure ChemicalIndustries (Tokyo, Japan). The molecular weight marker kit for 2DE was from Sigma (St. Louis, U.S.A.) and the isoelectric point marker kit for isoelectric focusing gel was from Pharmasia (Uppsala, Sweden). Concanavalin A (Con A) was purchased from Vector Laboratories (Burlingame, U.S.A.). Affi-Gel 10 (active ester agarose) was from BIO-RAD (Richmond, U.S.A.). For Con A-affinity chromatography,Con A-agarosewas coupled with Con A and Affi-Ge110according to an instructionof BIO-RAD. AffiGel 10 conjugatedprotein A was from BIO-RAD (Richmond,U.S.A.). a-Methyl-D-mannoside (a MM) was from Nacalai Tesque (Kyoto,
1 Division of Biochemist;~, Institute of Medical Science, The Jikei University School of Medicine, 3-25-8 Nishi-shinbashi, Minato-ku, Tokyo 105, Japan. 1011
0364-3190/92/1000-1011506.50/09 1992HcnumPublishingCorporation
1012 Japan). Peroxidase was from Sigma (St. Louis, U.S.A.). 3,3'-Diaminobenzidine tetrahydrochloride was from Nacalai Tesque (Kyoto, Japan).
Preparation of Synaptosomal Membrane Fraction and Extraction of Membrane Proteins. Fresh whole bovine brains were rapidly homogenized (10%; w/v) in 0.32 M sucrose. The crude mitochondrial fraction (P2) was subfractionated essentially as described by Natake et al. (13) and Ishioka et al. (14) into myelin, synaptosome, and mitochondria fractions. Synaptosomal plasma membranes were prepared from the synaptosome fraction and membrane proteins were solubilized with CHAPS and were extracted as described in detail by Ishioka et al. (14). The solubilized membrane proteins were dialyzed against 50 mM Tris-HC1, pH 7.5, containing 0.05% CHAPS, extensively. After centrifugation at 100,000 g for 15 min, the supematant was passed through a column of Con A-agarose. Con A- and Protein A-Affinity Chromatography. Affinity chromatography with Con A-agarose of the extracted synaptosomal membrane proteins was performed by the modified methods of Kaflsson (15) and Strocchi et al. (16). The Con A-agarose column was equilibrated with 50 mM Tris-HC1,pH 7.5, containing 0.05% CHAPS (buffer A). After the extracted membrane proteins was applied onto the column, the column was extensively washed with buffer A and nonspecifically adsorbed proteins were then eluted with 0.2 M galactose in buffer A. Con A-binding proteins were specifically eluted with 0.2 M ~xMMin buffer A. The protein A-agarose column was equilibrated with buffer A. After application of the Con A-binding proteins fraction, the column was extensively washed with buffer A. Protein A-binding proteins were eluted with 0.1 M sodium citrate, pH 3.0.
Two-Dimensional Electrophoresis and Con A-Peroxidase Staining. Two-dimensional electrophoresis (2DE) was performed by the methods of Natake et al. (13) and Ishioka et al. (14). The pH gradient value and the molecular weight value were measured by the 2DE method using pI marker proteins kit and MW marker kit. Proteins on 2DE gel were electrophoretically transferred to nitrocellulose paper using the Sartorius Semi-Dry Electroblotter. After transfer, the nitrocellulose paper was subjected to Con A-peroxidase staining. The Con A-peroxidase staining was modified from the method of Shimizu et al. (17). The transferred nitrocellulose paper was soaked in 0.15M NaCI, 0.01M Tris-HC1, pH 7.4, containing 1% bovine serum albumin. After 30 min, the paper was incubated with 5 ml of COn A solution (0.1 mg/ml in 0.5mM CaC12) for 30 min. After washing three times with 0.01M Tris-HCl, pH 7.4, containing 0.15M NaC1, the paper was reacted with 5 ml of peroxidase solution (0.1 mg/ml in the washing buffer) for 30 min. After washing as above, the Con A conjugates was visualized by reaction for 1-5 min with 10 ml of 0.05% 3, 3'-diaminobenzidine tetrahydrochloride and 0.01% I-I202 in 0.1M Tris-HCl, pH 7.4, and the stained paper was extensively washed with 1% NaCI. All of the above was performed at room temperature.
