Structure and function of myelin protein genes.

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MYELIN PROTEIN GENES Katsuhiko Mikoshiba, *t Hideyuki Okano, * Taka-aki Tamura, t and Kazuhiro Ikenaka* * Institute for Protein Research, Osaka University, Suita, Osaka 565, Japan; t Division of Behavior and Neurobiology, Department of Biological Regulation, National Institute for Basic Biology, Okazaki, Aichi 444, Japan

KEY

WORDS:

myelin basic protein, myelin proteolipid protein, myelin-associ ated glycoprotein, mutant mice, gene expression

Myelin is a unique cellular organelle produced in both the central (eNS) and peripheral (PNS) nervous systems. It plays an important role in saltatory conduction along the axon in both the eNS and PNS. eNS oligodendrocytes send out processes to recognize the axons nearby and wrap them to form compact lamellae. In the PNS, Schwann cells move around to form compact myelin lamellae. Myelination includes the process of neuron-glia cell recognition, molecular assembly of myelin components, and compaction of membranes to form lamellar structures. The expression of several myelin proteins is highly specific to the nervous system, and, therefore, the genes coding these proteins provide good models by which to analyze gene expression in the nervous system. We focus here on the structure and function of genes encoding myelin basic protein (MBP), proteolipid protein (PLP), and myelin-associated glycoprotein (MAG), which are abundantly expressed in eNS myelin. MYELIN BASIC PROTEIN

Myelin basic protein is one of the major eNS myelin proteins, comprising 30% of all myelin proteins (Lees & Brostoff 1 984). MBP is a highly cationic membrane-associated protein that interacts with negative charge groups of phospholipids in the myelin membrane. MBP is thought to make myelin 201 0147-006X/9 1/0301-0201$02.00

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MIKOSHIBA ET AL

lamellae compact by fusing the cytoplasmic surfaces of oligodendrocytes into the form of major dense lines (Lees & Brostoff 1984).

Annu. Rev. Neurosci. 1991.14:201-217. Downloaded from www.annualreviews.org by University of Oklahoma - Norman on 07/26/13. For personal use only.

Transcriptional Regulation of the Mouse MBP Gene Mouse MBP is encoded by a single gene that is composed of seven exons and spans over 30 kilobase (kb) (Figure I ) (Takahashi et a11985, deFerra et al 1985, Kimura et al 1985). The transcription of the MBP gene is nervous-tissue specific (oligodendrocytes in CNS and Schwann cells in PNS). Ontogenic studies have demonstrated that MBP gene expression occurs in a stage-specific manner (Zeller et al 1984, Okano et al 1987). Studies in transgenic mice have specified a 1. 3 kb region of the MBP upstream sequence that is necessary and sufficient for nervous tissue specific expression of the MBP gene (Katsuki et al 1988, Kimura et al 1989). Miura et al ( 1989) have determined the nucleotide sequence of this 1. 3 kb upstream region, introduced the 1. 3 kb MBP promoter-lacZ (MBP lacZ) chimeric gene into various cell lines, and defined the various cis elements within this 1. 3 kb region. The TATA box-like sequence (TTCAAA) is at - 34 map position, the CAAT box-like sequence (CACCT) at - 85, and the GC box (CCCGCC) at - 92. Sequences similar to the SV40 enhancer core (Weiher et al 1983) and NFljCTF-binding site (Leegwater et a11986) are found at -648 and - 125, respectively. Deletion studies of the MBP promoter region suggest that the region between - 1 3 18 and -254 contains sequences that repress MBP promoter normal

2 1

shiverer

345 6 I.. 1

7 •

2 1 1 -_II�....; ===============-

--------

myelin deficient 1 2 T

"\

a c�e

TT

2

345 6

7

----�I-�I�------------I.--�I�II�I��--+-��-----+I+I�I��. __--

x

I

Figure

1

10kb

I

Gene organization of the MBP gene in normal, shiverer, and myelin deficient mice.

Boxes with the number on top show exons and their numbers.

Dotted line with an arrowhead

at each side shows where the gene is deleted in shiverer. Arrowheads in myelin deficient MBP gene indicate the recombination points, which had apparently been taken place in mId. Inverted region in the mId MBP gene is illustrated with inverted numbers on the top of exons.


