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Shark CNS myelin contains four polypeptides related to the PNS protein Po of higher classes
Brain Research, 278 (1983) 350-353 Elsevier
350
Shark CNS myelin contains four polypeptides related to the PNS protein Po of higher classes F. LIN TAI and ROSS SMITH*
Department of Biochemistry, La Trobe University, Bundoora, Victoria 3083 (Australia) (Accepted June 21st, 1983)
Key words': myelin - - myelin proteins - - P,, protein - - shark - - immunoblotting - - Schwann cells - - oligodendroglialcells
The relationship between the proteins of shark central nervous system (CNS) myelin and those of myelin from higher classes has been investigated using antibodies raised against a 31,500 molecular weight polypeptide from shark myelin. The antibodies cross-reacted with 3 shark CNS polypeptides apart from the original antigen, with 2 major polypeptides from shark peripheral nervous system myelin, with the Po protein from chicken and sheep peripheral nervous system myelin, but with none of the components of bovine CNS myelin. It appears that the oligodendroglial cells of the shark synthesize a protein closely related to the P,, protein produced by Schwann cells of vertebrate classes above and including chondrichthytes. In the higher vertebrates myelin is formed by different classes of cells in the central (CNS) and peripheral (PNS) nervous systems. The oligodendroglial cells of the CNS and the Schwann cells of the PNS produce myelins that are easily distinguished by their polypeptide compositions. Although both myelins contain the same basic protein (designated P1 in the PNS and MBP in the CNS), the m a j o r intrinsic membrane proteins, the proteolipid protein (CNS) and the Po protein (PNS), a p p e a r unrelated 2. However, Franz et al.5 recently observed that the CNS myelin of fish does not have the polypeptide composition typical of higher animals, and in particular, that it has a major c o m p o n e n t with an apparent molecular weight close to that of Po. By making antibodies to this component we have now shown that it is indeed related to Po. Myelin was p r e p a r e d from the brains and cranial nerves of the shark, Mustelus antarticus, by standard methods j-12. PNS myelin proteins of chicken (Gallus gallus) and sheep (Ovis ovis) were kindly supplied by Ms. 1. M. Roberts of the School of Agriculture, La Trobe University. Bovine CNS myelin was p r e p a r e d as previously described m. Myelin samples were prepared for electrophoresis by taking about 25 ~1 of
suspension containing about 4 mg/ml of protein (assessed colorimetrically9) and adding 40/,1 of a solution containing 20% (w/v) sodium dodecyl sulphate, 0.1% (v/v) 2-mercaptoethanol, and b r o m o p h e n o l blue. Aliquots of these solutions were run on 15% polyacrylamide gels containing dodecyl sulphate, following LaemmliS. Following electrophoresis gels were stained with Coomassie blue or used for protein transfers. Antibodies were raised to a polypeptide from shark CNS myelin. This polypeptide was excised from unstained gels in which the samples had been labeled with fluorescaminea. U n d e r ultraviolet light the 31,500 molecular weight band was cut from the gels, the gel slices mascerated, and the protein extracted by stirring the gel in a small volume of 0.04% sodium dodecyl sulphate for 25 h. The extracted protein was freed of detergent by dialysis against watm and then used to raise antibodies in rabbits by intradermal injection. The antiserum was diluted 250-fold with 35% bovine serum albumin in phosphate-buffered saline (PBS) for electroblotting. Electrophoretic transfer of proteins in unstained gels to nitrocellulose p a p e r (Schleicher and Schiill) followed Burnette 3, except that 25 mM phosphate
* To whom correspondence should be addressed at: Department of Biochemistry, University of Queensland, St. Lucia, Queenshmd 4067, Australia. 0006-8993/83/$03.00 © 1983 Elsevier Science Publishers B.V.
351
r
1 2 3 4
ca
b
C
d
Fig. 1. Electrophoretic patterns of: 20/xg sheep PNS myelin proteins (a); 30/~g shark CNS myelin proteins (b); 30 ~g bovine CNS myelin proteins (c); and 30/~g shark PNS myelin proteins (d), stained with Coomassie blue. The apparent molecular weights of the shark polypeptides, deduced by comparison of mobilities with those of standard proteins, were 31,500 (marked as band 1 at left), 27,000 (2), 26,000 (3) and 21,000 (4).