RESULTS
Con A-Affinity Chromatography of Synaptosomal Membrane Proteins. A typical pattern of Con A-affinity chromatogram of synaptosomal membrane proteins was shown in Fig. 1. Con A-binding proteins were eluted in a broad peak with (x MM (shaded area in Figure 1). All of Con A-binding proteins of the extracted synaptosomal membrane proteins were recovered in the c~ MM frae-
Ishioka and Kurioka 0.1 E c r,l
Z
0.05
0.2M Galactose
0.2M =-MM
50
100
150
TUBE NO.
Fig. 1. Elution profile of synaptosomal membrane proteins on Con Aagarose affinity column. The membrane proteins extracted from synaptosomal fraction were dialyzed against buffer A. The dialysate (50 mg proteins/100 ml) were applied to a 20 ml-column of Affi-Gel 10 coupled with Con A and elnted as described under Materials and Methods, at a flow rate of 10 ml/min. The o~MM fraction (shaded area in figure) were collected and subjected to 2DE analysis after dialysis and lyophilization.
tion, and a crashed peak (an arrow in Figure 1) contained no Con A-binding proteins, judging by Con A-peroxidase staining of 2DE gels (data not shown). The (x MM fraction was pooled and dialyzed against 50mM TrisHC1, pH 7.5, containing 0.05% CHAPS. The dialysate in dialysis tube was dipped in 40% polyethylene glycol #20000 for concentration. The concentrated (x MM fraction was used for 2DE analysis.
2DE Analysis of Con A-Binding Proteins of Synaptosomal Membrane. A few proteins were detected on 2DE electrophoretogram stained with Coomassie Brilliant Blue, and the majority were only weakly indicated (data not shown). To obtain more information of the Con A-binding proteins of synaptosomal membrane, the proteins on 2DE gel were indicated by Con A-peroxidase staining after electroblot from 2DE gel. As shown in Figure 2A, more than 40 protein spots were visualized. Most of the blots detected by Con A-peroxidase staining were streaked across the pH gradient and/or exhibited as multiple spots, indicating broad charge and molecular weight heterogeneity. The strongly stained blots were classified into two protein spots (a and b in Figure 2A) and two groups (boxed c and d in Figure 2A). One of the two spots (a in Figure 2A) showed the highest molecular weight and was a streaked band across pI from 5.8 to 7.5 with MW 116 kDa (named GPll6). The spot of pI 5.2 and of MW 90 kDa (b in Figure 2A and named GP90) was deeply stained in brown color, indicating high mannose content. The group of a low molecular weight (d in Figure 2A) contained three spots. The mo-
Synaptosomal Membrane Glycoproteins
1013
Fig. 2. Indication of Con A-binding proteins and of protein A-binding proteins in the Con A-binding proteins fraction by Con A-peroxidase staining. The COn A-binding proteins (24 ~g) were indicated by COn A-peroxidase staining after electroblot from 2DE gel (A). At least 40 spots were observed, and, among them, the strongly stained spots were classified into two proteins (a; GPll6 and b; GP90) and two groups (boxed c; GP51 and d; GP13). The pattern of protein A-binding proteins (8 Izg) in Con A-binding proteins fraction showed in the (B), and the boxed indicated GP51 corresponded to c in the (A).
lecular weight of these spots was 13 kDa (named GP13). Three spots of GP13 separated horizontally (pi5.5-6.0) and exhibited microheterogeneity of MW with difference of about 0.5 kDa in each spot. GP13 was also detected in a deep brown color with Con A-peroxidase staining. The group of MW 51 kDa species (c in Figure 2A and named GP51) was a single band on SDS-PAGE gel, however it was found that GP51 consisted of seven main spots which separated horizontally in charge heterogeneity (pi5.85-7.5) shifted pI 0.15 each, and GP51 was quite interesting because of its binding ability to protein A. Figure 2B showed GP51 visualized by Con A-peroxidase staining, which were obtained by protein A-affinity chromatography of the Con A-binding proteins fraction. The GP51 was a major species of protein Abinding proteins in Con A-binding proteins.
