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Chapter 31 Structure and function of peripheral nerve myelin proteins
A.C.H. Yu,L.F. Eng,U.J. McMahan, H. Schulman, E.M. Shooter and A. Stadlin(Eds.) Progress in Bruin Resecirch, Vol. 105 0 1995 Elsevier Science BV. All rights reserved.
311
CHAPTER 31
Structure and function of peripheral nerve myelin proteins Keiichi Uyemura, Hiroaki Asou and Yasuo Takeda Department ofPhysiology, Keio University School of Medicine, Shinanornachi, Shinjuku-ku, Tokyo 160, Japan
Introduction Myelin glycoproteinsexpressed in Schwann cells appear to play unique roles in myelination, neurite outgrowth, and cell growth. In addition, recent studies revealed that mutation of these glycoproteins induce some hereditary neuropathies. In this paper, we describe recent studies on the structure and function of three functional glycoproteins, PASII/PMP22, PO, and Llcs, which are characteristically expressed in Schwann cell.
Progress of PASIUPMP22 protein studies Kitamura et al. (1976) first reported the existence of two characteristic glycoproteins in mammalian peripheral myelin, showing the protein and carbohydrate staining profiles of SDS-PAGE.Each peripheral myelin from bovine, pig, rabbit and guinea pig contained two distinct PAS positive glycoproteins, PO and PASII, indicating they are essential peripheral myelin components in mammals. Amino-terminal and carbohydrate-attaching peptide sequences of PO and PASII proteins were reported as shown in Fig. 1 (Kitamura et al., 1981). The results showed clearly that PASII is not a degradation product, but is different from PO. This is be-
cause both the amino-terminal and glycopeptide sequence of the two proteins are clearly different. However, analysis of the complete amino acid sequence of PASII had been difficult owing to its high hydrophobicity. Recently, Shooter’s group found the SR131 PMP22 protein, which was down-regulated specifically after nerve damage (Welcher et al., 1991). Interestingly, the partial amino acid sequence of PMP22 is quite similar not only t o PASII, but also t o the growth arrest specific protein (Gas3). Therefore PMP22 was considered to be a homologue of PASII and Gas3. By comparative studies of amino acid sequences of PASII/PMP22/SR13/Gas3 from bovine, human and rat, mouse and human revealed that this protein is highly conserved as shown in Fig. 2 (Welcher et al., 1991; Hayasaka et al., 1992; Patel et al., 1992; Uyemura et al., 1993). The gene locus of this protein, 17~12-p11.2,corresponds well t o the locus of Charcot-MarieTooth disease type 1A determined by linkage studies (Patel et al., 1992; Takahashi et al., 1992). Finally, in patients of this disease, gene duplication or a point mutation was detected, which indicated that PASIIPMP22 is a responsible protein for Charcot-Marie-Tooth 1A type disease (Patel et al., 1992; Suter et al., 1993; Uyemura et al., 1994).
312
PO
-
-
I1 e - V a l Va 1 T y 2- T h r Asp- X - X - V a l - X G 1 y -Ala - V a l -G1 y - d l a L e u Va1
N- terminal
sequence
-
-
-
PAS-11
-
-
Met L e u- L e u- L e u L e u Leu-Gly-Ile-lle-ValLeu- X Va1 A1a ValLeu- Val
-- -
---_----------------__________________c_---------------------
Carbohydrate1 i n k e d region
1
-Glx-A n - C y s - S e r - T h r -
-Gly-Asp-Asn-Gly-Thr-
B
c o
c o
Fig. 1. The N-terminal sequence and carbohydrate-linked regions of bovine PO protein and PASII protein.
BOVl N E PAS I1 HUMAN P M P - 2 2 RAT S R - 1 3 M O U S E Gas-3
M L L L L L G I I V LHVAV L V L LFV ST I V SQWMVGNGHA TDLWQ MLLLLLSIIVLHVAVLVLLFVSTIVSQWIVGNGHATDLWQ . . . . . .G . L F . . I . . . . . . . . . . . . . . . L . . . . . .G. LF. . I . ,. . . . . . . . . . . . . L
BOVINE P A S I I HUMAN P M P - 2 2 RAT SR-13 MOUSE Gas-3
NCST N CST S S SGNVHHCFSS SPNEWLQSVQATMI L S I IF SI L S L . . T . . A L . A . Q . , Y . . , V S , . . . . . . . . . . .. V . . . V . . . . .T. . A L . A . Q . y . . .VS . . . . . . . . . . . . . V . . . V . A .
BOVINE PAS11 HUMAN P M P - 2 2 RAT SR-13 MOUSE Gas-3
GGR FY I T GV F Q I L AV L CVMS A AS I Y T V R F L F F C Q L F T L T KGGRFY I T G I F 4 1 LAGLCVMSAAAI YTVR
BOVINE P A S I I HUMAN P M P - 2 2 RAT SR-13 MOUSE Gas-3
40
- - - - - - - - - - - - - - _ - _ _ - - _ - -------
*
.
---- - - - - - _ - - _ - - -
.v.. . . . . . .
.............. ..............
-- - - - - - - - - HLE HPEWHLNS .s.. .V.N 5 . . .V. T
.
80
.F
120
.............
- _ - - - - - - - - --------
--------------
- ------ --
Fig. 2. Amino acid sequence of bovine PASII, human PMP22, Rat SR13 and mouse Gas3 proteins. - - - -: Transmembrane segments; :I: A glycosylation site; .: Identical residues to human PMP22.
