Vol. 180, No. 2, 1991 October 31, 1991
BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS Pages 5] 5-518
Isolation and sequence determination of cDNA encoding the major structural protein of human peripheral myelin Kiyoshi Hayasaka 1, Kenji Nanao 1, Marie Tahara 1, Wataru Sato ~, Goro Takada I , Masayuki Miura 2 and Keiichi Uyemura2 1 Department of Pediatrics, Akita University School of Medicine, Akita, Japan 2Department of Physiology, Keio University School of Medicine, Tokyo, Japan Received September ii, 1991
Summary: A full length cDNA of the major structural protein of peripheral myelin ( P0 protein ) has been isolated from a cDNA library of human fetus spinal cord. The clone is 1948 base pairs ( bp ) in length and contains a 744 bp open reading frame encoding a polypeptide of 248 residues including 29 signal peptide. The deduced amino acid sequence is highly homologous to P0 protein from other species. ~ 1991 A~ademic ~..... ~nc.
Myelin is a multilamellar compacted membrane structure that surrounds and insulates the axon, facilitating the conduction of nerve impulses. Schwann cells are responsible for myelin formation in the peripheral nerve. P0, a small integral membrane glycoprotein, is the most abundant protein component of mammalian peripheral myelin. The primary structure of P0 protein has been deduced from cloned cDNAs for rat(l), shark(2), and chicken(3) as well as directly determined by sequencing of the bovine protein(4). Analysis of this structure has suggested a transmembrane structure and a function in which P0 protein plays essential roles in both the elaboration and the subsequent compaction of the myelin sheath.
The
primary structure of human P0 protein has not been reported; this information is necessary for the study of genetic disorders. In the present study we report the isolation and sequence analysis of a cDNA encoding the entire precursor form of the P0 protein from a human fetus spinal cord library. Materials and Methods Materials Restriction nucleases and DNA-modifying enzymes were purchased from Takara Shuzo (Kyoto,Japan). Radiolabeled nucleotides were obtained from New England Nuclear 0006-291X/91 $1.50 515
Copyright © 1991 by Academic Press, Inc. All rights of reproduction in any form reserved.
Vol. 180, No. 2, 1991
BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS
(Boston,MA). Oligonucleotide primers were synthesized on an Applied Biosystems 381A DNA synthesizer. Screening of cDNA Library A lambda gtll library from human fetus spinal cord(5) was screened with a radiolabelled cDNA of rat P0 protein(l). Filters were hybridized with 32p_ labelled probe overnight at 65°C in 6X SSC containing 5X Denhardt's solution, 10 % dextran sulfate, 10 mM EDTA, 0.5 % SDS, and 100 ~tg/ml salmon sperm DNA. They were then washed at room temperature in 2X SSC containing 0.1% SDS, followed by washing for 30 min at 65°C in 2X SSC containing 0 . 1 % SDS, and exposed to X-ray film. Twenty two positive clones were isolated from 1 x 106 plaques. Three of them were more than 1500 bp long. DNA Sequencing DNA inserts liberated by complete or partial digestion with EcoRI were isolated, purified and subcloned in pUC 19 using standard procedures. Nucleotide sequences were determined by the dideoxy method(6). The nucleotide sequences, the deduced primary structures and homologies were analyzed using a computer program, DNASIS(Hitachi Software Engineering Co., Ltd.).
Results and Discussion o f the three clones that were isolated, two were 2000 bp long and the other was 1800 bp. One of the 2000 bp clones, N7t, was fully sequenced according to the strategy outlined in Fig.l; the results are presented in Fig. 2. The other two clones were partially sequenced and these sequences were found to be present within the fully sequenced clone. Clone N7t is 1948 bp in length and has a 744 bp open reading frame flanked by a 5', 44 bp and a 3', 1160 bp, noncoding region. The nueleotide sequence of human P0 protein is 90 % identical to that of rat P0 protein(l). The amino acid sequence of mature peptide shows 94 % homology to both rat(l) and bovine P0 protein(4)(Fig.3). As reported for other P0 proteins, the sequence of mature protein includes a highly
hydrophobic, probably transmembrane segment, a
relatively hydrophobic excellular domain in the amino terminal region, and a basic domain in carboxyl-terminal region which may protrude from the cytoplasmic side of the membrane.