DISCUSSION The glycoproteins of synaptosomal membranes have been analyzed with a number of different methods (12,1821) using lectins which bind to mono- or disaccharide units within some specific parts of the glycoproteins. In this study, we employed Con A for preparation and separation of glycoproteins from synaptosomal membranes.
Con A has been commonly used, and its biochemical properties to attract and to bind glycoproteins has been well understood. The omission of major glycoproteins in synaptosomal membranes was highly unlikely (22) and the majority of the proteins were comprehensively detected by 2DE analysis. As reported previously, the 2DE method has been very useful for analyzing synaptosomal membrane proteins (14), and this method was also effective for analyzing membrane glycoproteins. At least 40 protein spots were visualized by Con A-peroxidase staining. Most of the Con A-binding proteins were streaked across the pH gradient and/or exhibited as multiple spots. Such a microheterogeneity of glycoproteins, indicating broad charge and small molecular weight heterogeneity, has been often revealed by high-resolution of 2DE analysis (23,24). The microheterogeneity is probably due to deamidation of glutamine and/or asparagine, phosphorylation, sulfation, variation in sialic acid content and so forth leading to charge heterogeneity, and due to variation in attachment of prefabricated neutral sugar structure (23,25) to the protein resulting in shift of molecular weight from 0.5 kDa to 1 kDa. Among the glycoprotein groups (GPll6, GP90, GP51 and GP13) which were densely indicated by Con A-peroxidase staining, GP13 and GP51 were relatively hard to detect on the gel by Coomassie- or
1014
silver-staining (data not shown). GP51 was characteristic of the charge heterogeneity and of the interaction with an immobilized protein A-gel. Protein A and immunoglobulin G have a mutual interaction, and the latter is a glycoprotein which is characteristic of microheterogeneity of the charge, The discovery of GP51 implicates that GP51 has a chemical structure similar to Fc region of immunoglobulin G, and this strongly implied that GP51 is related to immunoglobulins and/or to immunoglobulin supergene family.
REFERENCES 1. Hemperly, J. J., Murray, B. A., Edelman, G. M., and Cunningham, B. A. 1986. Sequence of a eDNA clone encoding the polysialic acid-rich and cytoplasmic domains of the neural cell adhesion molecule N-CAM. Proe. Natl. Acad. Sci. USA 83:30373041. 2. Martini, R., and Schachner, M. 1988. Immunoeleetron microscopic localization of neural cell adhesion molecules (L1, N-CAM, and Myelin-associated Glycoprotein) in regenerating adult mouse sciatic nerve. J. Cell Biol. 106:1735-1746. 3. Rathjen, F. G., and Schachner, M. 1984. Immunological and biochemical characterization of a new neuronal cell surface component (L1 antigen) which is involved in cell adhesion. EMBO J. 3:1-10. 4. Miura, M., Kobayashi, M., Asou, H., and Uyemura, K. 1991. Molecular cloning of eDNA encoding the rat neural cell adhesion molecule LI. Two L1 isoforms in the cytoplasmic region are produced by differential splicing. FEBS Lett. 289:91-95. 5. Salzer, J. L., Holmes, W. P., and Colman, D. R. 1987. The amino acid sequences of the myelin-associated glycoproteins: Homology to the immunoglobulin gene superfamily. J. Cell Biol. 104:957-965. 6. Harrelson, A. L., and Goodman, C. S. 1988. Growth cone guidance in insects: Fasciclin II is a member of the immunoglobulin superfamily. Science 242:700--708. 7. Snow, P. M., Zinn, K., Harrelson, A. L., McAllister, L., Schilling, J., Bastiani, M. J., Makk, G., and Goodman, C. S. 1988. Characterization and cloning of fasciclin I and fasciclin II glycoproteins in the grasshopper. Proc. Natl. Aead. Sci. USA 85:52915295. 8. Hood, L., Kronenberg, M., and Hunkapiller, T. 1985. T cell antigen receptor and the immunoglobulin supergene family. Cell 40:225-229. 9. Williams, A. F. 1987. A year in the life of the immunoglobulin superfamily. Immunol. Today 8:298-303.