Functional analysis of PO protein PO is a major glycoprotein of 28 kD in mammalian peripheral myelin and a member of the immunoglobulin superfamily (Uyemura et al., 1987; 1992). To address PO functions, we established a cultured cell line expressing PO by cDNA transfection and analyzed its physiological activities. PO cDNA with a beta-actin promoter was transfected and expressed in C6 rat astrocytoma cell. After incubation, PO expressing cells made great aggregates, while most of
the control cells remained as a single cell or small aggregates (Yazaki et al., 1992). This confirmed previous reports by Filbin et al. (1990) and D’Urso et al. (1990). PO mediated aggregation is homophilic, this is because DiIlabeled PO expressing cells made aggregates selectively. The time course of cell aggregation was shown by NtINo in Fig. 3. Nt and No are the total number of particles a t incubation times t and 0, respectively. The total particle number of PO expressing cells decreased markedly, compared to control cells. Effects of the PO
313
0.2
Fig, 3 . Short-term aggregation of C6PO (PO expressing CS) cells and C6 (control) cells. Nt and No are the total number of particles at incubation time t and 0, respectively. Rate of aggregation is represented by the index Nt/No.
TABLE 1 Peptide None 1 2 3 4
5
Sequence
(43)GGRDAISI(50) (83)IVIHNLDY(90) (90)YSDNGTF(96) (100)VKNPPDIV(107) CHO
I
(91)SDNGT(95)"'
% Inhibition
0 20.3 7.6 50.2 5.1
85.1
"'Glycopeptide.
peptides or a glycopeptide t o PO mediated aggregation were examined, as shown in Fig. 4. The glycopeptide showed the strongest inhibition (85%) followed by the peptide 3 (500/0), while other peptides showed low inhibition rates as shown in Table 1.The results indicated that the oligosaccharide chain and its neighbor-
I
0
I
l
20
(
I
40
'
1 l
60
l. I
80
S
Time (rnin)
1 0
Fig. 4.Effect of synthetic peptides and a PO glycopeptide on PO mediated cell aggregation. One of four synthetic peptides (pep.1- pep.4) or a glycopeptide purified from bovine PO (GP) was suspended a t 500 pg/ml in advance of making cell suspension.
ing peptide are the most important for the cell aggregation mediated PO. When the dorsal root ganglion cells were cocultured with the PO expressing C6 cells, marked neurite extension is observed (Yazaki et al., 1991).A similar effect of neurite extension was also observed in the case of coculture of young cortical neurons with PO expressing cells (Yazaki et al., 1994). The neurite extension was clearly inhibited by the addition of monoclonal anti-PO antibody, which recognized the peptide of PO protein. These results suggested that PO promotes neurite extension and the active site for neurite extension of PO is different from the site for cell aggregation.
Chromosomal locus of PO gene and its relation to hereditary neuropathy The chromosomal locus of PO was examined by the spot hybridization of the flow-sorted human
314
Rh-null hermolytic anemia Erythroblastosis fetalis
36.3 36.2
Elliptocytosis-1
36.1
P
Erythrokeratoderrnia variabilis
35 34.3 34.2 34.1 33 32.3 32.2 32.1 31.3 31.2
Galactose epirnerase deficiency Fucosidosis Porphyria cutanea tarda
31.1 22.3 22.2 22.1 21 13.3 132 13.1
I?-ii
Pk deficiency hemolytic anem ia
12
H e r e d i t a r y cataract (1 f o r m )
21.1 21.2 21.3
Charcot-Marie-Tooth disease (1 B )
PO
Gaucher disease, type I 24
A nt it h r o m b in
25
III deficiency
Elli ptocytoses - 2 31
Spherocytoses, recessive
32.1
Glycogenosis
32.2 32.3 41
42.1 42.2 42.3 43 44
H
'CR1 deficiency
U
Fig. 5. Chromosome locus of PO gene. The locus of PO gene was chromosome lq22-23.
chromosomes (Hayasaka et al., 1993).The metaphase chromosomes of human diploid B-lymphoblastoid GM130B cells were stained with fluorescence dyes and sorted on a FACS440 sorter. The P-32 labeled PO cDNA hybridized with the fraction corresponding to chromosome 1 by the two different methods, indicating the location of the PO gene on human chromosome 1.By FISH analysis of the human chromosome for the regional assignment using digoxigenenin-labeled genomic DNA, distinct fluorescent signals were detected on the long arm of chromosome 1.Measurement of the signal position made clear the PO gene in the lq22-q23, which corresponds well t o the locus of Charcot-
Marie-Tooth disease type 1B determined by linkage studies of the disease (Fig. 5). The result suggests a close relationship between PO and Charcot-Marie-Tooth disease type 1B. Recently our group found the point mutations of PO gene in patients of Charcot-MarieTooth disease type 1B (Hayasaka et al., 1993). Point mutations of a nucleotide in the gene caused exchange from Asp to Glu in position 61 in case 1 and from Lys t o Glu in 67 in case 2 (Fig. 6 ) . Probable configuration changes of PO due to point mutations may disturb PO mediated cell adhesion, while the locus is not in the vicinity of the carbohydrate attaching site of PO.
315 TABLE 2 Responsible gene/protein of hereditary motor and sensory neuropathy (HMSN) Disease
Type
Gene locus
HMSN (CMT) HMSN (CMT) HMSN (CMT) HMSN (CMT) HMSN (CMT) HMSN HMSN (DS) HMSN HMSN HMSN HMSN HNPP
IA
17~12-p11.2 lq21.1-q23.3 Xql l-q13 xq22.2 Xq26 ?
IB XI
x2 x3 I1 111 IV V VI VII
?
? ? ? ? 17~12-~11.2
Mutated protein
Mode of mutation
PASIIPMP22
Duplication; Point mutation Point mutation Point mutation
PO Connexin 32 ? ?
? ?
?
PO or PASIIPMPZ:! ? ? ? ? PASIWMP22
? Point mutation ? ? ? ? Deletion
CMT: Charcot-Marie-Tooth disease; DS: Dejerine-Sottas disease; HNPP: hereditary neuropathy with liability to pressure palsies.