buffer, pH 6.8, containing 0.01% sodium dodecyl sulphate was used as buffer. After the transfer the pape r was washed with a 3% solution of bovine serum albumin in PBS. It was next incubated for 1 h at room temperature with the diluted antiserum. Excess antibody was washed from the paper with 3 changes of PBS and one of 0.05% Nonidet P-40 in PBS, and the paper subsequently incubated with radio-iodinated protein A (from Staphylococcus aureas), containing 25/~Ci/~g. Excess protein A was removed by further washing as described above. The dried paper was autoradiographed for 10 h on Kodak X-ray film. As shown in Fig. 1, the protein patterns for shark and bovine CNS myelins differ substantially, although most of the proteins are of low molecular weight. The shark proteins correspond closely with those for other cartilaginous fish 5 and, in particular, include a polypeptide (marked 1 in Fig. 1) with the same apparent molecular weight as the PNS Po pro-
tein of higher classes. This 31,500 molecular weight polypeptide was isolated from gels and used to raise antibodies. The cross-reactivity of these antibodies with the proteins from several other myelin samples was examined by electroblotting. The antibody bound to 4 shark CNS myelin polypeptides (Fig. 2A) which are labeled 1-4 in Fig. 1. Thus several of the bands on the gel appear to contain closely related polypeptides. These bands may represent the products of translation of several related messenger RNAs (as with the CNS basic protein6), or be conformational variants of a single polypeptide chain (as appears to be the case with the proteolipid and DM20 proteins11). No cross-reaction was observed with any of the bovine CNS proteins (Fig. 2A, lanes b, c and d), implying the absence of common antigenic determinants. This lack of immunological cross-reaction could result from divergence of the sequences of a common protein, or from the absence from the CNS myelin of higher vertebrates of a protein related to this major shark protein. Our subsequent results favour the latter alternative. Shark PNS myelin showed several cross-reactive polypeptides, the largest corresponding in electrophoretic mobility to the CNS antigen (Fig. 2B, lanes c and d). But the sole labeledcomponent of sheep and chicken PNS myelins was found, by comparison of the autoradiograms with stained gels, to be the Po protein (Fig. 2B, lanes a and b). Thus the oligodendroglial and Schwann cells of the shark both synthesize this major protein, which is produced only by the Schwann cells of higher vertebrate classes. The shark CNS myelin seems not to contain the proteolipid protein, as all of the polypeptides with apparent molecular weights below 32,000 are labeled with antibodies to either the 31,500 molecular weight antigen or are related to the encephalitogenic basic protein of higher classes (unpublished observations). Sharks are phylogenetically the lowest vertebrates in which the CNS and PNS can be distinguished. Although the polypeptide composition of the CNS and PNS myelins of cartilaginous fish are not identicaP, it is now clear that several of their components are closely related. By contrast, in higher classes the two cell types have only the Pi basic protein as a common myelin component. Hence, although the oligodendroglial cells of higher animals possess the gene coding for the Po protein, this gene is not expressed. These cells ap-
352
B
A
a
b
c
d
! i i~ii~iil :
a b cd
e
Fig. 2. Autoradiographs of myelin proteins labeled with [L'5l]protein A. A: the samples are 50 #g whole shark CNS myelin protein (a): 35 ug whole bovine CNS myelin protein (b); 511ug bovine CNS myelin protein (c); 75 l~g bovine CNS myelin protein (d): 20 l~g of proteins extracted from shark CNS myelin following Kitamura et al. 7. This method is normally used to isolate the Po protein from PNS myelin: the fact that it also isolates the shark CNS myelin polypeptides shows the similarity in solubility properties of these polypeptides and Po (e). B:
the samples are 50 ~g chicken PNS myelin proteins (a); 50/~g sheep PNS myelin proteins (b); and 50 ~g of whole shark PNS myelin proteins (c) and (d). In both autoradiographs electrophoretic migration was from top to bottom, and the shark CNS bands marked 1-4 correspond to those labeled in Fig. 1.
p e a r to have u n d e r g o n e an e v o l u t i o n a r y d i v e r g e n c e
W e are grateful to Dr. C. C. A. B e r n a r d for sup-
away f r o m S c h w a n n cells with the Po p r o t e i n being
plying i o d i n a t e d p r o t e i n A . R. Smith is s u p p o r t e d by
r e p l a c e d by the p r o t e o l i p i d p r o t e i n as the m a j o r in-
grants f r o m the A u s t r a l i a n R e s e a r c h G r a n t s S c h e m e
trinsic m e m b r a n e p r o t e i n .
and the
National
Health
and
Medical
Research
Council.