1
I
0
i
500
1
I0'00
111
15'00
Fig.l. Restriction endonuclease map of N7t clone and strategy for nucleotide sequence analysis of human P0 protein cDNA. The open box represents the coding region and the solid lines represent the untranslated regions. The horizontal arrows indicate the direction and the extent of sequence analyses. The scale at the bottom is nucleotide base pairs. 516
Vol. 180, No. 2, 1991
BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS
I 1
GC TCA ACC CCA CAG ATG CTC CGG GCC CCT GCC CCT GCC CCA GCT ATG Met
47 1
48 2
GCT CCT GGG GCT CCC TCA TCC AGC CCC AGC CCT ATC CTG GCT GTG CTG Ala Pro GIy Ala Pro Set Ser Ser Pro Ser Pro Ile Leu hla Val Leu
95 17
96 18
CTC TTC TCT TCT TTG GTG CTG TCC CCG GCC CAG GCC ATC GTG GTT TAC Leu Phe S e t S e t L e u V a l Leu S e r P r o A1a G l n A l a I l e V a l V a l T y r
143 33
144 34
ACC GAC AGG GAG GTC CAT GGT GCT GTG GGC TCC CGG GTG ACC CTG CAC Thr Asp Arg Glu Val Ills Gly Ala Val Gly Ser Arg Val Thr Leu Ills
191 49
192 50
TGC TCC TTC TGG TCC AGT GAG TGG GTC TCA GAT GAC ATC TCC TTC ACC Cys S e t Phe T r p S e t S e t G l u T r p V a l S e r Asp Asp l l e S e r Phe T h r
239 65
240 66
TGG CGC TAC CAG CCC GAA GGA GGC AGA GAT GCC ATT TCG ATC TTC CAC T r p A r g T y r G l n P r o Glu G l y G l y A r g Asp A l a I l e S e r l i e Phe H i s
287 81
288 82
TAT GCC AAG GGA CAA CCC TAC ATT GAC GAG GTG GGG ACC TTC AAA GAG T y r A l a Lys G l y Gln P r o T y r I l e Asp G l u Val G l y T h r Phe L y s Glu
335 97
336 98
CGC ATC CAG TGG GTA GGG GAC CCT CGC TGG AAG GAT GGC TCC ATT GTC A r g l l e Gln T r p Val G l y Asp P r o A r g T r p Lys Asp G l y S e r l l e V a l
383 113
384 ll4
ATA CAC AAC CTA GAC TAC AGT GAC AAT GGC ACG TTC ACT TGT GAC GTC l l e I l l s Ash Leu Asp T y r S e t Asp Asn G l y T h r Phe T h r Cys Asp V a l
431 129
432 130
AAA AAC CCT CCA GAC ATA GTG GGC AAG ACC TCT CAG GTC ACG CTG TAT L y s A s n P r o P r o A s p l l e V a l G l y L y s T h r S e r G l n V a l T h r L e u TyF
479 145
480 146
GTC TTT GAA AAA GTG CCA ACT A G G T A C G G G G T C G T T CTG G G A G C T G T G Val Phe Glu Lys Val Pro Thr Arg Tyr GIy Val Val Leu Gly Ala Val
527 161
528 162
ATC GGG GGT GTC CTC GGG GTG GTG C T G T T G C T G CTG CTG C T T T T C T A C l l e G l y G l y V a l L e u G l y V a l Val Leu Leu Leu Leu Leu L e u Phe T y r
575 177
576 178
GTG GTT CGG TAC TGC TGG CTA CGC A G G CAG GCG GCC C T G C A G A G G A G G V a l V a l A r g T y r Cys T r p Leu A r g A r g Gln A l a A l a Leu Gln A r g A r g
623 193
624 194
CTC AGT GCT ATG GAG AAG GGG A A A T T G CAC A A G C C A GGA A A G G A C G C G L e u S e r A l a M e t G l u L y s G l y Lys Leu Hls Lys Pro Gly Lys Asp A l a
671 209
672 210