Responsible proteins and mutations of each hereditary sensory and motor neuropathy are summarized in Table 2. Concerning CharcotMarie-Tooth disease type lA, the genomic locus is 17~12-p11.2and the responsible protein is PASIWMP22. Mainly gene duplications, but also point mutations, have been reported. The genomic locus of Charcot-Marie-Tooth Disease type 1B is lq22-q23, which corresponds t o the PO locus. In this case, a point mutation of PO is responsible for the disease. And several other neuropathies linked t o the X chromosome are reported and recently connexin 32 has been identified as their responsible proteins (Bergoffen et al., 1993). In addition, there are many other hereditary neuropathies, which have not yet been well characterized. Further studies will be required t o understand the genetic background of these diseases.
Phylogenetic studies of myelin proteins
PO protein is a major myelin protein in peripheral nervous system (PNS), but not in the central nervous system (CNS) of mammals. HoweverJower vertebrates such as fish con-
Normal mRNA (Normal Amino Acid (No.))
CASEI:
CASEI:
Mutated mRNA: (Mutaled Amino Acid (No.)}
GAC (Asp(61)) AAA
{Lys(67))
Fig. 6 . Point mutations of PO gene/protein in patients of hereditary motor and sensory neuropathy type 1B (Charcot-Marie-Tooth disease 1B).
tained PO-like proteins in both the PNS and CNS (Waehneldt and Malotka, 1989; Takei et al., 1993). In the case of amphibians, adult axolotl and African craw frog contained both PO-like proteins and proteolipid protein (PLP) in the CNS. On the other hand, adult bullfrog contained only PLP in the CNS, but the young animal such as tadpole contained both PLP and PO-like protein in the CNS (Takei and Uyemura, 1993) (Fig. 7). Mammalian PNS shows regenerative activity, while the CNS does not. However, both CNS and PNS are able to regenerates in fish. And in general young animals have more ability for regeneration. The results suggest that PO expression may be related t o regenerative activity.
3 16
Osteiclithyes
MammaIia
Amphibia
Rodentla
Anum
Cyprlnlformer
\
I
h
Urodela
Axolotd
I
Prlmatea
Rat
i3Ldfrog
i
a
African clawad hvg
I
I
I
Fig. 7 . PO and PLP expression in myelin of central nervous system in various animals
An L1 isoform expressed in Schwann cell The complete form of L1 is a large 200 kD molecule of the immunoglobulin superfamily. It contained 6 immunoglobulin domains and 5 fibronectin type I11 domains. Recently we determined the complete sequence of rat and human L1 and analyzed their functions by transfection of L1 cDNA into cultured cells. L1 cDNA was transfected and expressed in L cells (Miura et al., 1992). When the dissociated neurons of a young cerebellum were cultured on L1 expressing L cells, the neurons showed strong adhesions t o the L1 expressing cells, while adhesion was hardly observed between neurons and the control L1 non-expressing cells. When the L1 expressing cell was cocultured with the neuronal reaggregate prepared from a young cerebellum, the neurons showed remarkable neurite outgrowth and cell migration from the reaggregate. These results confirmed the function of L1 for cell adhesion, neurite outgrowth, and neural migration in the nervous system. In the CNS L1 localized exclusively in neurons, but non-neuronal Schwann cell also con-
tained L1 in PNS. Herper et al. (1991) first reported an isoform of L1, which lacks 4 amino acid in the intracellular domain, is expressed in human melanoma cells. We examined distribution of complete L1 and its isoform by two-step PCR methods. While rat and mouse brains contained only the complete form of L1, the short isoform of L1 is found in their sciatic nerves (Miura et al., 1991; Takeda et al., 1993). The results indicated clearly that L1 in Schwann cell is a short form, different from complete L1 in neurons. The L1 isoform is probably formed by alternative splicing of L1 mRNA. By this deletion of 4 amino acids in the intracellular domain, two phosphorylation sites by casein b a s e I and I1 may be lost, suggesting a change of Ll’s function. Summary (1) Two glycoproteins, PO and PASII, are
widely distributed in the peripheral myelin, but not in the central myelin of mammals. POlike protein is expressed in both peripheral and central myelins of some lower vertebrates, such as fish and tadpoles. A close relationship is
317
suggested between PO expression and neural regenerative activity. (2) PMP22 was reported to show high sequence homology, not only to PASII, but also to the growth arrest specific protein. Human PASIWMP22 sequence was deduced and the locus of its gene, chromosome 17~12-p11.2,is similar to the region linked to Charcot-MarieTooth disease type 1A. (3) PO expressed on cultured cells mediated strong homophilic cell adhesion and neurite outgrowth. Addition of the PO glycopeptide inhibited cell adhesion markedly, indicating that the oligosaccharide with peptide is essential for PO mediated cell adhesion. The active site for neurite outgrowth in PO appears t o be different from the adhesion site. (4) We determined the human chromosomal locus of the PO gene, lq22-q23, which corresponded to the locus of hereditary motor and sensory neuropathy, Charcot-Marie-Tooth disease type 1B. Point mutations in the extracellular domain of PO are found in the patient’s chromosome. (5) L1 is a large multifunctional adhesive glycoprotein of 200 kD. Rat and human L1 sequences confirmed a common structure for the mammalian nervous systems. An isoform of L1 (Llcs), lacking four amino acids, appears to localize in non-neuronal cells such as Schwann cells, while the complete L1 is exclusively found in neurons. Llcs in Schwann cells may be functionally different from L1 in neurons.
Acknowledgements This work was partially supported by grants from the Ministry of Education, Science and Culture and of Health and Welfare in Japan.
References Bergoffen, J., Scherer, S.S., Wang, S., Oronzi Scott, M., Bone, L.J., Pul, D.L., Chen, K., Lensch, M.W., Chance, P.F. and Fischbeck, K.H. (1993) Connexin mutations in X-linked Charcot-Marie-Tooth disease. Science, 262: 2039-2042.
