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Immunogenicity of a membrane surface glycoprotein associated with central nervous system myelin
234
Brain Research, 159 (1978) 234-238
O Elsevier/North-HollandBiomedicalPress
Immunogenicity of a membrane surface glycoprotein associated with central nervous system myelin
JOSEPH F. PODUSLOand DALE E. McFARLIN Neuroimmunology Branch, National Institute of Neurological and Communicative Disorders attd Stroke, National Institutes of Health, Bethesda, Md. 20014 (U.S.A.)
(Accepted August24th, 1978)
The surface components of the myelin sheath and the oligodendroglial plasma membrane should provide important clues in defining the alterations which occur in certain human nervous system disorders involving demyelination, such as multiple sclerosis. Such components may not only play important recognition roles in the process of myelination or myelin maintenance, but they may also be readily susceptible to immunological damage or even act as specific viral receptors. Consequently, the identification, isolation, and immunological characterization of these surface membrane components should permit elucidation of their role in the initial mechanism of both the myelinating and demyelinating process. Several of the external surface components of this myelin sheath complex have been identified using the enzymatic membrane probe, galactose oxidase, on an intact spinal cord preparation s. A major glycoprotein (MyGP1) associated with central nervous system myelin was shown to have an external surface localization. Using a similar approach as developed by Marchesi and Andrews for isolating the major glycoprotein of the human erythrocyte membrane 4, this myelin-associated glycoprotein has been isolated in a water-soluble form. The experiments reported here demonstrate the immunogenicity of MyGP1 in rabbits and, therefore, define a membrane surface antigen associated with central nervous system myelin or the closely associated oligodendroglial plasma membrane. Lewis rat myelin was isolated according to the procedure of Norton and Poduslo 6 from 18 day old rats after a 48 h incorporation of 8.3 nmole (100/tCi per rat) of L-[6-3H]fucose (New England Nuclear) (intracranial injection; 50 #Ci per hemisphere). Fig. I A illustrates the radioactive fucose profile after separation of the myelin proteins by sodium dodecyl-sulfate polycrylamide gel electrophoresis. At least 3 radioactive peaks are observed. These quantitatively minor components of myelin have been partially characterized 10. At least 94~o of the fucose label was solubilized by using 0.1 M lithium diiodosalicylate (4) as a membrane perturbant of the isolated myelin. Treatment of this extract with 50 ~ phenol resulted in the selective partitioning into the aqueous phase of only MyGP1 (Fig. 1B). The other myelin glycoproteins remain in the phenol phase
235 -15.000
I
1
I
I
M;GP1 A: 18 Day Old Lewis Rat Myelin 10,000
After 48 Hour Fucose Incorporation
5,000
MyGP2 MyGP3 '
J 30,000 B: Isolation of the Major
"O
Myelin Glycoprotein (MyGPI)
o 25,000 ,,= - - 20.000
15,000
10,000 5,00(I/ 0
10
15
20
! 25 30 Sample Number
I
35
I
40
I
45
50
Fig. 1. Radioactive fucose pattern after sodium dodecyl-sulfate polyacrylamide gel electrophoresis of a glycoprotein preparation obtained by extraction of isolated myelin with lithium diiodosalicylate. A: typical [aH]fucose profile of isolated myelin from 18 day old Lewis rats after 48 h fucose incorporation (100 pg protein). B: radioactive fucose profile of the isolated glycoprotein preparation (30 pg protein). Discontinuous gel system: 6 % (w/v) acrylamide spacer gel (slices 1-3); 15 % (w/v) acrylamide separating gel (slices 4-50); Tris-glycine buffer system9,1°. The radioactive fucose peaks (MyGPI, MyGP2, MyGP3) are designated as myelin-associated glycoproteins. Such an association could include the myelin membrane, the oligodendroglial plasma membrane and its region of transition to myelin, or the axolemma of myelinated axons. along with the rest of the myelin proteins. The reasons for the selective partitioning