TCG AAG CGC GGG CGG CAG ACG CCA GTG CTG TAT GCA ATG CTG GAC CAC S e t L y s A r g G l y A r g G l n T h r P r o V a l L e u T y r A l a Met L e u A s p I l l s
719 225
720 226
AGC AGA AGC ACC AAA GCT GTC AGT GAG AAG AAG GCC AAG GGG CTG GGG Ser Arg Ser Thr Lys Ala Val Set Glu Lys Lys Ala Lys GIy Leu Gly
767 241
768 242
GAG TCT CGC AAG GAT AAG AAA TAG CGG TTA GCG GGC CGG GCG GGG GAT G l u S e r A r g L y s A s p L y s L y s • **
815 249
816 864 912 960 1008 1056 1104 1152 1200 1248 1296 1344 1392 1440 1488 1536 1584 1632 1680 1728 1.776 1824 1872 1920
CGG GAG GTC ATG TGC TCC GTA ACC ACC GAA ATT CCC CCC TAT CCT TCA CCA GGT AGG GGG AAA GGC ATA GAC
GGG ATG AAG GGC CAG CTC AGT TTC TGC GAA CCC GTC CCA TTA GTG TGT GCC TCC AGA AGG AGC TGC TTT ATT
TTA GAG TCC CTG AGC TTT TTC ACC CCC AGG ATC CCT GCT GGT CTC GGC CAA CCC GAG GGG AGG ACT CTT TCC
GGG CTA CCC AAC AGG GCT ATT CCT GGG GAC TCC CCC AAT TAT ATC TTT GTA TGA ACA CAA AAA GCA CTC AGA
GTG CGA AGC TCG GGG CCC CCA GGC GGC TTT TGC TCC ACC TAT CTC TGC GAG CCA GAG ATG AAC GCC AAA AAA
GAG AAG AGA GAC ACC CCG GAA TTT TGT GAT ATC CCT CAG TTT TGC AAT GGG CTT AGA AGC AAA ACA CCT AAA
TCC GAT ACC AAG TAG CCC TTC CTG GTG ACC CCC CTA AGC TTA CCT TCA CCC TGG GAG CTT TGG GGG CCA AAA
GCC GAG AGC TGA GCT TGC ATT GGA TTT CTC CAG CCA ACC TTT TAC CGC CTG AGC AAT AAG GAA GAT AAT AAA
AAA CAG CTC TCG CCT CCT CCC GCC GGT TGC CCT GGT CAG TTT ACC CCC ATC ACT GGA AAA ATG TCT GTC AAA
GGC AGC AAA CCA CTT GCC CAG CAG GCC CTC GTC GGC ATC AAT CAT CCT CTC GTT TCC TGG GGG TAG CTT AA
CCA CCG AAC CCC ACC CTC GCA GAG TGT AAG CCC CCA AGA CCA GAC TAA ACA CTG TCA TTT GGG GAT AAA
ILAG GTG ATG GTC GAG CTC CGG CCT GCC CTC AAG AAC CCC CAC CCC AGG CCC GTC TAG GTG ACC TCC CTT TGA ATT GTA TTC TCC CTA ATC CTA CCC CCA CCT GAG CAC TCC AGG CCA CCT CTG GCT GTT TCC GCT CCA TAC TCT CTC TCC TTC AGG TTC TTT GTT TGT T G A G G A CCA A T G GTC C~F ItAT GAA CTT CAG TAT AGC GGA CTC CAG GTC TAG GTC AGG GAG TTA AAC AAC CAA CGG GGG G G A G G A TTT TCT ACA TTC TGT Tq~-A~--A~C
ATC GCT ATG CCC CTT GAC CCC CTC TGA GGC TCT GTC GTT TTA ACG GAG ATT TTG TGG ACA AGA TGT ACT
863 911 959 1007 1055 1103 1151 1199 1247 1295 1343 1391 1439 1487 1535 1583 1631 1679 1727 1775 1823 1871 1919 1948
Fig.2. Nucleotide and deduced amino acid sequences for human P0 protein. The residues confirmed by the amino acid sequencing of purified protein(unpublished data) are underlined. The polyadenylation signals are boxed.
517
Vol. 180, No. 2, 1991
BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS
Signal
sequence
protein
Mature 1
HUMAN
31
MAPGAPSSSPSPILAVLLFSSLVLSPAQAIVVYTDREVHGAVGSRVTLHCSFWSSEWVSD
BOVINE
...... A ..........
TL
K ........
RAT
...............
..........
Y .....
HUMAN
DISFTWRYQPEGGRDAISIFHYAKGQPYIDEVGTFKERIQWVGDP-RWKDGSIVIHNLDYS
Q...Y
...........
Q ...............