D’Urso, D., Brophy, P.J., Staugaitis, S.M., Gillespie, C.S., Frey, A.B., Stempak, G. and Colman, D.R. (1990) Protein zero of peripheral nerve myelin: biosynthesis, membrane insertion, and evidence for homotypic interaction. Neuron, 2: 449-460. Filbin, M.T., Walsh, F.K, Trapp, B.D. Pizzey, J.A. and Tennekoon, G.I. (1990) Role of myelin PO protein as homophilic adhesion molecule. Nature, 344: 871-872. Harper, J.R., Prince, J.T., Healy, P.A., Stuart, J.K., Nauman, S.J. and Stallcup, W.B. (1991) Isolation and sequence of partial cDNA clones of human L1: homology of human and rodent L1 in the cytoplasmic region. J. Neurochem., 56: 797-804. Hayasaka, K., Himoro, M., Nanao, K., Sato, W., Miura, M., Uyemura, K., Takahashi, E. and Takada, G. (1992) Isolation and sequence determination of cDNA encoding PMP-22(PAS-II/SR13/GAS-3) of human peripheral myelin. Biochem. Biophys. Res. Commun., 186: 827831. Hayasaka, K., Himura, M., Wang, Y., Takata, M., Minoshima, S.,Shimizu, N., Miura, M., Uyemura, K. and Takada, G. (1993) Structure and chromosomal localization of the gene encoding the human myelin protein zero (MPZ). Genomics, 17: 755-758. Hayasaka, K., Himoro, M., Sato, W., Takada, G., Uyemura, K., Shimizu, N., Bird, T.D., Conneally, P.M. and Chance, P.F. (1993) Charcot-Marie-Tooth neuropathy type 1B is associated with mutations of the myelin PO gene. Nature Genetics, 5: 31-34. Kitamura, K., Suzuki, M. and Uyemura, K. (1976) Purification and partial characterization of two glycoproteins in bovine peripheral nerve myelin membrane. Biochim. Biophys. Actu, 455: 806-816. Kitamura, K., Sakamoto, Y., Suzuki, M. and Uyemura, K. (1981) Microhetrogeneity of carbohydrate in PO protein from bovine peripheral nerve myelin. In: T. Yamakawa, T. Osawa and S. Handa (Eds.), Glycoconjugates. Japan Scientic Society Press, pp. 273-274. Miura, M., Asou, H., Kobayashi, M. and Uyemura, K. (1992) Functional expression of a full-length cDNA coding for rat neural cell adhesion molecule L1 mediates homophilic intercellular adhesion and migration of cerebellar neurons. J . Biol. Chem., 267: 1075210758. Miura, M., Kobayashi, M., Asou, H. and Uyemura, K.(1991) Molecular cloning of cDNA encoding of rat neural cell adhesion molecule L1. FEBS Lett., 289: 91-95. Patel, P.I., Roa, B.B., Welcher, A.A., Schoener-Scott, R., Trask, B.J., Pentao, L., Snipes, G.J., Garcia, C.A., Francke, U., Shooter, E.M., Lupski, J.R. and Suter, U. (1992) The gene for the peripheral myelin protein PMP22 is a candidate for Charcot-Marie-Tooth disease type 1A. Nature Genet., 1: 159-165.
3 18
Suter, U., Welcher, A.A. and Snipes, J. (1993) Progress in the molecular understanding of hereditary peripheral neuropathies reveals new insights into the biology of the peripheral nervous system. Trends Neurosci., 16: 50-56. Takahashi, E., Takeda, O., Himoro, M., Nanao, K., Takada, G., and Hayasaka, K. (1992) Localization of PMP22 gene (candidate gene for the Charcot-Marie-Tooth disease 1A) to band 1 7 ~ 1 1 . 2by direct r-banding fluorescence in situ hybridization. Jpn. J. Human Genet., 37: 303-306. Takeda, Y., Asou, H., Miura, M., Kobayashi, M. and Uyeniura, K. (1993) Evidence for the cell-type dependent alternative splicing of cell adhesion molecule L1 in mammals. Bull. Jpn. Neurochem. Soc., 32: 560-561. Talcei, K. and Uyemura, K. (1993) Expression of PO-like glycoprotein in central nervous system myelin of the amphibians. Comp. Biochem. Physiol., 106B: 873-882. Talcei, K., Kitarnura, K., Banno, K. and Uyemura, K. (1993) Major glycoproteins in carp CNS myelin: homology to protein zero with HNK-l/L2 carbohydrate etitope. Neirrochein. Iiit., 23: 239-248. Uyemura, K. (1993) Functional glycoproteins expressed in Schwann cell membrane. Neurosci. Res., 16: 9-13. Uyemura, K., Suzuki, M., Sakamoto, Y. and Tanaka, S. (1987) Structure of PO protein: Homology to immunoglobulin superfamily. Biomed. Res., 8: 353-357.
Uyemura, K., Kitamura, K. and Miura, M. (1992) Structure and molecular biology of PO protein. In: R.E. Martenson (Ed.), Myelin: Biology and Chemistry. CRC Press, Boca Raton, FL, pp. 481-508. Uyemura, K., Takeda, Y., Asou, H. and Hayasaka, K. (1994) Neural cell adhesion proteins and neurological diseases. J.Biochem., 116: 1187-1192. Waehneldt, T.V. and Malotka, J. (1989) Presence of proteolipid protein in coelacanth brain myelin demonstrates tetrapod affinities and questions a chondrichthyan. J. Neurochem., 52: 1941-1943. Welcher, A.A., Suter, U., De Leon, M., Snipes, G.J. and Shooter, E.M. (1991) A myelin protein is encoded by a homologue of a growth arrest specific gene. Proc. Natl. Acad. Sci. USA, 88: 7195-7199. Yazaki, T., Miura, M., Asou, H., Toya, S. and Uyemura, K. (1991) Myelin PO protein expressed in C6 cells promotes neurite outgrowth. Biomed. Res., 12: 223-230. Yazaki, T., Miura, M., Asou, H., Kitamura, K., Toya, S. and Uyernura, K. (1992) Glycopeptide of PO protein inhibits hornophilic cell adhesion: Competition assay with transformant 8 and peptides. FEBS Lett., 307: 361366. Yazaki, T., Miura, M., Asou, H., Toya, S. and Uyemura, K. (1994) Peripheral myelin PO protein mediates neurite outgrowth of cortical neurons in vitro and axonal regeneration in vivo. Neurosci. Lett., 176: 13-16.
