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Expression of the large myelin-associated glycoprotein isoform during the development in the mouse peripheral nervous system
Brain Research, 562 (1991) 173-175 © 1991 Elsevier Science Publishers B.V. All rights reserved. 0006-8993/91/$03.50 ADONIS 000689939124876B
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BRES 24876
Expression of the large myelin-associated glycoprotein isoform during the development in the mouse peripheral nervous system Takashi Inuzuka, Nobuya Fujita, Shuzo Sato, Hiroko Baba, Ryoichi Nakano, Hideaki Ishiguro and Tadashi Miyatake Department of Neurology, Brain Research Institute, Niigata University, Niigata (Japan) (Accepted 9 July 1991) Key words: Myelin-associated glycoprotein; Isoform; Developmental change; Myelination
The developmental maximum expression of the large myelin-associated glycoprotein isoform (L-MAG) protein prior to that of the small myelin-associated glycoprotein isoform (S-MAG) in both the central and peripheral nervous systems (CNS, PNS) in mice was shown by immunobiotting techniques using specific antibodies to the L-MAG protein and the S-MAG protein. Both the L-MAG protein and the S-MAG protein were expressed earlier in the PNS than in the CNS, which reflects earlier myelination in the PNS. The peak of the L-MAG protein concentration was 8 days in the sciatic nerve and 15 days in the brainstem. The concentration of the S-MAG protein in the sciatic nerve reached a peak at 15 days, whereas in the brainstem it increased rapidly between 15 and 20 days and gradually thereafter. Thus, the preceding maximum expression of the L-MAG during active myelination in the PNS demonstrated here as well as in the CNS strongly suggests an important role for L-MAG in myelin formation. Myelin-associated glycoprotein (MAG) is a myelin protein thought to be involved in myelin forming cellaxon interaction 11. In rodents, two M A G isoforms that differ in their C-termini are produced as a result of alternative splicing of exon 12 (ref. 7). The large MAG isoform (L-MAG) is produced by m R N A lacking exon 12 whereas the small MAG isoform (S-MAG) is produced by m R N A containing this exon. Expression of these two mRNAs is supposed to be regulated by myelinogenesis in the CNS. The L-MAG m R N A is characteristically induced at the time of most active myelination, including remyelination in the CNS 3'4. Our previous Northern blotting and immunohistochemical studies in the quaking mutant mouse brain, which lacks compaction of the myelin sheath, have demonstrated the scarce expression of the L-MAG mRNA and protein 3'5. These results suggest that the L-MAG protein has an important role in myelin formation at an early developmental stage in the CNS. The developmental change described has been known, with respect to CNS, as change of immature to mature M A G proteins 11. On the other hand, the developmental changes of MAG mRNAs and proteins in the PNS are controversial. It is difficult to obtain enough m R N A from the PNS and nobody has succeeded to get the specific antibodies to distinguish L-MAG protein and S-MAG protein. We have succeeded
in preparing these antibodies, and our immunoblotting study clearly demonstrated the maximum expression of L-MAG protein prior to that of the S-MAG protein during development in the PNS as well as in the CNS. Charles River Co. Japan supplied the ddY mice which were sacrificed at postnatal days 3 (10 mice), 5 (10), 8 (10), 10 (8), 15 (5), 20 (5), 30 (3), 40 (3) and 180 (3). The number in parentheses denote the number of mice sacrificed. The brainstem and sciatic nerves were dissected out. All tissues were stored frozen at -70 °C until used. Three separate experiments were done in the same manner. Brainstem or sciatic nerve homogenates were prepared in cold 10 mM Tris-HC1 (pH 7.5) and appropriate aliquots were taken for the assay described as follows. Lyophilized homogenate aliquots were delipidized with diethyl ether:ethanol 3:2 (v/v) then electrophoresed on polyacrylamide slab gels according to the method of Laemmli and Favre 6. The running gel used was 11% polyacrylamide with 5 and 40/~g of total protein being applied for L-MAG protein in the CNS and in the PNS, respectively. Thirty and 20/~g of total protein was applied for S-MAG protein in the CNS and in the PNS, as the quantitative data could be obtained within the linear response range of densitometric evaluation of the Western blots. Protein concentrations of the homogenates were determined by the method of Lowry et al.S. Pro-
Correspondence: T. Inuzuka, Department of Neurology, Brain Research Institute, Niigata University, Niigata 951, Japan. Fax: (81) (25) 223-3620.