Ishioka and Kurioka 10. Zinn, K., McAllister, L., and Goodman, C. S. 1988. Sequence analysis and neuronal expression of fasciclin I in grasshopper and drosophila. Cell 53:577-587. 11. Ellis, L., Wallis, I., Abreu, E., and Pfenninger, K. H. 1985. Nerve growth cones isolated from fetal rat brain. IV. Preparation of a membrane subfraction and identification of a membrane glycoprotein expressed on sprouting neurons. J. Cell Biol. 101:19771989. 12. Greenberger, L. M., and Pfenninger, K. H. 1986. Membrane glycoproteins of the nerve growth cone: Diversity and growth regulation of oligosaceharides. J. Cell Biol. 103:1369-1382. 13. Natake, Y., Udagawa, K., Ishioka, N., and Kurioka, S. 1988. Preparation of synaptosomal membranes from frozen brain or P2 fraction that was frozen and analysis of synaptosomal membrane proteins. Jikeikai Med. J. 35:297-303. 14. Ishioka, N., Oda, T., Natake, Y., and Kurioka, S. 1990. Analysis and separation of synaptosomal membrane proteins. Neurochem. Res. 15:475---481. 15. Karlsson, J. -O. 1980. Proteins of rapid axonal transport: Polypeptides interacting with the lectin from Lens culinaris. J. Neurochem. 34:1184-1190. 16. Strocchi, P., Brown, B. A., Young, J. D., Bonventre, J. A., and Gilbert, J. M. 1981. The characterization of tubulin in CNS membrane fractions. J. Neurochem. 37:1295-1307. 17. Shimizu, S., Ito, M., and Dean, J. 1982. Giyeoproteins of mouse zona pelheida: Analysis of their reactivity to lectins. Biochem. Biophys. Res. Commun. 109:449-454. 18. Gurd, J. W., and Mahler, H. R. 1974. Fractionation of synaptie plasma membrane glycoproteins by lectin affinity chromatography. Biochemistry 13:5193-5198. 19. Zanetta, J. P., Morgan, I. G., and Gombos, G. 1975. Synaptosomal plasma membrane glycoproteins: Fractionation by affinity chromatography on concanavalin A. Brain R,s. 83:337-348. 20. Gurd, J. W. 1977. Synaptic plasma membrane glycoproteins: Molecular identification of lectin receptors. Biochemistry 16:369374. 21. Kelly, P. T., and Cotman, C. W. 1977. Identification of glycoproteins, proteins at synapses in the central nervous system. J. Biol. Chem. 252:786--793. 22. Mahler, H. R. 1979. Glycoproteins of the synapse, Pages 165184, in Margolis, R. U., and Margolis, R. K., (eds), Complex Carbohydrates of Nervous Tissue. Plenum Press, New York. 23. Anderson, N. L., and Anderson, N. G. 1979. Microheterogeneity of serum transferrin, haptoglobin and 2HS glyeoprotein examined by high resolution two-dimensional electrophoresis. Bioehem. Biophys. Res. Commun. 88:258-265. 24. Anderson, N. L., and Hickman, B. J. 1979. Analytical techniques for cell fractions. XXIV. Isoelectrie point standards for two-dimensional eleetrophoresis. Anal. Biochem. 93:312-320. 25. Lennarz, W. J. 1975. Lipid linked sugars in glycoprotein synthesis. Science 188:986--991.