91
B O V I N E .L . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
H.-
RAT
.............................................
-S ..............
HUMAN
DNGTFTCDVKNPPDIVGKTSQVTLYVFEKVPTRYGVVLOAVIGGVLGVVLLLLLLFYVVR
BOVINE
...................................................
RAT
............................................
HUMAN
YCWLRRQAALQRRLSAMEKGKLHKPGKDASKRGRQTPVLYAMLDHSRSTKAVSEKKAKGL
BOVINE
..............
RAT
.................
HUMAN
GESRKDKK
BOVINE
........
RAT
........
H .........
TA
Q...F..SS..S
............
151 A ..... I ............
......................... ......................
G
LI. LI.
211 A ....
T...
A ....
S...
220
Fig.3. Comparison of the deduced amino acid sequences of the P0 proteins from human, bovine(Ref.4) and rat(Ref.1).
These different domains may be required together with basic proteins like P2 protein, to make a compact myelin sheath
in the peripheral nerve.
Our data support the hypothesis of
considerable conservation of the sequence of P0 protein during evolution. Acknowledgments We thank Dr. A. T. Campagnoni for a lambda gtll library from human fetus spinal cord, Dr. G. Lemke for a cDNA of rat P0 protein and Dr. T. Nakagomi for synthesizing the oligonucleotides. References 1. 2. 3. 4. 5. 6.
Lemke, G. and Axel, R. (1985) Cell 40, 501-508. Saavedra, R. R., Fors, L., Aebersold, R.H., Arden, B., Horvath, S., Sanders, J. and Hood, L. (1989) J. Mol. Evol. 29, 149-156. Barbu, M. (1990) J. Neurosci. Res. 25, 143-151. Sakamoto, Y., Kitamura, K., Yoshimura, K., Nishijima, T. and Uyemura K. (1987) J. Biol. Chem. 262, 4208-4214. Roth, H. J., Kronquist, K., Pretorius, P. J., Crandall, B. F. and Carnpagnoni, A. T. (1986) J. Neurosci. Res. 16, 227-238. Sanger, F., Nicklen, S. and Coulson, A.R. (1977) Proc. Natl. Acad. Sci. USA 74, 54635467.
518
BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS Pages 5] 5-518
Isolation and sequence determination of cDNA encoding the major structural protein of human peripheral myelin Kiyoshi Hayasaka 1, Kenji Nanao 1, Marie Tahara 1, Wataru Sato ~, Goro Takada I , Masayuki Miura 2 and Keiichi Uyemura2 1 Department of Pediatrics, Akita University School of Medicine, Akita, Japan 2Department of Physiology, Keio University School of Medicine, Tokyo, Japan Received September ii, 1991
Summary: A full length cDNA of the major structural protein of peripheral myelin ( P0 protein ) has been isolated from a cDNA library of human fetus spinal cord. The clone is 1948 base pairs ( bp ) in length and contains a 744 bp open reading frame encoding a polypeptide of 248 residues including 29 signal peptide. The deduced amino acid sequence is highly homologous to P0 protein from other species. ~ 1991 A~ademic ~..... ~nc.
Myelin is a multilamellar compacted membrane structure that surrounds and insulates the axon, facilitating the conduction of nerve impulses. Schwann cells are responsible for myelin formation in the peripheral nerve. P0, a small integral membrane glycoprotein, is the most abundant protein component of mammalian peripheral myelin. The primary structure of P0 protein has been deduced from cloned cDNAs for rat(l), shark(2), and chicken(3) as well as directly determined by sequencing of the bovine protein(4). Analysis of this structure has suggested a transmembrane structure and a function in which P0 protein plays essential roles in both the elaboration and the subsequent compaction of the myelin sheath.
The
primary structure of human P0 protein has not been reported; this information is necessary for the study of genetic disorders. In the present study we report the isolation and sequence analysis of a cDNA encoding the entire precursor form of the P0 protein from a human fetus spinal cord library. Materials and Methods Materials Restriction nucleases and DNA-modifying enzymes were purchased from Takara Shuzo (Kyoto,Japan). Radiolabeled nucleotides were obtained from New England Nuclear 0006-291X/91 $1.50 515
Copyright © 1991 by Academic Press, Inc. All rights of reproduction in any form reserved.