174
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5
810
15
20
30
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3'o
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180day
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Fig. 2. Developmental change of the L-MAG protein (closed circles) and the S-MAG protein (open circles) in the brainstem (C) or in the sciatic nerves (P). The values are expressed as percent of the maximum of each isoform in (C) or (P) and represent the average of 3 experiments ± 3 S.D.
Fig. 1. Representative immunoblots stained for thc L-MAG protein in the brainstem (C) or the sciatic nerves (P). The numbers on top of the blots are days after birth.
tein blotting was carried out essentially according to the method of Towbin et al. 13. The blots were immunostained with anti-L-MAG protein or anti-S-MAG protein antibody, then with peroxidase-conjugated anti-rabbit IgG (heavy and light chain) (Kirkegaad & Perry Lab., Gaithesburg, MD, U.S.A.) using 4-chloro-l-naphthol (Sigma Chemical, St. Louis, MO, U.S.A.) as the chromogen. The anti-L-MAG protein and the anti-S-MAG protein antibodies were diluted 1:800 and 1:400, respectively, in 3% bovine serum albumin. The blots were then scanned with a spectrodensitometer (CS-910, Shimadzu, Japan) at 580 nm. The amount of each isoform was expressed as a percentage of its maximum during development in the CNS or in the PNS. Antibodies to each isoform were prepared as previously described 5. Briefly, the synthesized peptides unique to the L-MAG-derived 21 amino acid residues of the C-terminus and to the S-MAG-derived 11 amino acid residues deduced from the exon 12 portion including an additional lysine were used as antigens. Each processed peptide was emulsified with Freund's adjuvant and injected into rabbits several times at 14-day intervals. The
antibodies were specific in that they were quantitatively adsorbed by an excess of the peptide used to generate them. The maximum expression of the S-MAG protein following the L-MAG protein in the CNS (Figs. 1C and 2C) was similar to those of the L-MAG m R N A and the S-MAG m R N A previously reported 3. The expression of mRNAs are more transient than that of proteins, which may be a result of the accumulation of the M A G protein in the CNS. The peak of concentration of the L-MAG protein in the PNS was demonstrated at day 8, when that of the S-MAG protein in the PNS was still low (Figs. 1P and 2P). The concentration of the S-MAG protein in the PNS reached a peak at day 15. The maximum expression of the L-MAG protein and the S-MAG protein in the PNS was earlier than those in the CNS, as myelination in the PNS begins earlier. The L-MAG protein in the rat sciatic nerve was not detectable by in vitro translation 2. Tropak et al. 14 have reported by RNAase protection experiments that S-MAG m R N A is predominant throughout development whereas the L - M A G m R N A was barely detectable in the PNS. Western blotting using antibodies to the L-MAG protein and to the common part of both M A G isoform proteins suggested that only S-MAG was in the PNS 9. Recently, Owen et al. t° have demonstrated that the L-MAG protein was transiently expressed at the ear-
175 liest stage of myelination in vitro by immunostaining myelinating cultures of Schwann cells and dorsal root ganglion cells with an antibody specific to L - M A G protein. O u r present study, however, is the first r e p o r t to show the d e v e l o p m e n t a l changes of two M A G isoform proteins in the sciatic nerve using a specific a n t i b o d y to each isoform of M A G . The concentration of the L - M A G protein r e m a i n e d relatively high after the p e a k in the CNS c o m p a r e d with that in PNS, which is compatible with changes of the L - M A G m R N A in both nervous systems TM. This might be due to a difference in L - M A G protein turnover b e t w e e n CNS and PNS. O u r previous data have suggested the i m p o r t a n c e of the L - M A G protein in the early stage of myelinogenesis in the CNS 3-5. E d w a r d et a l ) r e p o r t e d that L - M A G protein is phos-
p h o r y l a t e d in mouse and in vitro in myelin, and that the p h o s p h o r y l a t e d residues include tyrosine. Protein tyrosine phosphorylation plays i m p o r t a n t roles in the regulation, growth and development. H e r e we have e m p h a sized that the L - M A G protein is expressed p r e c e d e n t to the S - M A G protein also in the PNS, and suggest that the L - M A G protein plays an essential role for myelin formation in the PNS as well as in the CNS.