311
CHAPTER 31
Structure and function of peripheral nerve myelin proteins Keiichi Uyemura, Hiroaki Asou and Yasuo Takeda Department ofPhysiology, Keio University School of Medicine, Shinanornachi, Shinjuku-ku, Tokyo 160, Japan
Introduction Myelin glycoproteinsexpressed in Schwann cells appear to play unique roles in myelination, neurite outgrowth, and cell growth. In addition, recent studies revealed that mutation of these glycoproteins induce some hereditary neuropathies. In this paper, we describe recent studies on the structure and function of three functional glycoproteins, PASII/PMP22, PO, and Llcs, which are characteristically expressed in Schwann cell.
Progress of PASIUPMP22 protein studies Kitamura et al. (1976) first reported the existence of two characteristic glycoproteins in mammalian peripheral myelin, showing the protein and carbohydrate staining profiles of SDS-PAGE.Each peripheral myelin from bovine, pig, rabbit and guinea pig contained two distinct PAS positive glycoproteins, PO and PASII, indicating they are essential peripheral myelin components in mammals. Amino-terminal and carbohydrate-attaching peptide sequences of PO and PASII proteins were reported as shown in Fig. 1 (Kitamura et al., 1981). The results showed clearly that PASII is not a degradation product, but is different from PO. This is be-
cause both the amino-terminal and glycopeptide sequence of the two proteins are clearly different. However, analysis of the complete amino acid sequence of PASII had been difficult owing to its high hydrophobicity. Recently, Shooter’s group found the SR131 PMP22 protein, which was down-regulated specifically after nerve damage (Welcher et al., 1991). Interestingly, the partial amino acid sequence of PMP22 is quite similar not only t o PASII, but also t o the growth arrest specific protein (Gas3). Therefore PMP22 was considered to be a homologue of PASII and Gas3. By comparative studies of amino acid sequences of PASII/PMP22/SR13/Gas3 from bovine, human and rat, mouse and human revealed that this protein is highly conserved as shown in Fig. 2 (Welcher et al., 1991; Hayasaka et al., 1992; Patel et al., 1992; Uyemura et al., 1993). The gene locus of this protein, 17~12-p11.2,corresponds well t o the locus of Charcot-MarieTooth disease type 1A determined by linkage studies (Patel et al., 1992; Takahashi et al., 1992). Finally, in patients of this disease, gene duplication or a point mutation was detected, which indicated that PASIIPMP22 is a responsible protein for Charcot-Marie-Tooth 1A type disease (Patel et al., 1992; Suter et al., 1993; Uyemura et al., 1994).
312
PO
-
-
I1 e - V a l Va 1 T y 2- T h r Asp- X - X - V a l - X G 1 y -Ala - V a l -G1 y - d l a L e u Va1
N- terminal
sequence
-
-
-
PAS-11
-
-
Met L e u- L e u- L e u L e u Leu-Gly-Ile-lle-ValLeu- X Va1 A1a ValLeu- Val
-- -
---_----------------__________________c_---------------------
Carbohydrate1 i n k e d region
1
-Glx-A n - C y s - S e r - T h r -
-Gly-Asp-Asn-Gly-Thr-
B
c o
c o
Fig. 1. The N-terminal sequence and carbohydrate-linked regions of bovine PO protein and PASII protein.
BOVl N E PAS I1 HUMAN P M P - 2 2 RAT S R - 1 3 M O U S E Gas-3
M L L L L L G I I V LHVAV L V L LFV ST I V SQWMVGNGHA TDLWQ MLLLLLSIIVLHVAVLVLLFVSTIVSQWIVGNGHATDLWQ . . . . . .G . L F . . I . . . . . . . . . . . . . . . L . . . . . .G. LF. . I . ,. . . . . . . . . . . . . L
BOVINE P A S I I HUMAN P M P - 2 2 RAT SR-13 MOUSE Gas-3
NCST N CST S S SGNVHHCFSS SPNEWLQSVQATMI L S I IF SI L S L . . T . . A L . A . Q . , Y . . , V S , . . . . . . . . . . .. V . . . V . . . . .T. . A L . A . Q . y . . .VS . . . . . . . . . . . . . V . . . V . A .
BOVINE PAS11 HUMAN P M P - 2 2 RAT SR-13 MOUSE Gas-3
GGR FY I T GV F Q I L AV L CVMS A AS I Y T V R F L F F C Q L F T L T KGGRFY I T G I F 4 1 LAGLCVMSAAAI YTVR
BOVINE P A S I I HUMAN P M P - 2 2 RAT SR-13 MOUSE Gas-3
40
- - - - - - - - - - - - - - _ - _ _ - - _ - -------
*
.
---- - - - - - _ - - _ - - -
.v.. . . . . . .
.............. ..............
-- - - - - - - - - HLE HPEWHLNS .s.. .V.N 5 . . .V. T
.
80
.F
120
.............
- _ - - - - - - - - --------
--------------
- ------ --
Fig. 2. Amino acid sequence of bovine PASII, human PMP22, Rat SR13 and mouse Gas3 proteins. - - - -: Transmembrane segments; :I: A glycosylation site; .: Identical residues to human PMP22.
Functional analysis of PO protein PO is a major glycoprotein of 28 kD in mammalian peripheral myelin and a member of the immunoglobulin superfamily (Uyemura et al., 1987; 1992). To address PO functions, we established a cultured cell line expressing PO by cDNA transfection and analyzed its physiological activities. PO cDNA with a beta-actin promoter was transfected and expressed in C6 rat astrocytoma cell. After incubation, PO expressing cells made great aggregates, while most of
the control cells remained as a single cell or small aggregates (Yazaki et al., 1992). This confirmed previous reports by Filbin et al. (1990) and D’Urso et al. (1990). PO mediated aggregation is homophilic, this is because DiIlabeled PO expressing cells made aggregates selectively. The time course of cell aggregation was shown by NtINo in Fig. 3. Nt and No are the total number of particles a t incubation times t and 0, respectively. The total particle number of PO expressing cells decreased markedly, compared to control cells. Effects of the PO
313
0.2
Fig, 3 . Short-term aggregation of C6PO (PO expressing CS) cells and C6 (control) cells. Nt and No are the total number of particles at incubation time t and 0, respectively. Rate of aggregation is represented by the index Nt/No.