Vol. 180, No. 2, 1991
BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS
(Boston,MA). Oligonucleotide primers were synthesized on an Applied Biosystems 381A DNA synthesizer. Screening of cDNA Library A lambda gtll library from human fetus spinal cord(5) was screened with a radiolabelled cDNA of rat P0 protein(l). Filters were hybridized with 32p_ labelled probe overnight at 65°C in 6X SSC containing 5X Denhardt's solution, 10 % dextran sulfate, 10 mM EDTA, 0.5 % SDS, and 100 ~tg/ml salmon sperm DNA. They were then washed at room temperature in 2X SSC containing 0.1% SDS, followed by washing for 30 min at 65°C in 2X SSC containing 0 . 1 % SDS, and exposed to X-ray film. Twenty two positive clones were isolated from 1 x 106 plaques. Three of them were more than 1500 bp long. DNA Sequencing DNA inserts liberated by complete or partial digestion with EcoRI were isolated, purified and subcloned in pUC 19 using standard procedures. Nucleotide sequences were determined by the dideoxy method(6). The nucleotide sequences, the deduced primary structures and homologies were analyzed using a computer program, DNASIS(Hitachi Software Engineering Co., Ltd.).
Results and Discussion o f the three clones that were isolated, two were 2000 bp long and the other was 1800 bp. One of the 2000 bp clones, N7t, was fully sequenced according to the strategy outlined in Fig.l; the results are presented in Fig. 2. The other two clones were partially sequenced and these sequences were found to be present within the fully sequenced clone. Clone N7t is 1948 bp in length and has a 744 bp open reading frame flanked by a 5', 44 bp and a 3', 1160 bp, noncoding region. The nueleotide sequence of human P0 protein is 90 % identical to that of rat P0 protein(l). The amino acid sequence of mature peptide shows 94 % homology to both rat(l) and bovine P0 protein(4)(Fig.3). As reported for other P0 proteins, the sequence of mature protein includes a highly
hydrophobic, probably transmembrane segment, a
relatively hydrophobic excellular domain in the amino terminal region, and a basic domain in carboxyl-terminal region which may protrude from the cytoplasmic side of the membrane.
1
I
0
i
500
1
I0'00
111
15'00
Fig.l. Restriction endonuclease map of N7t clone and strategy for nucleotide sequence analysis of human P0 protein cDNA. The open box represents the coding region and the solid lines represent the untranslated regions. The horizontal arrows indicate the direction and the extent of sequence analyses. The scale at the bottom is nucleotide base pairs. 516
Vol. 180, No. 2, 1991
BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS
I 1
GC TCA ACC CCA CAG ATG CTC CGG GCC CCT GCC CCT GCC CCA GCT ATG Met
47 1
48 2
GCT CCT GGG GCT CCC TCA TCC AGC CCC AGC CCT ATC CTG GCT GTG CTG Ala Pro GIy Ala Pro Set Ser Ser Pro Ser Pro Ile Leu hla Val Leu
95 17
96 18
CTC TTC TCT TCT TTG GTG CTG TCC CCG GCC CAG GCC ATC GTG GTT TAC Leu Phe S e t S e t L e u V a l Leu S e r P r o A1a G l n A l a I l e V a l V a l T y r
143 33
144 34
ACC GAC AGG GAG GTC CAT GGT GCT GTG GGC TCC CGG GTG ACC CTG CAC Thr Asp Arg Glu Val Ills Gly Ala Val Gly Ser Arg Val Thr Leu Ills