1 Autilio, L. A., Norton, W. T. and Terry, R. D., The preparation and some properties of purified myelin from the CNS, J. Neurochem., 11 (1964) 17-27. 2 Braun, P. E. and Brostoff. S. W., Proteins of myelin, In P. Morell (Ed.). Myelin. Plenum Press, NY, 1977, pp. 201-231. 3 Burnette, W. N., "Western blotting': Electrophoretic transfer of proteins from SDS-polyacryla, mide gels to unmodified nitrocellulose and radiographic detection with antibodies and radioiodinated protein A, Analyt. Biochem., 112 (1981) 195-203. 4 Eng, P. R. and Parkes, C. O., Sodium dodecyl sulphate electrophoresis of fluorescamine-labelled proteins, Analvt. Biochem., 59 (1974) 323-325. 5 Franz, T., Waehneldt, T. V., Neuhoff, V. and Wachtler, K., Central nervous system myelin proteins and glycoproteins in vertebrates: a phylogenetic study, Brain Research. 226 (1981) 245-258.
6 Hall, C., Mahadevan, C., Whatley, S. A,, Ling, T.-S. and Lira, L., The polyadenylated RNA directing the synthesis of the rat myelin basic proteins is present in both free and membrane-bound forebrain polyribosomes, Biochem. J., 202 (1982) 407-417. 7 Kitamura, K., Suzuki, M. and Uyemura, K., Purification and partial characterization of two glycoproteins in bovine peripheral nerve myelin membrane, Biochem. Biophys. Acta. 455 11976) 806-816. 8 Laemmli, U. K., Cleavage of structural proteins during the assembly of the head of bacteriophage T4, Nature (Lond. ~. 227 (1970) 680-685. 9 Lowry, O. H., Rosebrough, N. J., Farr, A. L. and Randall, R. J., Protein measurement with the Folin phenol reagent, J. biol. Chem., 193 11951) 265-275. 10 Smith, R., The secondary structure of myelin basic protein extracted by deoxycholate, Biochim. biophys. Acta, 49t ( 19771 581-590.
353 11 Stoffel, W., Schr6der, W., Hillen, H. and Deutzmann, R., Analysis of the primary structure of the strongly hydrophobic brain myelin proteolipid apoprotein (lipophilin). Isolation and amino acid sequence determination of proteolytic fragments, Hoppe-Seyler's Z. Physiol. Chem., 363 (1982) 1117-1131.
12 Uyemura, K., Tobari, C., Hirano, S. and Tsukada, Y., Comparative studies on the myelin proteins of bovine peripheral nerve and spinal cord, J. Neurochem., 19 (1972) 2607-2614.
350
Shark CNS myelin contains four polypeptides related to the PNS protein Po of higher classes F. LIN TAI and ROSS SMITH*
Department of Biochemistry, La Trobe University, Bundoora, Victoria 3083 (Australia) (Accepted June 21st, 1983)
Key words': myelin - - myelin proteins - - P,, protein - - shark - - immunoblotting - - Schwann cells - - oligodendroglialcells
The relationship between the proteins of shark central nervous system (CNS) myelin and those of myelin from higher classes has been investigated using antibodies raised against a 31,500 molecular weight polypeptide from shark myelin. The antibodies cross-reacted with 3 shark CNS polypeptides apart from the original antigen, with 2 major polypeptides from shark peripheral nervous system myelin, with the Po protein from chicken and sheep peripheral nervous system myelin, but with none of the components of bovine CNS myelin. It appears that the oligodendroglial cells of the shark synthesize a protein closely related to the P,, protein produced by Schwann cells of vertebrate classes above and including chondrichthytes. In the higher vertebrates myelin is formed by different classes of cells in the central (CNS) and peripheral (PNS) nervous systems. The oligodendroglial cells of the CNS and the Schwann cells of the PNS produce myelins that are easily distinguished by their polypeptide compositions. Although both myelins contain the same basic protein (designated P1 in the PNS and MBP in the CNS), the m a j o r intrinsic membrane proteins, the proteolipid protein (CNS) and the Po protein (PNS), a p p e a r unrelated 2. However, Franz et al.5 recently observed that the CNS myelin of fish does not have the polypeptide composition typical of higher animals, and in particular, that it has a major c o m p o n e n t with an apparent molecular weight close to that of Po. By making antibodies to this component we have now shown that it is indeed related to Po. Myelin was p r e p a r e d from the brains and cranial nerves of the shark, Mustelus antarticus, by standard methods j-12. PNS myelin proteins of chicken (Gallus gallus) and sheep (Ovis ovis) were kindly supplied by Ms. 1. M. Roberts of the School of Agriculture, La Trobe University. Bovine CNS myelin was p r e p a r e d as previously described m. Myelin samples were prepared for electrophoresis by taking about 25 ~1 of
suspension containing about 4 mg/ml of protein (assessed colorimetrically9) and adding 40/,1 of a solution containing 20% (w/v) sodium dodecyl sulphate, 0.1% (v/v) 2-mercaptoethanol, and b r o m o p h e n o l blue. Aliquots of these solutions were run on 15% polyacrylamide gels containing dodecyl sulphate, following LaemmliS. Following electrophoresis gels were stained with Coomassie blue or used for protein transfers. Antibodies were raised to a polypeptide from shark CNS myelin. This polypeptide was excised from unstained gels in which the samples had been labeled with fluorescaminea. U n d e r ultraviolet light the 31,500 molecular weight band was cut from the gels, the gel slices mascerated, and the protein extracted by stirring the gel in a small volume of 0.04% sodium dodecyl sulphate for 25 h. The extracted protein was freed of detergent by dialysis against watm and then used to raise antibodies in rabbits by intradermal injection. The antiserum was diluted 250-fold with 35% bovine serum albumin in phosphate-buffered saline (PBS) for electroblotting. Electrophoretic transfer of proteins in unstained gels to nitrocellulose p a p e r (Schleicher and Schiill) followed Burnette 3, except that 25 mM phosphate