TABLE 1 Peptide None 1 2 3 4
5
Sequence
(43)GGRDAISI(50) (83)IVIHNLDY(90) (90)YSDNGTF(96) (100)VKNPPDIV(107) CHO
I
(91)SDNGT(95)"'
% Inhibition
0 20.3 7.6 50.2 5.1
85.1
"'Glycopeptide.
peptides or a glycopeptide t o PO mediated aggregation were examined, as shown in Fig. 4. The glycopeptide showed the strongest inhibition (85%) followed by the peptide 3 (500/0), while other peptides showed low inhibition rates as shown in Table 1.The results indicated that the oligosaccharide chain and its neighbor-
I
0
I
l
20
(
I
40
'
1 l
60
l. I
80
S
Time (rnin)
1 0
Fig. 4.Effect of synthetic peptides and a PO glycopeptide on PO mediated cell aggregation. One of four synthetic peptides (pep.1- pep.4) or a glycopeptide purified from bovine PO (GP) was suspended a t 500 pg/ml in advance of making cell suspension.
ing peptide are the most important for the cell aggregation mediated PO. When the dorsal root ganglion cells were cocultured with the PO expressing C6 cells, marked neurite extension is observed (Yazaki et al., 1991).A similar effect of neurite extension was also observed in the case of coculture of young cortical neurons with PO expressing cells (Yazaki et al., 1994). The neurite extension was clearly inhibited by the addition of monoclonal anti-PO antibody, which recognized the peptide of PO protein. These results suggested that PO promotes neurite extension and the active site for neurite extension of PO is different from the site for cell aggregation.
Chromosomal locus of PO gene and its relation to hereditary neuropathy The chromosomal locus of PO was examined by the spot hybridization of the flow-sorted human
314
Rh-null hermolytic anemia Erythroblastosis fetalis
36.3 36.2
Elliptocytosis-1
36.1
P
Erythrokeratoderrnia variabilis
35 34.3 34.2 34.1 33 32.3 32.2 32.1 31.3 31.2
Galactose epirnerase deficiency Fucosidosis Porphyria cutanea tarda
31.1 22.3 22.2 22.1 21 13.3 132 13.1
I?-ii
Pk deficiency hemolytic anem ia
12
H e r e d i t a r y cataract (1 f o r m )
21.1 21.2 21.3
Charcot-Marie-Tooth disease (1 B )
PO
Gaucher disease, type I 24
A nt it h r o m b in
25
III deficiency
Elli ptocytoses - 2 31
Spherocytoses, recessive
32.1
Glycogenosis
32.2 32.3 41
42.1 42.2 42.3 43 44
H
'CR1 deficiency
U
Fig. 5. Chromosome locus of PO gene. The locus of PO gene was chromosome lq22-23.
chromosomes (Hayasaka et al., 1993).The metaphase chromosomes of human diploid B-lymphoblastoid GM130B cells were stained with fluorescence dyes and sorted on a FACS440 sorter. The P-32 labeled PO cDNA hybridized with the fraction corresponding to chromosome 1 by the two different methods, indicating the location of the PO gene on human chromosome 1.By FISH analysis of the human chromosome for the regional assignment using digoxigenenin-labeled genomic DNA, distinct fluorescent signals were detected on the long arm of chromosome 1.Measurement of the signal position made clear the PO gene in the lq22-q23, which corresponds well t o the locus of Charcot-
Marie-Tooth disease type 1B determined by linkage studies of the disease (Fig. 5). The result suggests a close relationship between PO and Charcot-Marie-Tooth disease type 1B. Recently our group found the point mutations of PO gene in patients of Charcot-MarieTooth disease type 1B (Hayasaka et al., 1993). Point mutations of a nucleotide in the gene caused exchange from Asp to Glu in position 61 in case 1 and from Lys t o Glu in 67 in case 2 (Fig. 6 ) . Probable configuration changes of PO due to point mutations may disturb PO mediated cell adhesion, while the locus is not in the vicinity of the carbohydrate attaching site of PO.
315 TABLE 2 Responsible gene/protein of hereditary motor and sensory neuropathy (HMSN) Disease
Type
Gene locus
HMSN (CMT) HMSN (CMT) HMSN (CMT) HMSN (CMT) HMSN (CMT) HMSN HMSN (DS) HMSN HMSN HMSN HMSN HNPP
IA
17~12-p11.2 lq21.1-q23.3 Xql l-q13 xq22.2 Xq26 ?
IB XI
x2 x3 I1 111 IV V VI VII
?
? ? ? ? 17~12-~11.2
Mutated protein
Mode of mutation
PASIIPMP22
Duplication; Point mutation Point mutation Point mutation
PO Connexin 32 ? ?
? ?
?
PO or PASIIPMPZ:! ? ? ? ? PASIWMP22
? Point mutation ? ? ? ? Deletion
CMT: Charcot-Marie-Tooth disease; DS: Dejerine-Sottas disease; HNPP: hereditary neuropathy with liability to pressure palsies.
Responsible proteins and mutations of each hereditary sensory and motor neuropathy are summarized in Table 2. Concerning CharcotMarie-Tooth disease type lA, the genomic locus is 17~12-p11.2and the responsible protein is PASIWMP22. Mainly gene duplications, but also point mutations, have been reported. The genomic locus of Charcot-Marie-Tooth Disease type 1B is lq22-q23, which corresponds t o the PO locus. In this case, a point mutation of PO is responsible for the disease. And several other neuropathies linked t o the X chromosome are reported and recently connexin 32 has been identified as their responsible proteins (Bergoffen et al., 1993). In addition, there are many other hereditary neuropathies, which have not yet been well characterized. Further studies will be required t o understand the genetic background of these diseases.