of this glycoprotein into the aqueous phase are not clear although high levels of carbohydrate associated with the protein remain as a distinct possibility. This preparation of MyGP1 represented a 22-fold purification comparing the specific activities found in isolated myelin (58 cpm/pg total protein) with the isolated protein (1273 cpm/pg glycoprotein). The radioactive fucose profile shown in Fig. 1B indicated that a highly enriched preparation of MyGP1 has been obtained. This [3H]fucoselabeled glycoprotein was used in a radioimmunoassay to quantitate antibody in rabbits immunized with this protein and with isolated Lewis rat myelin. New Zealand white rabbits were injected with 100 pg of MyGP1 in Freund's complete adjuvant (FCA) (2.5 mg Mycobacterium butyricum/ml). Animals were boosted 21 days later with 280 p g of glycoprotein in incomplete Freund's adjuvant and
236 Antiserum Titration Curves: .80
I I
I
I
I
I
1
I
I I I
I
I
I
I
I
I
70
60
0.361 x 10 -7 M
, 0
-
~ 40 ~
_
O. 30 - -
33% Binding Level
" ~ , , ~
~
_
2O 10 I 0.1
I
I
I 0.06
I
I
I
II I I I I I I 0.01 0.005 Final Dilution of Rabbit Anti Myelin GP1 Serum
I 0.001
}rig. 2. Titration curves of the rabbit a n t i - M y G P l serum. Specific activity of the M y G P I preparation: 1480 cpm/pg protein. [ M y G P I ] = 0.361 x 10 7 M : total cpm -- 535, assay volume - 100/~1. [ M y G P I ] 1.526 x 10 -7 M : total c p m -- ! 130, assay volume = 50 ffl. Based on estimated molecular weight o f 105 daltons. D a t a points duplicate averages. Experimental binding - total binding - - nonspecific binding o f n o r m a l s e r u m a n d sheep anti-rabbit l g G serum/total c p m - - nonspecific binding o f n o r m a l s e r u m a n d sheep anti-rabbit l g G serum. -
-
Antiserum Absorption: 6(
5(
i
i
~X
•~ : Unabsorbed ~ Heart o----o Thymocytes
~,~
~
%\
-
Liver
oo
~= 3o
2 2O
10
1/10
1/20 1/40 1/80 Final Dilution of Rabbit Anti Myelin GP1 Serum
1/160
Fig. 3. Absorption of rabbit a n t i - M y O P l serum. The antisera (1 ml of 1/5 dilution) was absorbed twice with an equal v o l u m e o f Lewis rat h o m o g e n a t e s o f tissue in borate buffered saline, p H 8.0 (20 mg/ml) by i n c u b a t i o n at 37 °C for 30 rain a n d at 4 °C for 30 m i n followed by centrifugation at 12,000 x g for 10 rain. Heart, t h y m o c y t e , liver, a n d brain h o m o g e n a t e s were subjected to prior acetone extraction.
237 bled at day 30. These animals showed no evidence of EAE at day 30. The capacity of the antisera to bind [aH]fucose-MyGP1 was tested using a double antibody procedure with sheep anti-rabbit IgG as the second antibody. The antiserum to rabbit IgG, which was purified from rabbit y-globulin protein (Cappel Lab.) by DEAE-cellulose, was produced in sheep by primary immunization in CFA followed by successive secondary immunizations in incomplete Freund's adjuvant. Equivalence was determined in this double-antibody system at each dilution of both the immune and normal sera. After incubation of normal and immune rabbit serum with the labeled MyGP1 for 1 h at 37 and l h at 4 °C, an equivalent amount of this sheep anti-rabbit IgG was added. After an overnite incubation, the precipitate formed was washed 3 times with borate buffered saline (pH 8.0) and hydrolyzed for 30 min with 200/A 1 N NaOH. The radioactivity was then determined by liquid scintillation spectrometry to a 2-sigma counting error of 4- 4 ~ . Titration curves of one antiglycoprotein serum at two different antigen concentrations are shown in Fig. 2. The ratio between the two concentrations was 0.24 while the ratio between the titers at the 50 ~ maximal binding level was 0.29 which indicated that the immune precipitation was proportional to the antigen in this concentration range. At a dilution of 1:213, 33 ~ of the antigen was bound which represented an antigen binding capacity of 2.54 #M. Absorption of this antiglycoprotein antiserum with brain or myelin significantly reduced the antigen binding capacity, although absorption with heart, liver, or thymocyte homogenates did not affect its capacity to bind the labeled antigen (Fig. 3). Rabbits were also injected with isolated Lewis rat myelin (440 #g myelin protein) in CFA followed by successive secondary immunizations and bleedings. These rabbits developed EAE by day 12 and produced substantial levels of antibody to the glycoprotein. An experimental binding of 32 and 22 ~o was obtained at a dilution of 1/10 for two different rabbits immunized with whole myelin, while an experimental binding of 66 and 59 ~o was obtained at a similar dilution with rabbits immunized with the isolated glycoprotein preparation. The demonstration that a membrane surtace glycoprotein associated with the myelin sheath complex is antigenic should provide a basis for investigating its precise cytochemical localization. In addition, antisera against MyGP1 can be used to study differentiation in the nervous system as well as provide the means for searching for the release of MyGPI during the demyelinating process of certain human neurological diseases. Such an approach has recently been pursued with a less accessible component of myelin, the basic protein1,2, n. The accessible, surface membrane, location of MyGPI as well as its susceptibility to degradation after autolysis compared to other myelin proteins~ could provide a sensitive indication of disease activity. The present finding of antibody to MyGPI in animals with EAE that were challenged with isolated myelin supports the possibility that the immune response to other antigenic components in nervous tissue may contribute to the pathogenesis of this experimental disease. It is well known that, on a dry weight basis, the potency of CNS tissue to induce EAE is much greater than basic protein itself or its encephalitogenic determinant a,v. The presence of additional myelin-associated antigens operating in conjunction with the major encephalitogenic determinant of basic protein could
238 a c c o u n t for the greater encephalitogenicity seen with whole tissue in this disease process. The c a p a c i t y for a n t i b o d y p r o d u c t i o n against this myelin-associated glycop r o t e i n should facilitate f u r t h e r investigations o f b o t h f u n d a m e n t a l n e u r o b i o l o g i c a l a n d p a t h o g e n i c mechanisms operative in the nervous system.
1 Cohen, S. R., Herndon, R. M. and McKhann, G. M., Radioimmunoassay of myelin basic protein in spinal fluid, New Engl. J. Med., 295 (1976) 1455-1457. 2 Gutstein, H. S. and Cohen, S. R., Spinal fluid differences in experimental allergic encephalomyelitis and multiple sclerosis, Science, 199 (1978) 301-303. 3 Hoffman, P. M., Gaston, D. D. and Spitler, L. E., Comparison of experimental allergic encephalomyelitis induced with spinal cord, basic protein, and synthetic encephalitogenic peptide, Clin. Immunol. lmmunopath., 1 (1973) 364-371. 4 Marchesi,V. T. and Andrews, E. P., Glycoproteins: isolation from cell membranes with lithium diiodosalicylate,Science, 174 (1971) 1247-1248. 5 Matthieu, J.-M., Koellreutter, B. and Joyet, M.-L., Changes in CNS myelin proteins and glycoproteins after in situ autolysis, Brain Res. Bull., 2 (1977) 15-21. 6 Norton, W. T. and Poduslo, S. E., Myelination in rat brain: method of myelin isolation, J. Neurochem., 21 (1973) 749-757. 7 Paterson, P. Y., Autoimmune neurological disease: experimental animal systems and implications for multiple sclerosis. In N. Talal (Ed.), Autoimmunity, Academic Press, New York, 1978, pp. 643691. 8 Poduslo, J. F., The molecular architecture ofmyelin: identification ofthe external surface membrane components, Advanc. exp. med. BioL, 100 (1978) 189-206. 9 Poduslo, J. F. and Braun, P. E., Topographical arrangement of membrane proteins in the intact myelin sheath, J. bioL Chem., 250 (1975) 1099-1105. 10 Poduslo, J. F., Everly, J. L. and Quarles, R. H., A low molecular weight glycoprotein associated with isolated myelin : distinction from myelin proteolipid protein, J. Neurochem., 28 (1977) 977-986. 11 Whitaker, J. N., Myelin encephalitogenic protein fragments in cerebrospinal fluid of persons with multiple sclerosis, Neurology (Minneap.), 27 (1977) 911-920.