Analysis of Con A-Binding Glycoproteins in Synaptosomal Membranes Noriaki Ishioka 1 a n d S u s u m u Kllrioka I
(Accepted February 7, 1992) Concanavalin A (Con A)-binding proteins obtained from solubilized synaptosomal membranes of bovine brain were analyzed by two-dimensional electrophoresis (2DE), and were identified by peroxidase conjugated Con A (Con A-peroxidase staining), after transfer from 2DE gel to nitrocellulose paper. The Con A-binding proteins were resolved up to 40 spots, ranging in isoelectric points (pI) from 4.5 to 8.0 and molecular weight (MW) from 10 kDa to 120 kDa. Most of the Con A-binding proteins were streaked across a pH gradient and/or exhibited as multiple spots, indicating broad charge and molecular weight heterogeneity. The presence of protein groups that showed high affinities for Con A were revealed. Most interesting group (named GP51), which consisted of seven spots separated horizontally in charge heterogeneity (pi5.85-7.5) with MW 51kDa, was characterized by its binding to an immobilized protein A gel. This implies that GP51 is related to immunoglobulins and/or GP51 may be a new member of the immunoglobulin supergene family. KEY WORDS: Synaptosomes; membrane glycoproteins; Con A-binding; protein A-binding; two-dimensional electrophoresis; immunoglobulin supergene family.
INTRODUCTION
purpose, we analyze total Con A-binding proteins of the membranes and especially Con A-binding proteins which have ability to associate with protein-A molecule.
The membrane glycoproteins in nervous system such as N-CAM (1,2), 1,1 protein (2-4), MAG (2,5), and fasciclin II (6,7), are neural cell adhesion molecules, and belong to the immunoglobulin supergene family (8,9). It is assumed that tlhese membrane glycoproteins take part in neuronal cell-to-cell interaction as in the case of recognition in immune system. The others such as fasciclin I (7,10), 5B4 antigen (11), gp27-30 (12) are in nerve growth cone. These glycoproteins also play important roles on neural cells. In this study, we focused to seek for some undiscovered immunoglobulin supergene family proteins involved in synaptosomal membranes. To attain this
EXPERIMENTAL PROCEDURE Materials. All of the reagents were generally of analytical grade. Ampholines (pH 3.5-10 and pH 3.5-5.0) were obtained from LKB (Bromma, Sweden). 3-[(3-Cholamidopropyl)dimethylammonio]-lpropane-sulfonate (CHAPS) for sohbilization of membranes was purchased from Wako Pure ChemicalIndustries (Tokyo, Japan). The molecular weight marker kit for 2DE was from Sigma (St. Louis, U.S.A.) and the isoelectric point marker kit for isoelectric focusing gel was from Pharmasia (Uppsala, Sweden). Concanavalin A (Con A) was purchased from Vector Laboratories (Burlingame, U.S.A.). Affi-Gel 10 (active ester agarose) was from BIO-RAD (Richmond, U.S.A.). For Con A-affinity chromatography,Con A-agarosewas coupled with Con A and Affi-Ge110according to an instructionof BIO-RAD. AffiGel 10 conjugatedprotein A was from BIO-RAD (Richmond,U.S.A.). a-Methyl-D-mannoside (a MM) was from Nacalai Tesque (Kyoto,
1 Division of Biochemist;~, Institute of Medical Science, The Jikei University School of Medicine, 3-25-8 Nishi-shinbashi, Minato-ku, Tokyo 105, Japan. 1011
0364-3190/92/1000-1011506.50/09 1992HcnumPublishingCorporation
1012 Japan). Peroxidase was from Sigma (St. Louis, U.S.A.). 3,3'-Diaminobenzidine tetrahydrochloride was from Nacalai Tesque (Kyoto, Japan).
Preparation of Synaptosomal Membrane Fraction and Extraction of Membrane Proteins. Fresh whole bovine brains were rapidly homogenized (10%; w/v) in 0.32 M sucrose. The crude mitochondrial fraction (P2) was subfractionated essentially as described by Natake et al. (13) and Ishioka et al. (14) into myelin, synaptosome, and mitochondria fractions. Synaptosomal plasma membranes were prepared from the synaptosome fraction and membrane proteins were solubilized with CHAPS and were extracted as described in detail by Ishioka et al. (14). The solubilized membrane proteins were dialyzed against 50 mM Tris-HC1, pH 7.5, containing 0.05% CHAPS, extensively. After centrifugation at 100,000 g for 15 min, the supematant was passed through a column of Con A-agarose. Con A- and Protein A-Affinity Chromatography. Affinity chromatography with Con A-agarose of the extracted synaptosomal membrane proteins was performed by the modified methods of Kaflsson (15) and Strocchi et al. (16). The Con A-agarose column was equilibrated with 50 mM Tris-HC1,pH 7.5, containing 0.05% CHAPS (buffer A). After the extracted membrane proteins was applied onto the column, the column was extensively washed with buffer A and nonspecifically adsorbed proteins were then eluted with 0.2 M galactose in buffer A. Con A-binding proteins were specifically eluted with 0.2 M ~xMMin buffer A. The protein A-agarose column was equilibrated with buffer A. After application of the Con A-binding proteins fraction, the column was extensively washed with buffer A. Protein A-binding proteins were eluted with 0.1 M sodium citrate, pH 3.0.