* To whom correspondence should be addressed at: Department of Biochemistry, University of Queensland, St. Lucia, Queenshmd 4067, Australia. 0006-8993/83/$03.00 © 1983 Elsevier Science Publishers B.V.
351
r
1 2 3 4
ca
b
C
d
Fig. 1. Electrophoretic patterns of: 20/xg sheep PNS myelin proteins (a); 30/~g shark CNS myelin proteins (b); 30 ~g bovine CNS myelin proteins (c); and 30/~g shark PNS myelin proteins (d), stained with Coomassie blue. The apparent molecular weights of the shark polypeptides, deduced by comparison of mobilities with those of standard proteins, were 31,500 (marked as band 1 at left), 27,000 (2), 26,000 (3) and 21,000 (4).
buffer, pH 6.8, containing 0.01% sodium dodecyl sulphate was used as buffer. After the transfer the pape r was washed with a 3% solution of bovine serum albumin in PBS. It was next incubated for 1 h at room temperature with the diluted antiserum. Excess antibody was washed from the paper with 3 changes of PBS and one of 0.05% Nonidet P-40 in PBS, and the paper subsequently incubated with radio-iodinated protein A (from Staphylococcus aureas), containing 25/~Ci/~g. Excess protein A was removed by further washing as described above. The dried paper was autoradiographed for 10 h on Kodak X-ray film. As shown in Fig. 1, the protein patterns for shark and bovine CNS myelins differ substantially, although most of the proteins are of low molecular weight. The shark proteins correspond closely with those for other cartilaginous fish 5 and, in particular, include a polypeptide (marked 1 in Fig. 1) with the same apparent molecular weight as the PNS Po pro-
tein of higher classes. This 31,500 molecular weight polypeptide was isolated from gels and used to raise antibodies. The cross-reactivity of these antibodies with the proteins from several other myelin samples was examined by electroblotting. The antibody bound to 4 shark CNS myelin polypeptides (Fig. 2A) which are labeled 1-4 in Fig. 1. Thus several of the bands on the gel appear to contain closely related polypeptides. These bands may represent the products of translation of several related messenger RNAs (as with the CNS basic protein6), or be conformational variants of a single polypeptide chain (as appears to be the case with the proteolipid and DM20 proteins11). No cross-reaction was observed with any of the bovine CNS proteins (Fig. 2A, lanes b, c and d), implying the absence of common antigenic determinants. This lack of immunological cross-reaction could result from divergence of the sequences of a common protein, or from the absence from the CNS myelin of higher vertebrates of a protein related to this major shark protein. Our subsequent results favour the latter alternative. Shark PNS myelin showed several cross-reactive polypeptides, the largest corresponding in electrophoretic mobility to the CNS antigen (Fig. 2B, lanes c and d). But the sole labeledcomponent of sheep and chicken PNS myelins was found, by comparison of the autoradiograms with stained gels, to be the Po protein (Fig. 2B, lanes a and b). Thus the oligodendroglial and Schwann cells of the shark both synthesize this major protein, which is produced only by the Schwann cells of higher vertebrate classes. The shark CNS myelin seems not to contain the proteolipid protein, as all of the polypeptides with apparent molecular weights below 32,000 are labeled with antibodies to either the 31,500 molecular weight antigen or are related to the encephalitogenic basic protein of higher classes (unpublished observations). Sharks are phylogenetically the lowest vertebrates in which the CNS and PNS can be distinguished. Although the polypeptide composition of the CNS and PNS myelins of cartilaginous fish are not identicaP, it is now clear that several of their components are closely related. By contrast, in higher classes the two cell types have only the Pi basic protein as a common myelin component. Hence, although the oligodendroglial cells of higher animals possess the gene coding for the Po protein, this gene is not expressed. These cells ap-
352
B
A
a
b
c
d
! i i~ii~iil :
a b cd
e
Fig. 2. Autoradiographs of myelin proteins labeled with [L'5l]protein A. A: the samples are 50 #g whole shark CNS myelin protein (a): 35 ug whole bovine CNS myelin protein (b); 511ug bovine CNS myelin protein (c); 75 l~g bovine CNS myelin protein (d): 20 l~g of proteins extracted from shark CNS myelin following Kitamura et al. 7. This method is normally used to isolate the Po protein from PNS myelin: the fact that it also isolates the shark CNS myelin polypeptides shows the similarity in solubility properties of these polypeptides and Po (e). B:
the samples are 50 ~g chicken PNS myelin proteins (a); 50/~g sheep PNS myelin proteins (b); and 50 ~g of whole shark PNS myelin proteins (c) and (d). In both autoradiographs electrophoretic migration was from top to bottom, and the shark CNS bands marked 1-4 correspond to those labeled in Fig. 1.