Brain Research, 159 (1978) 234-238
O Elsevier/North-HollandBiomedicalPress
Immunogenicity of a membrane surface glycoprotein associated with central nervous system myelin
JOSEPH F. PODUSLOand DALE E. McFARLIN Neuroimmunology Branch, National Institute of Neurological and Communicative Disorders attd Stroke, National Institutes of Health, Bethesda, Md. 20014 (U.S.A.)
(Accepted August24th, 1978)
The surface components of the myelin sheath and the oligodendroglial plasma membrane should provide important clues in defining the alterations which occur in certain human nervous system disorders involving demyelination, such as multiple sclerosis. Such components may not only play important recognition roles in the process of myelination or myelin maintenance, but they may also be readily susceptible to immunological damage or even act as specific viral receptors. Consequently, the identification, isolation, and immunological characterization of these surface membrane components should permit elucidation of their role in the initial mechanism of both the myelinating and demyelinating process. Several of the external surface components of this myelin sheath complex have been identified using the enzymatic membrane probe, galactose oxidase, on an intact spinal cord preparation s. A major glycoprotein (MyGP1) associated with central nervous system myelin was shown to have an external surface localization. Using a similar approach as developed by Marchesi and Andrews for isolating the major glycoprotein of the human erythrocyte membrane 4, this myelin-associated glycoprotein has been isolated in a water-soluble form. The experiments reported here demonstrate the immunogenicity of MyGP1 in rabbits and, therefore, define a membrane surface antigen associated with central nervous system myelin or the closely associated oligodendroglial plasma membrane. Lewis rat myelin was isolated according to the procedure of Norton and Poduslo 6 from 18 day old rats after a 48 h incorporation of 8.3 nmole (100/tCi per rat) of L-[6-3H]fucose (New England Nuclear) (intracranial injection; 50 #Ci per hemisphere). Fig. I A illustrates the radioactive fucose profile after separation of the myelin proteins by sodium dodecyl-sulfate polycrylamide gel electrophoresis. At least 3 radioactive peaks are observed. These quantitatively minor components of myelin have been partially characterized 10. At least 94~o of the fucose label was solubilized by using 0.1 M lithium diiodosalicylate (4) as a membrane perturbant of the isolated myelin. Treatment of this extract with 50 ~ phenol resulted in the selective partitioning into the aqueous phase of only MyGP1 (Fig. 1B). The other myelin glycoproteins remain in the phenol phase
235 -15.000
I
1
I
I
M;GP1 A: 18 Day Old Lewis Rat Myelin 10,000
After 48 Hour Fucose Incorporation
5,000
MyGP2 MyGP3 '
J 30,000 B: Isolation of the Major
"O
Myelin Glycoprotein (MyGPI)
o 25,000 ,,= - - 20.000
15,000
10,000 5,00(I/ 0
10
15
20
! 25 30 Sample Number
I
35
I
40
I
45
50
Fig. 1. Radioactive fucose pattern after sodium dodecyl-sulfate polyacrylamide gel electrophoresis of a glycoprotein preparation obtained by extraction of isolated myelin with lithium diiodosalicylate. A: typical [aH]fucose profile of isolated myelin from 18 day old Lewis rats after 48 h fucose incorporation (100 pg protein). B: radioactive fucose profile of the isolated glycoprotein preparation (30 pg protein). Discontinuous gel system: 6 % (w/v) acrylamide spacer gel (slices 1-3); 15 % (w/v) acrylamide separating gel (slices 4-50); Tris-glycine buffer system9,1°. The radioactive fucose peaks (MyGPI, MyGP2, MyGP3) are designated as myelin-associated glycoproteins. Such an association could include the myelin membrane, the oligodendroglial plasma membrane and its region of transition to myelin, or the axolemma of myelinated axons. along with the rest of the myelin proteins. The reasons for the selective partitioning of this glycoprotein into the aqueous phase are not clear although high levels of carbohydrate associated