Phylogenetic studies of myelin proteins
PO protein is a major myelin protein in peripheral nervous system (PNS), but not in the central nervous system (CNS) of mammals. HoweverJower vertebrates such as fish con-
Normal mRNA (Normal Amino Acid (No.))
CASEI:
CASEI:
Mutated mRNA: (Mutaled Amino Acid (No.)}
GAC (Asp(61)) AAA
{Lys(67))
Fig. 6 . Point mutations of PO gene/protein in patients of hereditary motor and sensory neuropathy type 1B (Charcot-Marie-Tooth disease 1B).
tained PO-like proteins in both the PNS and CNS (Waehneldt and Malotka, 1989; Takei et al., 1993). In the case of amphibians, adult axolotl and African craw frog contained both PO-like proteins and proteolipid protein (PLP) in the CNS. On the other hand, adult bullfrog contained only PLP in the CNS, but the young animal such as tadpole contained both PLP and PO-like protein in the CNS (Takei and Uyemura, 1993) (Fig. 7). Mammalian PNS shows regenerative activity, while the CNS does not. However, both CNS and PNS are able to regenerates in fish. And in general young animals have more ability for regeneration. The results suggest that PO expression may be related t o regenerative activity.
3 16
Osteiclithyes
MammaIia
Amphibia
Rodentla
Anum
Cyprlnlformer
\
I
h
Urodela
Axolotd
I
Prlmatea
Rat
i3Ldfrog
i
a
African clawad hvg
I
I
I
Fig. 7 . PO and PLP expression in myelin of central nervous system in various animals
An L1 isoform expressed in Schwann cell The complete form of L1 is a large 200 kD molecule of the immunoglobulin superfamily. It contained 6 immunoglobulin domains and 5 fibronectin type I11 domains. Recently we determined the complete sequence of rat and human L1 and analyzed their functions by transfection of L1 cDNA into cultured cells. L1 cDNA was transfected and expressed in L cells (Miura et al., 1992). When the dissociated neurons of a young cerebellum were cultured on L1 expressing L cells, the neurons showed strong adhesions t o the L1 expressing cells, while adhesion was hardly observed between neurons and the control L1 non-expressing cells. When the L1 expressing cell was cocultured with the neuronal reaggregate prepared from a young cerebellum, the neurons showed remarkable neurite outgrowth and cell migration from the reaggregate. These results confirmed the function of L1 for cell adhesion, neurite outgrowth, and neural migration in the nervous system. In the CNS L1 localized exclusively in neurons, but non-neuronal Schwann cell also con-
tained L1 in PNS. Herper et al. (1991) first reported an isoform of L1, which lacks 4 amino acid in the intracellular domain, is expressed in human melanoma cells. We examined distribution of complete L1 and its isoform by two-step PCR methods. While rat and mouse brains contained only the complete form of L1, the short isoform of L1 is found in their sciatic nerves (Miura et al., 1991; Takeda et al., 1993). The results indicated clearly that L1 in Schwann cell is a short form, different from complete L1 in neurons. The L1 isoform is probably formed by alternative splicing of L1 mRNA. By this deletion of 4 amino acids in the intracellular domain, two phosphorylation sites by casein b a s e I and I1 may be lost, suggesting a change of Ll’s function. Summary (1) Two glycoproteins, PO and PASII, are
widely distributed in the peripheral myelin, but not in the central myelin of mammals. POlike protein is expressed in both peripheral and central myelins of some lower vertebrates, such as fish and tadpoles. A close relationship is
317
suggested between PO expression and neural regenerative activity. (2) PMP22 was reported to show high sequence homology, not only to PASII, but also to the growth arrest specific protein. Human PASIWMP22 sequence was deduced and the locus of its gene, chromosome 17~12-p11.2,is similar to the region linked to Charcot-MarieTooth disease type 1A. (3) PO expressed on cultured cells mediated strong homophilic cell adhesion and neurite outgrowth. Addition of the PO glycopeptide inhibited cell adhesion markedly, indicating that the oligosaccharide with peptide is essential for PO mediated cell adhesion. The active site for neurite outgrowth in PO appears t o be different from the adhesion site. (4) We determined the human chromosomal locus of the PO gene, lq22-q23, which corresponded to the locus of hereditary motor and sensory neuropathy, Charcot-Marie-Tooth disease type 1B. Point mutations in the extracellular domain of PO are found in the patient’s chromosome. (5) L1 is a large multifunctional adhesive glycoprotein of 200 kD. Rat and human L1 sequences confirmed a common structure for the mammalian nervous systems. An isoform of L1 (Llcs), lacking four amino acids, appears to localize in non-neuronal cells such as Schwann cells, while the complete L1 is exclusively found in neurons. Llcs in Schwann cells may be functionally different from L1 in neurons.
Acknowledgements This work was partially supported by grants from the Ministry of Education, Science and Culture and of Health and Welfare in Japan.
References Bergoffen, J., Scherer, S.S., Wang, S., Oronzi Scott, M., Bone, L.J., Pul, D.L., Chen, K., Lensch, M.W., Chance, P.F. and Fischbeck, K.H. (1993) Connexin mutations in X-linked Charcot-Marie-Tooth disease. Science, 262: 2039-2042.