1 Edwards, A.M., Braun, P.E. and Bell, J.C., Phosphorylation of myelin-associated glycoprotein in vivo and in vitro occurs only in the cytoplasmic domain of the large isoform, J. Neurochem., 52 (1989) 317-320. 2 Frail, D.E., Webster, H.F. and Braun, P.E., Developmental expression of the myelin-associated glycoprotein in peripheral nervous system is different from that in the central nervous system, J. Neurochem., 45 (1985) 1308-1310. 3 Fujita, N., Sato, S., Kurihara, T., Inuzuka, T., Takahashi, Y. and Miyatake, T., Developmentally regulated alternative splicing of brain myelin-associated glycoprotein mRNA is lacking in the quaking mouse, FEBS Lett., 232 (1988) 323-327. 4 Fujita, N., Ishiguro, H., Sato, S., Kurihara, T., Kuwano, R., Sakimura, K., Takahashi, Y. and Miyatake, T., Induction of myelin-associated glycoprotein mRNA in experimental remyelination, Brain Research, 513 (1990) 152-155. 5 Fujita, N., Sato, S., Ishiguro, H., Inuzuka, T., Baba, H., Kurihara, T., Takahashi, Y. and Miyatake, T., The large isoform of myelin-associated glycoprotein is scarcely expressed in the quaking mouse brain, J. Neurochem., 55 (1990) 1056-1059. 6 Laemmli, U.K. and Favre, M., Maturation of the head of bacteriophage T4. I. DNA packing events, J. Mol. Biol., 80 (1973) 575-600. 7 Lai, C., Brown, M.A., Nave, K.-A., Noronha, A.B., Quarles, R.H., Bloom, EE., Milner, R.J. and Sutcliffe, J.G., Two forms of 1B236/myelin-associated glycoprotein, a cell adhesion molecule for postnatal neural development, are produced by alter-
native splicing, Proc. Natl. Acad. Sci. U.S.A., 84 (1987) 43374341. Lowry, O.H., Rosebrough, N.J., Farr, A.L. and Randall, R.J., Protein measurement with the Folin phenol reagent, J. Biol. Chem., 193 (1951) 265-275. Noronha, A.B., Hammer, J.A., Lai, C., Kiel, M., Milner, R.J., Sutcliffe, J.G. and Quarles, R.H., Myelin-associated glycoprotein (MAG) and rat brain-specific 1B236 protein: mapping of epitopes and demonstration of immunological identity, J. Mol. Neurosci., 1 (1989) 159-170. Owens, G.C., Boyd, C.J., Bunge, R.P. and Salzer, J.L., Expression of recombinant myelin-associated glycoprotein in primary Schwann cells promotes the initial investment of axons by myelinating Schwann cells, J. Cell. Biol., 111 (1990) 1171-1182. Quades, R.H., Everly, J.L. and Brady, R.O., Myelin-associated glycoprotein: a developmental change, Brain Research, 58 (1973) 506-509. Quarles, R.H., Myelin-associated glycoprotein in development and disease, Dev. Neurosci., 6 (1984) 285-303. Towbin, H., Staehelin, T. and Gordon, J., Eleetrophoretic transfer of proteins from polyacrylamide gel to nitrocellulose sheets: procedure and applications, Proc. Natl. Acad. Sci. U.S.A., 76 (1979) 4350-4354. Tropak, M.B., Johnson, P.W., Dunn, R.J. and Roder, J.C., Differential splicing of MAG transcripts during CNS and PNS development, Mol. Brain Res., 4 (1988) 143-155.
The authors thank Ms. S. Nagasawa, Ms. Y. Yagi and Ms. I. Morohashi for their excellent technical assistance. This work was supported by grants from the Ministry of Health and Welfare, from the Ministry of Education, Science and Culture of Japan, from Japan foundation for health science, from Uehara memorial foundation and from the Taishou Pharmaceutical Co.