with the protein remain as a distinct possibility. This preparation of MyGP1 represented a 22-fold purification comparing the specific activities found in isolated myelin (58 cpm/pg total protein) with the isolated protein (1273 cpm/pg glycoprotein). The radioactive fucose profile shown in Fig. 1B indicated that a highly enriched preparation of MyGP1 has been obtained. This [3H]fucoselabeled glycoprotein was used in a radioimmunoassay to quantitate antibody in rabbits immunized with this protein and with isolated Lewis rat myelin. New Zealand white rabbits were injected with 100 pg of MyGP1 in Freund's complete adjuvant (FCA) (2.5 mg Mycobacterium butyricum/ml). Animals were boosted 21 days later with 280 p g of glycoprotein in incomplete Freund's adjuvant and
236 Antiserum Titration Curves: .80
I I
I
I
I
I
1
I
I I I
I
I
I
I
I
I
70
60
0.361 x 10 -7 M
, 0
-
~ 40 ~
_
O. 30 - -
33% Binding Level
" ~ , , ~
~
_
2O 10 I 0.1
I
I
I 0.06
I
I
I
II I I I I I I 0.01 0.005 Final Dilution of Rabbit Anti Myelin GP1 Serum
I 0.001
}rig. 2. Titration curves of the rabbit a n t i - M y G P l serum. Specific activity of the M y G P I preparation: 1480 cpm/pg protein. [ M y G P I ] = 0.361 x 10 7 M : total cpm -- 535, assay volume - 100/~1. [ M y G P I ] 1.526 x 10 -7 M : total c p m -- ! 130, assay volume = 50 ffl. Based on estimated molecular weight o f 105 daltons. D a t a points duplicate averages. Experimental binding - total binding - - nonspecific binding o f n o r m a l s e r u m a n d sheep anti-rabbit l g G serum/total c p m - - nonspecific binding o f n o r m a l s e r u m a n d sheep anti-rabbit l g G serum. -
-
Antiserum Absorption: 6(
5(
i
i
~X
•~ : Unabsorbed ~ Heart o----o Thymocytes
~,~
~
%\
-
Liver
oo
~= 3o
2 2O
10
1/10
1/20 1/40 1/80 Final Dilution of Rabbit Anti Myelin GP1 Serum
1/160
Fig. 3. Absorption of rabbit a n t i - M y O P l serum. The antisera (1 ml of 1/5 dilution) was absorbed twice with an equal v o l u m e o f Lewis rat h o m o g e n a t e s o f tissue in borate buffered saline, p H 8.0 (20 mg/ml) by i n c u b a t i o n at 37 °C for 30 rain a n d at 4 °C for 30 m i n followed by centrifugation at 12,000 x g for 10 rain. Heart, t h y m o c y t e , liver, a n d brain h o m o g e n a t e s were subjected to prior acetone extraction.
237 bled at day 30. These animals showed no evidence of EAE at day 30. The capacity of the antisera to bind [aH]fucose-MyGP1 was tested using a double antibody procedure with sheep anti-rabbit IgG as the second antibody. The antiserum to rabbit IgG, which was purified from rabbit y-globulin protein (Cappel Lab.) by DEAE-cellulose, was produced in sheep by primary immunization in CFA followed by successive secondary immunizations in incomplete Freund's adjuvant. Equivalence was determined in this double-antibody system at each dilution of both the immune and normal sera. After incubation of normal and immune rabbit serum with the labeled MyGP1 for 1 h at 37 and l h at 4 °C, an equivalent amount of this sheep anti-rabbit IgG was added. After an overnite incubation, the precipitate formed was washed 3 times with borate buffered saline (pH 8.0) and hydrolyzed for 30 min with 200/A 1 N NaOH. The radioactivity was then determined by liquid scintillation spectrometry to a 2-sigma counting error of 4- 4 ~ . Titration curves of one antiglycoprotein serum at two different antigen concentrations are shown in Fig. 2. The ratio between the two concentrations was 0.24 while the ratio between the titers at the 50 ~ maximal binding level was 0.29 which indicated that the immune precipitation was proportional to the antigen in this concentration range. At a dilution of 1:213, 33 ~ of the antigen was bound which represented an antigen binding capacity of 2.54 #M. Absorption of this antiglycoprotein antiserum with brain or myelin significantly reduced the antigen binding capacity, although absorption with heart, liver, or thymocyte homogenates did not affect its capacity to bind the labeled antigen (Fig. 3). Rabbits were also injected with isolated