191 49
192 50
TGC TCC TTC TGG TCC AGT GAG TGG GTC TCA GAT GAC ATC TCC TTC ACC Cys S e t Phe T r p S e t S e t G l u T r p V a l S e r Asp Asp l l e S e r Phe T h r
239 65
240 66
TGG CGC TAC CAG CCC GAA GGA GGC AGA GAT GCC ATT TCG ATC TTC CAC T r p A r g T y r G l n P r o Glu G l y G l y A r g Asp A l a I l e S e r l i e Phe H i s
287 81
288 82
TAT GCC AAG GGA CAA CCC TAC ATT GAC GAG GTG GGG ACC TTC AAA GAG T y r A l a Lys G l y Gln P r o T y r I l e Asp G l u Val G l y T h r Phe L y s Glu
335 97
336 98
CGC ATC CAG TGG GTA GGG GAC CCT CGC TGG AAG GAT GGC TCC ATT GTC A r g l l e Gln T r p Val G l y Asp P r o A r g T r p Lys Asp G l y S e r l l e V a l
383 113
384 ll4
ATA CAC AAC CTA GAC TAC AGT GAC AAT GGC ACG TTC ACT TGT GAC GTC l l e I l l s Ash Leu Asp T y r S e t Asp Asn G l y T h r Phe T h r Cys Asp V a l
431 129
432 130
AAA AAC CCT CCA GAC ATA GTG GGC AAG ACC TCT CAG GTC ACG CTG TAT L y s A s n P r o P r o A s p l l e V a l G l y L y s T h r S e r G l n V a l T h r L e u TyF
479 145
480 146
GTC TTT GAA AAA GTG CCA ACT A G G T A C G G G G T C G T T CTG G G A G C T G T G Val Phe Glu Lys Val Pro Thr Arg Tyr GIy Val Val Leu Gly Ala Val
527 161
528 162
ATC GGG GGT GTC CTC GGG GTG GTG C T G T T G C T G CTG CTG C T T T T C T A C l l e G l y G l y V a l L e u G l y V a l Val Leu Leu Leu Leu Leu L e u Phe T y r
575 177
576 178
GTG GTT CGG TAC TGC TGG CTA CGC A G G CAG GCG GCC C T G C A G A G G A G G V a l V a l A r g T y r Cys T r p Leu A r g A r g Gln A l a A l a Leu Gln A r g A r g
623 193
624 194
CTC AGT GCT ATG GAG AAG GGG A A A T T G CAC A A G C C A GGA A A G G A C G C G L e u S e r A l a M e t G l u L y s G l y Lys Leu Hls Lys Pro Gly Lys Asp A l a
671 209
672 210
TCG AAG CGC GGG CGG CAG ACG CCA GTG CTG TAT GCA ATG CTG GAC CAC S e t L y s A r g G l y A r g G l n T h r P r o V a l L e u T y r A l a Met L e u A s p I l l s
719 225
720 226
AGC AGA AGC ACC AAA GCT GTC AGT GAG AAG AAG GCC AAG GGG CTG GGG Ser Arg Ser Thr Lys Ala Val Set Glu Lys Lys Ala Lys GIy Leu Gly
767 241
768 242
GAG TCT CGC AAG GAT AAG AAA TAG CGG TTA GCG GGC CGG GCG GGG GAT G l u S e r A r g L y s A s p L y s L y s • **
815 249
816 864 912 960 1008 1056 1104 1152 1200 1248 1296 1344 1392 1440 1488 1536 1584 1632 1680 1728 1.776 1824 1872 1920
CGG GAG GTC ATG TGC TCC GTA ACC ACC GAA ATT CCC CCC TAT CCT TCA CCA GGT AGG GGG AAA GGC ATA GAC
GGG ATG AAG GGC CAG CTC AGT TTC TGC GAA CCC GTC CCA TTA GTG TGT GCC TCC AGA AGG AGC TGC TTT ATT
TTA GAG TCC CTG AGC TTT TTC ACC CCC AGG ATC CCT GCT GGT CTC GGC CAA CCC GAG GGG AGG ACT CTT TCC
GGG CTA CCC AAC AGG GCT ATT CCT GGG GAC TCC CCC AAT TAT ATC TTT GTA TGA ACA CAA AAA GCA CTC AGA
GTG CGA AGC TCG GGG CCC CCA GGC GGC TTT TGC TCC ACC TAT CTC TGC GAG CCA GAG ATG AAC GCC AAA AAA
GAG AAG AGA GAC ACC CCG GAA TTT TGT GAT ATC CCT CAG TTT TGC AAT GGG CTT AGA AGC AAA ACA CCT AAA
TCC GAT ACC AAG TAG CCC TTC CTG GTG ACC CCC CTA AGC TTA CCT TCA CCC TGG GAG CTT TGG GGG CCA AAA
GCC GAG AGC TGA GCT TGC ATT GGA TTT CTC CAG CCA ACC TTT TAC CGC CTG AGC AAT AAG GAA GAT AAT AAA
AAA CAG CTC TCG CCT CCT CCC GCC GGT TGC CCT GGT CAG TTT ACC CCC ATC ACT GGA AAA ATG TCT GTC AAA