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BRES 24876
Expression of the large myelin-associated glycoprotein isoform during the development in the mouse peripheral nervous system Takashi Inuzuka, Nobuya Fujita, Shuzo Sato, Hiroko Baba, Ryoichi Nakano, Hideaki Ishiguro and Tadashi Miyatake Department of Neurology, Brain Research Institute, Niigata University, Niigata (Japan) (Accepted 9 July 1991) Key words: Myelin-associated glycoprotein; Isoform; Developmental change; Myelination
The developmental maximum expression of the large myelin-associated glycoprotein isoform (L-MAG) protein prior to that of the small myelin-associated glycoprotein isoform (S-MAG) in both the central and peripheral nervous systems (CNS, PNS) in mice was shown by immunobiotting techniques using specific antibodies to the L-MAG protein and the S-MAG protein. Both the L-MAG protein and the S-MAG protein were expressed earlier in the PNS than in the CNS, which reflects earlier myelination in the PNS. The peak of the L-MAG protein concentration was 8 days in the sciatic nerve and 15 days in the brainstem. The concentration of the S-MAG protein in the sciatic nerve reached a peak at 15 days, whereas in the brainstem it increased rapidly between 15 and 20 days and gradually thereafter. Thus, the preceding maximum expression of the L-MAG during active myelination in the PNS demonstrated here as well as in the CNS strongly suggests an important role for L-MAG in myelin formation. Myelin-associated glycoprotein (MAG) is a myelin protein thought to be involved in myelin forming cellaxon interaction 11. In rodents, two M A G isoforms that differ in their C-termini are produced as a result of alternative splicing of exon 12 (ref. 7). The large MAG isoform (L-MAG) is produced by m R N A lacking exon 12 whereas the small MAG isoform (S-MAG) is produced by m R N A containing this exon. Expression of these two mRNAs is supposed to be regulated by myelinogenesis in the CNS. The L-MAG m R N A is characteristically induced at the time of most active myelination, including remyelination in the CNS 3'4. Our previous Northern blotting and immunohistochemical studies in the quaking mutant mouse brain, which lacks compaction of the myelin sheath, have demonstrated the scarce expression of the L-MAG mRNA and protein 3'5. These results suggest that the L-MAG protein has an important role in myelin formation at an early developmental stage in the CNS. The developmental change described has been known, with respect to CNS, as change of immature to mature M A G proteins 11. On the other hand, the developmental changes of MAG mRNAs and proteins in the PNS are controversial. It is difficult to obtain enough m R N A from the PNS and nobody has succeeded to get the specific antibodies to distinguish L-MAG protein and S-MAG protein. We have succeeded
in preparing these antibodies, and our immunoblotting study clearly demonstrated the maximum expression of L-MAG protein prior to that of the S-MAG protein during development in the PNS as well as in the CNS. Charles River Co. Japan supplied the ddY mice which were sacrificed at postnatal days 3 (10 mice), 5 (10), 8 (10), 10 (8), 15 (5), 20 (5), 30 (3), 40 (3) and 180 (3). The number in parentheses denote the number of mice sacrificed. The brainstem and sciatic nerves were dissected out. All tissues were stored frozen at -70 °C until used. Three separate experiments were done in the same manner. Brainstem or sciatic nerve homogenates were prepared in cold 10 mM Tris-HC1 (pH 7.5) and appropriate aliquots were taken for the assay described as follows. Lyophilized homogenate aliquots were delipidized with diethyl ether:ethanol 3:2 (v/v) then electrophoresed on polyacrylamide slab gels according to the method of Laemmli and Favre 6. The running gel used was 11% polyacrylamide with 5 and 40/~g of total protein being applied for L-MAG protein in the CNS and in the PNS, respectively. Thirty and 20/~g of total protein was applied for S-MAG protein in the CNS and in the PNS, as the quantitative data could be obtained within the linear response range of densitometric evaluation of the Western blots. Protein concentrations of the homogenates were determined by the method of Lowry et al.S. Pro-
Correspondence: T. Inuzuka, Department of Neurology, Brain Research Institute, Niigata University, Niigata 951, Japan. Fax: (81) (25) 223-3620.