MYELIN GENE EXPRESSION

20 3

activity. DNase I footprint analysis with NG108-15 whole cell extracts revealed a cellular factor(s) that bind(s) to the region between positions - 127 and - 108. This region contains the TGGCA motif that binds to cellular transcription factors and has been named MBTE (myelin basic protein transcription element). Tamura et al (1988a) showed that MBP DNA linear template is efficiently transcribed in HeLa whole cell extracts and that purified NFl from HeLa cells binds to MBTE. Therefore, MBTE is a critical element for efficient MBP gene transcription and plays an important role in tissue-specific transcription of the MBP gene, where nervous tissue-specific NF l s are presumed to be involved (Aoyama et al 1990). A homologue of the NFl-binding motif was also found within the promoter/enhancer region of human papovavirus lC, which is responsible for a progressive multifocal leukoencephalopathy (PML) (Nowock et al 1985). lC virus selectively degenerates oligodendrocytes (Small et al 1986), and its promoter/enhancer function works specifically in the oligo dendrocyte (Kenney et al 1984). In vitro studies showed that the NF1binding motif in the viral enhancer is indispensable for lC virus early gene transcription (Tamura et al 1988b). Cis-elements necessary for tissue-specific MBP gene transcription have been further characterized by a tissue-specific cell-free transcription system (Tamura et al 1989a,b). The proximal region downstream (from -5 3 to +60; core promoter region) can direct preferential transcription in brain nuclear extracts. The MBP promoter may utilize a specific TAT A box factor that offers tissue specificity. The distal promoter region (from - 254 and -54) directs preferential transcription in brain extracts in an orien tation-dependent manner. Cooperative interaction between the distal and proximal promoter regions may be required for higher specificity of MBP gene transcription.

Post-Transcriptional Events and Intracellular Transport Mouse MBP has at least five isoforms [21.5, 18.5, 17(a,b), and 14 kD] encoded by separate mRNAs (Figure 2) (Zeller et al 1984, Takahashi et al 1985, deFerra et al 1985, Kimura et al 1985, Newman et al 1987) produced by alternative splicing. The presence of a sixth form of mouse MBP mRNA, encoding a 20 kD MBP, has been expected from the results of immunoblotting (Roth et a1 1987, Campagnoni & Macklin 1988). MBP mRNAs belonging to a minor population and very low in abundance were identified by eDNA cloning (Newman et al 1988, Kitamura et al 1990). One seemed to utilize novel untranslated exons (exons 0 and l a) located upstream from exon 1. The 2 1.5 and 17 kD MBPs, which contain exon 2, are speculated to play an important role in the early stages of myelin

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MIKOSHIBA ET AL

MBP gene

(32 kb) EXON 1

2

3 45

6

7


Protein 21.5kD 18.5kD

17kD 17kD 14kD Figure 2

Exon usage in five forms of MBP mRNAs. Exons 2, 5 and 6 are alternatively used

to make five forms of MBP mRNAs.

wrapping, since they are relatively richer at this time than at later stages (Carson et al 1983). Kimura et al (1989) showed, however, that 14 kD MBP, which lacks exons 2 and 6, is sufficient for myelination in transgenic mice. It has recently been shown that each oligodendrocyte produces multiple forms of MBP (C. Shiota, K. Ikenaka, K. Mikoshiba, submitted). Thus, more intensive studies will be necessary to clarify the role of each MBP isoform. It has been suggested that MBPs are translated on free ribosomes, and these ribosomes, together with MBP mRNAs, enter the oligodendrocyte processes. Thus, translation of MBP mRNAs seems to occur close to the site at which MBP is assembled (Coleman et al 1982). . Migration of MBP mRNA into the oligodendrocyte process during development was demonstrated by in situ mRNA hybridization (Zeller et al 1985, Trapp et al 1987, Shiota et al 1989).

Molecular and Cellular Biology of Shiverer and Myelin Deficient (mId) Mice Shiverer and myelin deficient (mid) are two autosomal recessive murine mutants deficient in the expression of MBP gene (Doolittle & Schweikart


205


1977, Doolittle et al 1981). They are allelic to each other, and can be mapped to the same region as the MBP gene in the mouse genome (Roach et al 1985, Sidman et al 1985, Okano et al 1988a). They provide good systems by which to study the function and regulation of the MBP gene. SHIVERER CNS myelin in shiverer is completely devoid of MBP (Dupouey et a1 1979, Mikoshiba et al 1980a,b). Chimeric mice studies showed that the shiverer mutation is intrinsic to thc oligodcndrocytc (Mikoshiba et al 198 2a, Inoue et al 1986), the only cell-type expressing MBPs in the CNS. After these initial characterizations, it was demonstrated that a large portion of the MBP gene is deleted in shiverer (Figure 1) (Roach et al 198 3, 1985, Kimura et a11985, 1986, Molineaux et aI 1986). The "shiverer" phenotype and myelination in the CNS of the shiverer mutant were rescued by producing transgenic mice with an MBP minigene (1. 3 kb promoter region plus 14 kD MBP cDNA ; Kimura et a11989) or the whole MBP gene (Readhead et al 1987). Therefore, it was concluded that a deletion in the MBP gene is the only cause of hypomyelination and the "shivering" phenotype of the shiverer mouse. Katsuki et al (1988) suc ceeded in converting the phenotype from the normal to the mutant shiverer type by creating transgenic mice by means of the antisense minigene.