Two-Dimensional Electrophoresis and Con A-Peroxidase Staining. Two-dimensional electrophoresis (2DE) was performed by the methods of Natake et al. (13) and Ishioka et al. (14). The pH gradient value and the molecular weight value were measured by the 2DE method using pI marker proteins kit and MW marker kit. Proteins on 2DE gel were electrophoretically transferred to nitrocellulose paper using the Sartorius Semi-Dry Electroblotter. After transfer, the nitrocellulose paper was subjected to Con A-peroxidase staining. The Con A-peroxidase staining was modified from the method of Shimizu et al. (17). The transferred nitrocellulose paper was soaked in 0.15M NaCI, 0.01M Tris-HC1, pH 7.4, containing 1% bovine serum albumin. After 30 min, the paper was incubated with 5 ml of COn A solution (0.1 mg/ml in 0.5mM CaC12) for 30 min. After washing three times with 0.01M Tris-HCl, pH 7.4, containing 0.15M NaC1, the paper was reacted with 5 ml of peroxidase solution (0.1 mg/ml in the washing buffer) for 30 min. After washing as above, the Con A conjugates was visualized by reaction for 1-5 min with 10 ml of 0.05% 3, 3'-diaminobenzidine tetrahydrochloride and 0.01% I-I202 in 0.1M Tris-HCl, pH 7.4, and the stained paper was extensively washed with 1% NaCI. All of the above was performed at room temperature.
RESULTS
Con A-Affinity Chromatography of Synaptosomal Membrane Proteins. A typical pattern of Con A-affinity chromatogram of synaptosomal membrane proteins was shown in Fig. 1. Con A-binding proteins were eluted in a broad peak with (x MM (shaded area in Figure 1). All of Con A-binding proteins of the extracted synaptosomal membrane proteins were recovered in the c~ MM frae-
Ishioka and Kurioka 0.1 E c r,l
Z
0.05
0.2M Galactose
0.2M =-MM
50
100
150
TUBE NO.
Fig. 1. Elution profile of synaptosomal membrane proteins on Con Aagarose affinity column. The membrane proteins extracted from synaptosomal fraction were dialyzed against buffer A. The dialysate (50 mg proteins/100 ml) were applied to a 20 ml-column of Affi-Gel 10 coupled with Con A and elnted as described under Materials and Methods, at a flow rate of 10 ml/min. The o~MM fraction (shaded area in figure) were collected and subjected to 2DE analysis after dialysis and lyophilization.
tion, and a crashed peak (an arrow in Figure 1) contained no Con A-binding proteins, judging by Con A-peroxidase staining of 2DE gels (data not shown). The (x MM fraction was pooled and dialyzed against 50mM TrisHC1, pH 7.5, containing 0.05% CHAPS. The dialysate in dialysis tube was dipped in 40% polyethylene glycol #20000 for concentration. The concentrated (x MM fraction was used for 2DE analysis.
