Neuroscience Letters, 138 (1992) 49-52 © 1992 Elsevier Scientific Publishers Ireland Ltd. All rights reserved 0304-3940/92/$ 05.00

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NSL 08522

Origin of brain 2',3'-cyclic-nucleotide 3'-phosphodiesterase doublet Tadashi Kurihara, Yoko Tohyama, Junko Yamamoto, Tomoyuki Kanamatsu, Shigemi Kitajima

Rihito Watanabe and

Institute of Life Science, Soka University, Tokyo (Japan)

(Received 16 October 1991; Revised version received 9 January 1992; Accepted 13 January 1992) Key words: 2',3'-Cyclic-nucleotide 3'-phosphodiesterase; Isoform; Alternative splicing; Oligodendrocyte; Myelin

The present study established that 2',3'-cyclic-nucleotide 3'-phosphodiesterase doublet common to mammalian brain originates from an alternative splicing. Peptides specific to the predicted larger translation product were synthesized and antisera against these peptides were prepared. Immunostaining of SDS/PAGE blots showed that the antisera react with the larger protein, but not with the smaller protein, of 2',3'-cyclic-nucleotide 3'-phosphodiesterase doublet in all mammals studied.

Extremely high 2',3'-cyclic-nucleotide 3'-phosphodiesterase (CNP; EC 3.1.4.37) activity is f o u n d in brain and spinal cord, where the enzyme protein and its m R N A are expressed exclusively by oligodendrocytes [4, 11, 14]. C N P m R N A in brain increases with age, reaches the m a x i m u m at the time o f active myelination and then decreases gradually [6]. A study with cultured oligodendrocytes suggests that C N P is involved in m e m b r a n e extension prior to myelin formation [13]. C N P in m a m malian brain consists o f two protein c o m p o n e n t s (doublet) with slightly different mobilities in SDS/ P A G E ; in mouse brain, however, C N P consists o f m o r e than two protein c o m p o n e n t s [3]. Two forms o f C N P m R N A alternatively spliced within exon 1 were shown recently in mouse brain [3]. The alternative splicing is predicted to generate two C N P proteins with and without 20 amino acid residues at the a m i n o terminal, explaining the possible origin o f C N P doublet. In the present study, peptides specific to the predicted larger translation p r o d u c t were synthesized and antisera against these peptides were prepared. I m m u n o s t a i n i n g o f SDS/ P A G E blots for mouse, rat, h u m a n , and bovine myelin preparations indicates that C N P doublet c o m m o n to m a m m a l i a n brain originates f r o m an alternative splicing d e m o n s t r a t e d in mouse. Peptides were synthesized with an Excell Peptide Synthesizer (MilliGen/Biosearch) and purified by H P L C . Antisera against these peptides were prepared as follows. Correspondence: T. Kurihara, Institute of Life Science, Soka University, Tangi-cho, Hachioji, Tokyo 192, Japan.

A synthetic peptide (12 mg) and bovine serum albumin (9 mg) were dissolved in 2 ml o f 0.1 M N a H C O 3 ( p H 8.5); 15 g l o f 1% (v/v) glutaraldehyde were then added. The mixture was incubated for 15 min at 22°C and dialysed overnight at 4°C against 0.1 M phosphate-buffered

Mouse

CNP

(M) S S S G A K E K . . . (M)NTSFTRKSHTFLPKLFFRKMSSSGAKEK... Peptide A Rat

CNP

(M) S S S G A K D K . . . (M) S T S F A R K S H T F L P K I F F R K M S S S G A K D K . . . Human

CNP

(M) S S S G A K D K . . . (M) N R G F S R K S H T F L P K I F F R K M S S S G A K D K . . . Peptide B Bovine

CNP (M) S S S G A K D K . . . (M) S R G F S R K S Q T F L P K V F F R K M S S S G A K D K . . . Fig. 1. Amino-terminal sequences of predicted CNP isoforms. Translation of alternatively spliced mouse CNP mRNA is predicted to result in two CNP isoforms with different amino-terminal sequences [3]. Rat, human and bovine sequences were deduced respectively from rat cDNA [1], human gene [10] and bovine cDNA [2, 15], on the assumption that rat, human and bovine CNP isoforms originate from the same alternative splicing as that demonstrated in mouse. Translation initiation methionine shown in parenthesis is probably removed after translation. Peptides of underlined sequences (peptides A and B) were synthesized, coupled to bovine serum albumin and used as immunogens.