p e a r to have u n d e r g o n e an e v o l u t i o n a r y d i v e r g e n c e
W e are grateful to Dr. C. C. A. B e r n a r d for sup-
away f r o m S c h w a n n cells with the Po p r o t e i n being
plying i o d i n a t e d p r o t e i n A . R. Smith is s u p p o r t e d by
r e p l a c e d by the p r o t e o l i p i d p r o t e i n as the m a j o r in-
grants f r o m the A u s t r a l i a n R e s e a r c h G r a n t s S c h e m e
trinsic m e m b r a n e p r o t e i n .
and the
National
Health
and
Medical
Research
Council.
1 Autilio, L. A., Norton, W. T. and Terry, R. D., The preparation and some properties of purified myelin from the CNS, J. Neurochem., 11 (1964) 17-27. 2 Braun, P. E. and Brostoff. S. W., Proteins of myelin, In P. Morell (Ed.). Myelin. Plenum Press, NY, 1977, pp. 201-231. 3 Burnette, W. N., "Western blotting': Electrophoretic transfer of proteins from SDS-polyacryla, mide gels to unmodified nitrocellulose and radiographic detection with antibodies and radioiodinated protein A, Analyt. Biochem., 112 (1981) 195-203. 4 Eng, P. R. and Parkes, C. O., Sodium dodecyl sulphate electrophoresis of fluorescamine-labelled proteins, Analvt. Biochem., 59 (1974) 323-325. 5 Franz, T., Waehneldt, T. V., Neuhoff, V. and Wachtler, K., Central nervous system myelin proteins and glycoproteins in vertebrates: a phylogenetic study, Brain Research. 226 (1981) 245-258.
6 Hall, C., Mahadevan, C., Whatley, S. A,, Ling, T.-S. and Lira, L., The polyadenylated RNA directing the synthesis of the rat myelin basic proteins is present in both free and membrane-bound forebrain polyribosomes, Biochem. J., 202 (1982) 407-417. 7 Kitamura, K., Suzuki, M. and Uyemura, K., Purification and partial characterization of two glycoproteins in bovine peripheral nerve myelin membrane, Biochem. Biophys. Acta. 455 11976) 806-816. 8 Laemmli, U. K., Cleavage of structural proteins during the assembly of the head of bacteriophage T4, Nature (Lond. ~. 227 (1970) 680-685. 9 Lowry, O. H., Rosebrough, N. J., Farr, A. L. and Randall, R. J., Protein measurement with the Folin phenol reagent, J. biol. Chem., 193 11951) 265-275. 10 Smith, R., The secondary structure of myelin basic protein extracted by deoxycholate, Biochim. biophys. Acta, 49t ( 19771 581-590.
353 11 Stoffel, W., Schr6der, W., Hillen, H. and Deutzmann, R., Analysis of the primary structure of the strongly hydrophobic brain myelin proteolipid apoprotein (lipophilin). Isolation and amino acid sequence determination of proteolytic fragments, Hoppe-Seyler's Z. Physiol. Chem., 363 (1982) 1117-1131.
12 Uyemura, K., Tobari, C., Hirano, S. and Tsukada, Y., Comparative studies on the myelin proteins of bovine peripheral nerve and spinal cord, J. Neurochem., 19 (1972) 2607-2614.