2DE Analysis of Con A-Binding Proteins of Synaptosomal Membrane. A few proteins were detected on 2DE electrophoretogram stained with Coomassie Brilliant Blue, and the majority were only weakly indicated (data not shown). To obtain more information of the Con A-binding proteins of synaptosomal membrane, the proteins on 2DE gel were indicated by Con A-peroxidase staining after electroblot from 2DE gel. As shown in Figure 2A, more than 40 protein spots were visualized. Most of the blots detected by Con A-peroxidase staining were streaked across the pH gradient and/or exhibited as multiple spots, indicating broad charge and molecular weight heterogeneity. The strongly stained blots were classified into two protein spots (a and b in Figure 2A) and two groups (boxed c and d in Figure 2A). One of the two spots (a in Figure 2A) showed the highest molecular weight and was a streaked band across pI from 5.8 to 7.5 with MW 116 kDa (named GPll6). The spot of pI 5.2 and of MW 90 kDa (b in Figure 2A and named GP90) was deeply stained in brown color, indicating high mannose content. The group of a low molecular weight (d in Figure 2A) contained three spots. The mo-
Synaptosomal Membrane Glycoproteins
1013
Fig. 2. Indication of Con A-binding proteins and of protein A-binding proteins in the Con A-binding proteins fraction by Con A-peroxidase staining. The COn A-binding proteins (24 ~g) were indicated by COn A-peroxidase staining after electroblot from 2DE gel (A). At least 40 spots were observed, and, among them, the strongly stained spots were classified into two proteins (a; GPll6 and b; GP90) and two groups (boxed c; GP51 and d; GP13). The pattern of protein A-binding proteins (8 Izg) in Con A-binding proteins fraction showed in the (B), and the boxed indicated GP51 corresponded to c in the (A).
lecular weight of these spots was 13 kDa (named GP13). Three spots of GP13 separated horizontally (pi5.5-6.0) and exhibited microheterogeneity of MW with difference of about 0.5 kDa in each spot. GP13 was also detected in a deep brown color with Con A-peroxidase staining. The group of MW 51 kDa species (c in Figure 2A and named GP51) was a single band on SDS-PAGE gel, however it was found that GP51 consisted of seven main spots which separated horizontally in charge heterogeneity (pi5.85-7.5) shifted pI 0.15 each, and GP51 was quite interesting because of its binding ability to protein A. Figure 2B showed GP51 visualized by Con A-peroxidase staining, which were obtained by protein A-affinity chromatography of the Con A-binding proteins fraction. The GP51 was a major species of protein Abinding proteins in Con A-binding proteins.
DISCUSSION The glycoproteins of synaptosomal membranes have been analyzed with a number of different methods (12,1821) using lectins which bind to mono- or disaccharide units within some specific parts of the glycoproteins. In this study, we employed Con A for preparation and separation of glycoproteins from synaptosomal membranes.
Con A has been commonly used, and its biochemical properties to attract and to bind glycoproteins has been well understood. The omission of major glycoproteins in synaptosomal membranes was highly unlikely (22) and the majority of the proteins were comprehensively detected by 2DE analysis. As reported previously, the 2DE method has been very useful for analyzing synaptosomal membrane proteins (14), and this method was also effective for analyzing membrane glycoproteins. At least 40 protein spots were visualized by Con A-peroxidase staining. Most of the Con A-binding proteins were streaked across the pH gradient and/or exhibited as multiple spots. Such a microheterogeneity of glycoproteins, indicating broad charge and small molecular weight heterogeneity, has been often revealed by high-resolution of 2DE analysis (23,24). The microheterogeneity is probably due to deamidation of glutamine and/or asparagine, phosphorylation, sulfation, variation in sialic acid content and so forth leading to charge heterogeneity, and due to variation in attachment of prefabricated neutral sugar structure (23,25) to the protein resulting in shift of molecular weight from 0.5 kDa to 1 kDa. Among the glycoprotein groups (GPll6, GP90, GP51 and GP13) which were densely indicated by Con A-peroxidase staining, GP13 and GP51 were relatively hard to detect on the gel by Coomassie- or
1014
silver-staining (data not shown). GP51 was characteristic of the charge heterogeneity and of the interaction with an immobilized protein A-gel. Protein A and immunoglobulin G have a mutual interaction, and the latter is a glycoprotein which is characteristic of microheterogeneity of the charge, The discovery of GP51 implicates that GP51 has a chemical structure similar to Fc region of immunoglobulin G, and this strongly implied that GP51 is related to immunoglobulins and/or to immunoglobulin supergene family.
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