50

(a)

(b)

"~" O

v" "o

Human

Rat

Mouse

Bovine

--6

~'. -o

~-. O

~-O

v" -o

-" 6

~ -o

v O

Z "0

¢~ ~

Z ~

¢' ~

Z "0

Z "0

~ ~

Z -0

¢~ ~

Z "o

2 3

> ~ (D 2 3

2 3

> (~ (D 2 3

2 3

2 3

oo (~ • 2 3

2 3

oo ~ (D 2 3

2 3

(c)

Fig. 2. Parallel staining of mouse, rat, human, and bovine CNPs with anti-peptide A or B serum and anti-CNP serum. (a) Mouse and rat CNPs; (b) human and bovine CNPs; (c) mouse C N E magnified. An SDS/PAGE blot of mouse and rat myelin preparations was stained with anti-peptide A serum (diluted 1:200) and with anti-bovine CNP serum (diluted 1: 1000). Peptide A is specific to the predicted larger isoform of mouse CNP (Fig. 1). An SDS/PAGE blot of human and bovine myelin preparations was stained with anti-peptide B serum (diluted 1:200) and with anti-bovine CNP serum (diluted l:1000). Peptide B is specific to the predicted larger isoform of human CNP (Fig. 1). See the text for anti-peptide A and B sera, and reference 5 for anti-bovine CNP serum.

saline. The volume of the solution was adjusted to 6 ml with 0.1 M phosphate-buffered saline. The conjugate was then emulsified with Freund's complete adjuvant and injected into two rabbits three times at 14-day intervals. Anti-bovine CNP serum [5] was also used. Myelin was prepared from adult brain by the procedure of Norton [12]. Strain ddY mouse and albino rat were used. A sample of myelin (50/21) containing 250300/2g protein was mixed with 50/21 of the solution containing 0.15 M Tris-HCl (pH 6.8), 4% (w/v) SDS and 20% (v/v) glycerol; 2/21 each of 0.5 M dithiothreitol and 0.1% Bromophenol blue were then added. The mixture was heated for 5 min in boiling water and applied to the top of the polyacrylamide slab gel (5.5-cm wide). The electorophoresis conditions were those of Laemmli [8];

10% (w/v) separation gel was used. Transfer to nitrocellulose filters was perfomed electrophoretically with a simplified blotting apparatus (Nihon Eido, Tokyo, Japan) in a buffer containing 25 mM Tris, 150 m M glycine and 20% (v/v) methanol. The nitrocellulose filters were stained by a biotin-avidin system (Vector Labs, Burlingame, CA, USA) after blocking in 1% (w/v) skim milk. Colour was developed with 4-chloro-l-naphthol. Two CNP protein isoforms with and without 20 amino acid residues at the amino terminal are predicted from an alternative splicing shown recently in mouse [3]. Fig. 1 shows the amino-terminal sequences of the predicted isoforms for mouse, rat, human, and bovine CNPs. Peptides specific to the predicted larger isoforms of mouse and human (peptides A and B, respectively, underlined

51

(a)

(b)

Fig. 3. Parallel staining of rat cerebral sections with anti-peptide A serum (a) and with anti-bovine CNP serum (b). Coronal sections of paraffinembedded rat cerebrumwere used. Anti-peptideA serum was diluted 1:50and anti-bovineCNP serum was diluted 1:250. Bar=100 am. See the text for anti-peptideA serum and reference5 for anti-bovineCNP serum. Anti-peptideA serum was passed through a column of bovine serum albuminagarose before use.

in Fig. 1) were synthesized, coupled to bovine serum albumin and injected to rabbits to raise their antibodies. An SDS/PAGE blot of mouse and rat myelin preparations was stained with anti-peptide A serum as well as with anti-CNP serum (Fig. 2a). In rat, the larger protein component (CNP II) reacted, but the smaller protein component (CNP I) did not react, with anti-peptide A serum. Mouse CNP was found by the parallel staining with the two antisera to consist o f four protein components, designated CNP II, CNP II', CNP I, and CNP I', in the order of decreasing size (Fig. 2c). The intermediate bands (CNP II' and CNP I) were very close and could not distinguish from each other previously [3]. In mouse, the larger two components (CNP II and CNP II') reacted, but the smaller two components (CNP I and CNP I') did not react, with anti-peptide A serum. The structural differences between CNP II and CNP II' and between CNP I and CNP I' are probably the same, but remain unknown. An SDS/PAGE blot of human and bovine myelin preparations was stained similarly with anti-peptide B serum as well as with anti-CNP serum (Fig. 2b). Only the larger protein component (CNP II) of CNP doublet reacted with anti-peptide B serum. Immunostaining of SDS/PAGE blots thus showed that in all mammals studied peptide A or B portion is present in the larger protein component, CNP II (and CNP II' in mouse), but not in the smaller protein component, CNP I (and CNP I' in mouse). The results indicate that CNP doublet common to mammalian brain originates from an alternative splicing demonstrated in mouse brain.