GGC AGC AAA CCA CTT GCC CAG CAG GCC CTC GTC GGC ATC AAT CAT CCT CTC GTT TCC TGG GGG TAG CTT AA
CCA CCG AAC CCC ACC CTC GCA GAG TGT AAG CCC CCA AGA CCA GAC TAA ACA CTG TCA TTT GGG GAT AAA
ILAG GTG ATG GTC GAG CTC CGG CCT GCC CTC AAG AAC CCC CAC CCC AGG CCC GTC TAG GTG ACC TCC CTT TGA ATT GTA TTC TCC CTA ATC CTA CCC CCA CCT GAG CAC TCC AGG CCA CCT CTG GCT GTT TCC GCT CCA TAC TCT CTC TCC TTC AGG TTC TTT GTT TGT T G A G G A CCA A T G GTC C~F ItAT GAA CTT CAG TAT AGC GGA CTC CAG GTC TAG GTC AGG GAG TTA AAC AAC CAA CGG GGG G G A G G A TTT TCT ACA TTC TGT Tq~-A~--A~C
ATC GCT ATG CCC CTT GAC CCC CTC TGA GGC TCT GTC GTT TTA ACG GAG ATT TTG TGG ACA AGA TGT ACT
863 911 959 1007 1055 1103 1151 1199 1247 1295 1343 1391 1439 1487 1535 1583 1631 1679 1727 1775 1823 1871 1919 1948
Fig.2. Nucleotide and deduced amino acid sequences for human P0 protein. The residues confirmed by the amino acid sequencing of purified protein(unpublished data) are underlined. The polyadenylation signals are boxed.
517
Vol. 180, No. 2, 1991
BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS
Signal
sequence
protein
Mature 1
HUMAN
31
MAPGAPSSSPSPILAVLLFSSLVLSPAQAIVVYTDREVHGAVGSRVTLHCSFWSSEWVSD
BOVINE
...... A ..........
TL
K ........
RAT
...............
..........
Y .....
HUMAN
DISFTWRYQPEGGRDAISIFHYAKGQPYIDEVGTFKERIQWVGDP-RWKDGSIVIHNLDYS
Q...Y
...........
Q ...............
91
B O V I N E .L . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
H.-
RAT
.............................................
-S ..............
HUMAN
DNGTFTCDVKNPPDIVGKTSQVTLYVFEKVPTRYGVVLOAVIGGVLGVVLLLLLLFYVVR
BOVINE
...................................................
RAT
............................................
HUMAN
YCWLRRQAALQRRLSAMEKGKLHKPGKDASKRGRQTPVLYAMLDHSRSTKAVSEKKAKGL
BOVINE
..............
RAT
.................
HUMAN
GESRKDKK
BOVINE
........
RAT
........
H .........
TA
Q...F..SS..S
............
151 A ..... I ............
......................... ......................
G
LI. LI.
211 A ....
T...
A ....
S...
220
Fig.3. Comparison of the deduced amino acid sequences of the P0 proteins from human, bovine(Ref.4) and rat(Ref.1).
These different domains may be required together with basic proteins like P2 protein, to make a compact myelin sheath
in the peripheral nerve.
Our data support the hypothesis of
considerable conservation of the sequence of P0 protein during evolution. Acknowledgments We thank Dr. A. T. Campagnoni for a lambda gtll library from human fetus spinal cord, Dr. G. Lemke for a cDNA of rat P0 protein and Dr. T. Nakagomi for synthesizing the oligonucleotides. References 1. 2. 3. 4. 5. 6.
Lemke, G. and Axel, R. (1985) Cell 40, 501-508. Saavedra, R. R., Fors, L., Aebersold, R.H., Arden, B., Horvath, S., Sanders, J. and Hood, L. (1989) J. Mol. Evol. 29, 149-156. Barbu, M. (1990) J. Neurosci. Res. 25, 143-151. Sakamoto, Y., Kitamura, K., Yoshimura, K., Nishijima, T. and Uyemura K. (1987) J. Biol. Chem. 262, 4208-4214. Roth, H. J., Kronquist, K., Pretorius, P. J., Crandall, B. F. and Carnpagnoni, A. T. (1986) J. Neurosci. Res. 16, 227-238. Sanger, F., Nicklen, S. and Coulson, A.R. (1977) Proc. Natl. Acad. Sci. USA 74, 54635467.
518