174
C,o 50
5
810
15
20
30
t's 2'o
3'o
40
180day
P ~0c~
o ~ ~ ~b
4'o -
t~OUay
Fig. 2. Developmental change of the L-MAG protein (closed circles) and the S-MAG protein (open circles) in the brainstem (C) or in the sciatic nerves (P). The values are expressed as percent of the maximum of each isoform in (C) or (P) and represent the average of 3 experiments ± 3 S.D.
Fig. 1. Representative immunoblots stained for thc L-MAG protein in the brainstem (C) or the sciatic nerves (P). The numbers on top of the blots are days after birth.
tein blotting was carried out essentially according to the method of Towbin et al. 13. The blots were immunostained with anti-L-MAG protein or anti-S-MAG protein antibody, then with peroxidase-conjugated anti-rabbit IgG (heavy and light chain) (Kirkegaad & Perry Lab., Gaithesburg, MD, U.S.A.) using 4-chloro-l-naphthol (Sigma Chemical, St. Louis, MO, U.S.A.) as the chromogen. The anti-L-MAG protein and the anti-S-MAG protein antibodies were diluted 1:800 and 1:400, respectively, in 3% bovine serum albumin. The blots were then scanned with a spectrodensitometer (CS-910, Shimadzu, Japan) at 580 nm. The amount of each isoform was expressed as a percentage of its maximum during development in the CNS or in the PNS. Antibodies to each isoform were prepared as previously described 5. Briefly, the synthesized peptides unique to the L-MAG-derived 21 amino acid residues of the C-terminus and to the S-MAG-derived 11 amino acid residues deduced from the exon 12 portion including an additional lysine were used as antigens. Each processed peptide was emulsified with Freund's adjuvant and injected into rabbits several times at 14-day intervals. The
antibodies were specific in that they were quantitatively adsorbed by an excess of the peptide used to generate them. The maximum expression of the S-MAG protein following the L-MAG protein in the CNS (Figs. 1C and 2C) was similar to those of the L-MAG m R N A and the S-MAG m R N A previously reported 3. The expression of mRNAs are more transient than that of proteins, which may be a result of the accumulation of the M A G protein in the CNS. The peak of concentration of the L-MAG protein in the PNS was demonstrated at day 8, when that of the S-MAG protein in the PNS was still low (Figs. 1P and 2P). The concentration of the S-MAG protein in the PNS reached a peak at day 15. The maximum expression of the L-MAG protein and the S-MAG protein in the PNS was earlier than those in the CNS, as myelination in the PNS begins earlier. The L-MAG protein in the rat sciatic nerve was not detectable by in vitro translation 2. Tropak et al. 14 have reported by RNAase protection experiments that S-MAG m R N A is predominant throughout development whereas the L - M A G m R N A was barely detectable in the PNS. Western blotting using antibodies to the L-MAG protein and to the common part of both M A G isoform proteins suggested that only S-MAG was in the PNS 9. Recently, Owen et al. t° have demonstrated that the L-MAG protein was transiently expressed at the ear-
175 liest stage of myelination in vitro by immunostaining myelinating cultures of Schwann cells and dorsal root ganglion cells with an antibody specific to L - M A G protein. O u r present study, however, is the first r e p o r t to show the d e v e l o p m e n t a l changes of two M A G isoform proteins in the sciatic nerve using a specific a n t i b o d y to each isoform of M A G . The concentration of the L - M A G protein r e m a i n e d relatively high after the p e a k in the CNS c o m p a r e d with that in PNS, which is compatible with changes of the L - M A G m R N A in both nervous systems TM. This might be due to a difference in L - M A G protein turnover b e t w e e n CNS and PNS. O u r previous data have suggested the i m p o r t a n c e of the L - M A G protein in the early stage of myelinogenesis in the CNS 3-5. E d w a r d et a l ) r e p o r t e d that L - M A G protein is phos-
p h o r y l a t e d in mouse and in vitro in myelin, and that the p h o s p h o r y l a t e d residues include tyrosine. Protein tyrosine phosphorylation plays i m p o r t a n t roles in the regulation, growth and development. H e r e we have e m p h a sized that the L - M A G protein is expressed p r e c e d e n t to the S - M A G protein also in the PNS, and suggest that the L - M A G protein plays an essential role for myelin formation in the PNS as well as in the CNS.