Serial sections of rat cerebrum were incubated with anti-peptide A serum or with anti-bovine CNP serum, followed by anti-rabbit Ig and rabbit PAP [11]. With both sera, Ig deposition was found associated with myelin (Fig. 3). This is further evidence for the presence of the predicted larger CNP isoform in rat brain. As far as we examined, there was no difference in staining between the two sera, indicating that the distributions of the two CNP isoforms are identical or similar. R N A blot hybridization shows two m R N A bands for rat [1] and mouse [6] CNPs, but an apparently single m R N A band for bovine [2, 15] and human [7] CNPs. The two m R N A forms of mouse shown on the R N A blot are produced by an alternative splicing [3]. The present study indicates that the CNP doublet of bovine and human originates from the same alternative splicing as that demonstrated in mouse. We suppose that the two m R N A forms of bovine and human may overlap on the R N A blot due to the larger size of m R N A and/or the smaller difference in size between the two m R N A forms. Studies in progress indicate that two human genomic probes specific respectively to the two predicted m R N A forms can both hybridize on the human R N A blot (Monoh and Kurihara, unpublished work). We thank Dr. Shuzo Sato, Department of Neurology, Brain Research Institute, Niigata University, for supplying a human myelin preparation. This study was supported by grants from the Ministry of Education, Science and Culture of Japan (02220207;03833030) and from the Yamada Science Foundation.

52 1 Bernier, L., Alvarez, F., Norgard, E.M., Raible, D.W.. Mentaberry, A., Shembri, J.G., Sabatini, D.D. and Colman, D.R., Molecular cloning of a 2',3'-cyclic nucleotide 3'-phosphodiesterase: mRNAs with different 5' ends encode the same set of proteins in nervous and lymphoid tissues, J. Neurosci., 7 (1987) 2703-2710. 2 Kurihara, T., Fowler, A.V. and Takahashi, Y., cDNA cloning and amino acid sequence of bovine brain 2',Y-cyelic-nucleotide 3'- phosphodiesterase, J. Biol. Chem., 262 (1987) 3256 3261: 16754. 3 Kurihara, T., Monoh, K., Sakimura, K. and Takahashi, Y., Alternative splicing of mouse brain 2',3'-cyclic-nucleotide 3'- phosphodiesterase mRNA, Biochem. Biophys. Res. Commun., 170 (1990) 1074-1081. 4 Kurihara, T., Monoh, K., Takahashi, Y., Goto, K. and Kondo, H., 2',3'-Cyclic-nucleotide 3'-phosphodiesterase. Complementary DNA and gene cloning for mouse enzyme and in situ hybridization of the messenger RNA in mouse brain, Adv. Second Messengers Phosphoprotein Res., 25 (1992) 101 110. 5 Kurihara, T., Nishizawa, Y, Takahashi, Y. and Odani, S., Chemical immunological and catalytic properties of 2',3'-cyelic-nucleotide 3'phosphodiesterase purified from brain white matter, Biochem. J., 195 (1981) 153 157. 6 Kurihara, T., Takahashi, Y., Fujita, N., Sato, S. and Miyatake, T., Developmental expression of 2',Y-cyclic-nucleotide 3"-phosphodiesterase mRNA in brains of normal and quaking mice, Mol. Brain Res., 5 (1989) 247 -250. 7 Kurihara, T., Takabashi, Y., Nishiyama, A. and Kumanishi. T., cDNA cloning and amino acid sequence of human brain 2',3'cyclic-nucleotide 3'-phosphodiesterase, Biochem. Biophys. Res. Commun., 152 (1988) 837 842.

8 Laemmli, U.K., Cleawtgc of structural proteins duriug the assembly of the head of bacteriophage T4, Nature, 227 (1970) 680 -685. 9 Monoh, K., Kurihara, T., Sakimura, K. and Takahashi, Y., Structure of mouse 2',3'-cyclic-nucleotide 3'-phosphodiesterase gene, Biochem. Biophys. Res. Commun., 165 (1989) 1213 1220. 10 Monoh, K., Kurihara, T. and Takahashi, Y., Structure of human 2',3'-cyclic-nucleotide Y-phosphodiesterase gene, Trans. Am. Soc. Neurochem., 21 (1990) 228. 11 Nishizawa, Y., Kurihara, T., Masuda, T. and Takahashi, Y., lmmunohistochemical localization of 2',3'-cyclic-nucleotide 3'- phosphodiesterase in adult bovine cerebrum and cerebellum, Neurochem. Res., 10 (1985) 1107 1118. 12 Norton, W.T., Isolation of myelin from nerve tissue, Methods Enzymol., 31 (1974) 435-444. 13 Reynolds, R., Carey, E.M. and Herschkowiz, N., Immunohistochemical localization of myelin basic protein and 2',3'-cyclic nucleotide Y-phosphohydrolase in flattened membrane expansions produced by cultured oligodendrocytes, Neuroscience, 28 (1989) 181 188. 14 Trapp, B.D., Bernier, k.. Andrews, S.B. and Colman, D.R., Cellular and subcellular distribution of 2',3'-cyclic-nucleotide 3'- phosphodiesterase and its mRNA in the rat central nervous system, J. Neurochem., 51 (1988) 859 868. 15 Vogel, LI.S. and Thompson. R.J., Molecular cloning of lhc myelinspecific enzyme 2",3'-cyclic-nucleotide 3'-phosphodiesterasc, FEBS Left.,218(1987)261 265.

Origin of brain 2',3'-cyclic-nucleotide 3'-phosphodiesterase doublet.

The present study established that 2',3'-cyclic-nucleotide 3'-phosphodiesterase doublet common to mammalian brain originates from an alternative splic...
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