D’Urso, D., Brophy, P.J., Staugaitis, S.M., Gillespie, C.S., Frey, A.B., Stempak, G. and Colman, D.R. (1990) Protein zero of peripheral nerve myelin: biosynthesis, membrane insertion, and evidence for homotypic interaction. Neuron, 2: 449-460. Filbin, M.T., Walsh, F.K, Trapp, B.D. Pizzey, J.A. and Tennekoon, G.I. (1990) Role of myelin PO protein as homophilic adhesion molecule. Nature, 344: 871-872. Harper, J.R., Prince, J.T., Healy, P.A., Stuart, J.K., Nauman, S.J. and Stallcup, W.B. (1991) Isolation and sequence of partial cDNA clones of human L1: homology of human and rodent L1 in the cytoplasmic region. J. Neurochem., 56: 797-804. Hayasaka, K., Himoro, M., Nanao, K., Sato, W., Miura, M., Uyemura, K., Takahashi, E. and Takada, G. (1992) Isolation and sequence determination of cDNA encoding PMP-22(PAS-II/SR13/GAS-3) of human peripheral myelin. Biochem. Biophys. Res. Commun., 186: 827831. Hayasaka, K., Himura, M., Wang, Y., Takata, M., Minoshima, S.,Shimizu, N., Miura, M., Uyemura, K. and Takada, G. (1993) Structure and chromosomal localization of the gene encoding the human myelin protein zero (MPZ). Genomics, 17: 755-758. Hayasaka, K., Himoro, M., Sato, W., Takada, G., Uyemura, K., Shimizu, N., Bird, T.D., Conneally, P.M. and Chance, P.F. (1993) Charcot-Marie-Tooth neuropathy type 1B is associated with mutations of the myelin PO gene. Nature Genetics, 5: 31-34. Kitamura, K., Suzuki, M. and Uyemura, K. (1976) Purification and partial characterization of two glycoproteins in bovine peripheral nerve myelin membrane. Biochim. Biophys. Actu, 455: 806-816. Kitamura, K., Sakamoto, Y., Suzuki, M. and Uyemura, K. (1981) Microhetrogeneity of carbohydrate in PO protein from bovine peripheral nerve myelin. In: T. Yamakawa, T. Osawa and S. Handa (Eds.), Glycoconjugates. Japan Scientic Society Press, pp. 273-274. Miura, M., Asou, H., Kobayashi, M. and Uyemura, K. (1992) Functional expression of a full-length cDNA coding for rat neural cell adhesion molecule L1 mediates homophilic intercellular adhesion and migration of cerebellar neurons. J . Biol. Chem., 267: 1075210758. Miura, M., Kobayashi, M., Asou, H. and Uyemura, K.(1991) Molecular cloning of cDNA encoding of rat neural cell adhesion molecule L1. FEBS Lett., 289: 91-95. Patel, P.I., Roa, B.B., Welcher, A.A., Schoener-Scott, R., Trask, B.J., Pentao, L., Snipes, G.J., Garcia, C.A., Francke, U., Shooter, E.M., Lupski, J.R. and Suter, U. (1992) The gene for the peripheral myelin protein PMP22 is a candidate for Charcot-Marie-Tooth disease type 1A. Nature Genet., 1: 159-165.
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Suter, U., Welcher, A.A. and Snipes, J. (1993) Progress in the molecular understanding of hereditary peripheral neuropathies reveals new insights into the biology of the peripheral nervous system. Trends Neurosci., 16: 50-56. Takahashi, E., Takeda, O., Himoro, M., Nanao, K., Takada, G., and Hayasaka, K. (1992) Localization of PMP22 gene (candidate gene for the Charcot-Marie-Tooth disease 1A) to band 1 7 ~ 1 1 . 2by direct r-banding fluorescence in situ hybridization. Jpn. J. Human Genet., 37: 303-306. Takeda, Y., Asou, H., Miura, M., Kobayashi, M. and Uyeniura, K. (1993) Evidence for the cell-type dependent alternative splicing of cell adhesion molecule L1 in mammals. Bull. Jpn. Neurochem. Soc., 32: 560-561. Talcei, K. and Uyemura, K. (1993) Expression of PO-like glycoprotein in central nervous system myelin of the amphibians. Comp. Biochem. Physiol., 106B: 873-882. Talcei, K., Kitarnura, K., Banno, K. and Uyemura, K. (1993) Major glycoproteins in carp CNS myelin: homology to protein zero with HNK-l/L2 carbohydrate etitope. Neirrochein. Iiit., 23: 239-248. Uyemura, K. (1993) Functional glycoproteins expressed in Schwann cell membrane. Neurosci. Res., 16: 9-13. Uyemura, K., Suzuki, M., Sakamoto, Y. and Tanaka, S. (1987) Structure of PO protein: Homology to immunoglobulin superfamily. Biomed. Res., 8: 353-357.
Uyemura, K., Kitamura, K. and Miura, M. (1992) Structure and molecular biology of PO protein. In: R.E. Martenson (Ed.), Myelin: Biology and Chemistry. CRC Press, Boca Raton, FL, pp. 481-508. Uyemura, K., Takeda, Y., Asou, H. and Hayasaka, K. (1994) Neural cell adhesion proteins and neurological diseases. J.Biochem., 116: 1187-1192. Waehneldt, T.V. and Malotka, J. (1989) Presence of proteolipid protein in coelacanth brain myelin demonstrates tetrapod affinities and questions a chondrichthyan. J. Neurochem., 52: 1941-1943. Welcher, A.A., Suter, U., De Leon, M., Snipes, G.J. and Shooter, E.M. (1991) A myelin protein is encoded by a homologue of a growth arrest specific gene. Proc. Natl. Acad. Sci. USA, 88: 7195-7199. Yazaki, T., Miura, M., Asou, H., Toya, S. and Uyemura, K. (1991) Myelin PO protein expressed in C6 cells promotes neurite outgrowth. Biomed. Res., 12: 223-230. Yazaki, T., Miura, M., Asou, H., Kitamura, K., Toya, S. and Uyernura, K. (1992) Glycopeptide of PO protein inhibits hornophilic cell adhesion: Competition assay with transformant 8 and peptides. FEBS Lett., 307: 361366. Yazaki, T., Miura, M., Asou, H., Toya, S. and Uyemura, K. (1994) Peripheral myelin PO protein mediates neurite outgrowth of cortical neurons in vitro and axonal regeneration in vivo. Neurosci. Lett., 176: 13-16.