1 Edwards, A.M., Braun, P.E. and Bell, J.C., Phosphorylation of myelin-associated glycoprotein in vivo and in vitro occurs only in the cytoplasmic domain of the large isoform, J. Neurochem., 52 (1989) 317-320. 2 Frail, D.E., Webster, H.F. and Braun, P.E., Developmental expression of the myelin-associated glycoprotein in peripheral nervous system is different from that in the central nervous system, J. Neurochem., 45 (1985) 1308-1310. 3 Fujita, N., Sato, S., Kurihara, T., Inuzuka, T., Takahashi, Y. and Miyatake, T., Developmentally regulated alternative splicing of brain myelin-associated glycoprotein mRNA is lacking in the quaking mouse, FEBS Lett., 232 (1988) 323-327. 4 Fujita, N., Ishiguro, H., Sato, S., Kurihara, T., Kuwano, R., Sakimura, K., Takahashi, Y. and Miyatake, T., Induction of myelin-associated glycoprotein mRNA in experimental remyelination, Brain Research, 513 (1990) 152-155. 5 Fujita, N., Sato, S., Ishiguro, H., Inuzuka, T., Baba, H., Kurihara, T., Takahashi, Y. and Miyatake, T., The large isoform of myelin-associated glycoprotein is scarcely expressed in the quaking mouse brain, J. Neurochem., 55 (1990) 1056-1059. 6 Laemmli, U.K. and Favre, M., Maturation of the head of bacteriophage T4. I. DNA packing events, J. Mol. Biol., 80 (1973) 575-600. 7 Lai, C., Brown, M.A., Nave, K.-A., Noronha, A.B., Quarles, R.H., Bloom, EE., Milner, R.J. and Sutcliffe, J.G., Two forms of 1B236/myelin-associated glycoprotein, a cell adhesion molecule for postnatal neural development, are produced by alter-
native splicing, Proc. Natl. Acad. Sci. U.S.A., 84 (1987) 43374341. Lowry, O.H., Rosebrough, N.J., Farr, A.L. and Randall, R.J., Protein measurement with the Folin phenol reagent, J. Biol. Chem., 193 (1951) 265-275. Noronha, A.B., Hammer, J.A., Lai, C., Kiel, M., Milner, R.J., Sutcliffe, J.G. and Quarles, R.H., Myelin-associated glycoprotein (MAG) and rat brain-specific 1B236 protein: mapping of epitopes and demonstration of immunological identity, J. Mol. Neurosci., 1 (1989) 159-170. Owens, G.C., Boyd, C.J., Bunge, R.P. and Salzer, J.L., Expression of recombinant myelin-associated glycoprotein in primary Schwann cells promotes the initial investment of axons by myelinating Schwann cells, J. Cell. Biol., 111 (1990) 1171-1182. Quades, R.H., Everly, J.L. and Brady, R.O., Myelin-associated glycoprotein: a developmental change, Brain Research, 58 (1973) 506-509. Quarles, R.H., Myelin-associated glycoprotein in development and disease, Dev. Neurosci., 6 (1984) 285-303. Towbin, H., Staehelin, T. and Gordon, J., Eleetrophoretic transfer of proteins from polyacrylamide gel to nitrocellulose sheets: procedure and applications, Proc. Natl. Acad. Sci. U.S.A., 76 (1979) 4350-4354. Tropak, M.B., Johnson, P.W., Dunn, R.J. and Roder, J.C., Differential splicing of MAG transcripts during CNS and PNS development, Mol. Brain Res., 4 (1988) 143-155.
The authors thank Ms. S. Nagasawa, Ms. Y. Yagi and Ms. I. Morohashi for their excellent technical assistance. This work was supported by grants from the Ministry of Health and Welfare, from the Ministry of Education, Science and Culture of Japan, from Japan foundation for health science, from Uehara memorial foundation and from the Taishou Pharmaceutical Co.
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