Lewis rat myelin (440 #g myelin protein) in CFA followed by successive secondary immunizations and bleedings. These rabbits developed EAE by day 12 and produced substantial levels of antibody to the glycoprotein. An experimental binding of 32 and 22 ~o was obtained at a dilution of 1/10 for two different rabbits immunized with whole myelin, while an experimental binding of 66 and 59 ~o was obtained at a similar dilution with rabbits immunized with the isolated glycoprotein preparation. The demonstration that a membrane surtace glycoprotein associated with the myelin sheath complex is antigenic should provide a basis for investigating its precise cytochemical localization. In addition, antisera against MyGP1 can be used to study differentiation in the nervous system as well as provide the means for searching for the release of MyGPI during the demyelinating process of certain human neurological diseases. Such an approach has recently been pursued with a less accessible component of myelin, the basic protein1,2, n. The accessible, surface membrane, location of MyGPI as well as its susceptibility to degradation after autolysis compared to other myelin proteins~ could provide a sensitive indication of disease activity. The present finding of antibody to MyGPI in animals with EAE that were challenged with isolated myelin supports the possibility that the immune response to other antigenic components in nervous tissue may contribute to the pathogenesis of this experimental disease. It is well known that, on a dry weight basis, the potency of CNS tissue to induce EAE is much greater than basic protein itself or its encephalitogenic determinant a,v. The presence of additional myelin-associated antigens operating in conjunction with the major encephalitogenic determinant of basic protein could
238 a c c o u n t for the greater encephalitogenicity seen with whole tissue in this disease process. The c a p a c i t y for a n t i b o d y p r o d u c t i o n against this myelin-associated glycop r o t e i n should facilitate f u r t h e r investigations o f b o t h f u n d a m e n t a l n e u r o b i o l o g i c a l a n d p a t h o g e n i c mechanisms operative in the nervous system.
1 Cohen, S. R., Herndon, R. M. and McKhann, G. M., Radioimmunoassay of myelin basic protein in spinal fluid, New Engl. J. Med., 295 (1976) 1455-1457. 2 Gutstein, H. S. and Cohen, S. R., Spinal fluid differences in experimental allergic encephalomyelitis and multiple sclerosis, Science, 199 (1978) 301-303. 3 Hoffman, P. M., Gaston, D. D. and Spitler, L. E., Comparison of experimental allergic encephalomyelitis induced with spinal cord, basic protein, and synthetic encephalitogenic peptide, Clin. Immunol. lmmunopath., 1 (1973) 364-371. 4 Marchesi,V. T. and Andrews, E. P., Glycoproteins: isolation from cell membranes with lithium diiodosalicylate,Science, 174 (1971) 1247-1248. 5 Matthieu, J.-M., Koellreutter, B. and Joyet, M.-L., Changes in CNS myelin proteins and glycoproteins after in situ autolysis, Brain Res. Bull., 2 (1977) 15-21. 6 Norton, W. T. and Poduslo, S. E., Myelination in rat brain: method of myelin isolation, J. Neurochem., 21 (1973) 749-757. 7 Paterson, P. Y., Autoimmune neurological disease: experimental animal systems and implications for multiple sclerosis. In N. Talal (Ed.), Autoimmunity, Academic Press, New York, 1978, pp. 643691. 8 Poduslo, J. F., The molecular architecture ofmyelin: identification ofthe external surface membrane components, Advanc. exp. med. BioL, 100 (1978) 189-206. 9 Poduslo, J. F. and Braun, P. E., Topographical arrangement of membrane proteins in the intact myelin sheath, J. bioL Chem., 250 (1975) 1099-1105. 10 Poduslo, J. F., Everly, J. L. and Quarles, R. H., A low molecular weight glycoprotein associated with isolated myelin : distinction from myelin proteolipid protein, J. Neurochem., 28 (1977) 977-986. 11 Whitaker, J. N., Myelin encephalitogenic protein fragments in cerebrospinal fluid of persons with multiple sclerosis, Neurology (Minneap.), 27 (1977) 911-920.