MYELIN DEFICIENT (mid) The myelin deficient (mid) mutant shows symptom similar to the shiverer. In mid, the MBP gcnc was shown to bc duplicatcd (Figure 1) (Akowitz et a11987, Okano et a1 1987, 1988a, Popko et a11987, 1988). An intact copy and another one with certain rearrangements seemed to be present (Akowitz et al 1987). In vitro transcription studies as well as nucleotide sequencing analysis revealed that the promoter region is not responsible for the reduced MBP expression in mid (Okano et al 1988a). Popko et al (1988) demonstrated that a large portion is inverted in the upstream copy and that an intact MBP gene is located downstream in the mid locus (Figure 1). From this genome structure of mid, it was speculated that antisense RNA or transcriptional interference might be responsible for the reduced MBP expression. The antisense RNA corresponding to the invertcd segment was detected by nuclear run-on (Popko et al 1988, Tosic et al 1990) and RNase protection (Okano et al 1988b, 1991). The antisense RNA seemed, from many lines of evidence (Okano et a1 1988b, Roch et al 1986, Tosic et al 1990), to play a more important role in repression than transcriptional interference. The major puzzle remaining in mid is the mosaic expression of the MBP gene. MBP-positive myelin forms patches in the CNS (Mikoshiba et al 1987 , Shen et al 1985, Inoue et al 1988). The amount of MBP expressed in each oligodendrocytc in culture seems to vary considerably (Akowitz et al 1987). The degree of gene expression repressed by transcriptional

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MIKOSHIBA ET AL

interference (Emerman & Termin 1984) or anti-sense RNA is known to vary from cell to cell as a result of environmental factors. This cell-to-cell variation might result in mosaic expression. Another possibility is that somatic genetic alteration of the mid locus caused "revertant" oligo dendrocytes expressing MBP. Further study is required to solve this problem.


MYELIN PROTEOLIPID PROTEIN

Myelin proteolipid protein is one of the most abundant proteins in eNS myelin, constituting 50% of the myelin membrane proteins in the eNS. It is called "proteolipid" because of its unusual property of dissolving in organic solvents (Folch & Lees 1951). There is another less abundant proteolipid, DM20, which shows immunological cross-reactivity with the major proteolipid protein (Lees & Brostoff 1984). Lipids are covalently attached to PLP via an O-ester linkage (Stoffyn & Folch 1971). Because the amino acid and nucleotide sequences of PLP are remarkably well conserved in the mouse, rat, cow, and human (Diehl et a11988, Gardinier et al 1986, Hudson et al 1987, Ikenaka et al 1988, Macklin et al 1987a, Milner et al 1985, Naismith et al 1985, Nave et a11986, Puckett et a11987) and because scvere dysmyelination is observed in animals whose PLP synthesis is affected (Hogan & Greenfield 1984), PLP is thought to play a crucial role in myelination in the eNS, probably by promoting the appo sition of extracellular surfaces of the myelin lamellae. Recently, molecular genetic studies of PLP have made great advances, and many interesting aspects of the synthesis and function of PLP have been reported. We focus on these recent advances in PLP-molecular genetics and the characterization of the X-linked dysmyelinating mutant animals, whose PLP synthesis is affected.

Structure of PLP and DM20-mRNAs Northern blot analysis of rat brain RNA in which labeled PLP cDNA was used as a probe indicated that rat PLP-mRNAs occur as two abundant families of approximately 3.2 and l.6 kb, and a less abundant family of approximately 2.4 kb in length (Milner et al 1985). It has been shown that 3.2 and 1.6 kb, and probably 2.4 kb PLP-mRNAs are produced by alter nate usage of polyadenylation sites (Milner et al 1985) (see Figure 3). The relative abundance of the three PLP-mRNAs seems to differ among species; i.e. the mouse has a much lower level of l.6 kb and higher level of 2.4 kb RNA (Dautigny et a11986, Gardinier et a11986, Hudson et a11987, Milner et al 1985, Nave et al 1986), whereas the human apparently has only the longest 2.8 kb RNA (Puckett et al 1987).



PLP GENE

207

EXON 1

�------�

NORMAL PLP

DM20

JIMPY PLP

DM20 Figure 3

Exon and polyadenylation site usage of the PLP gene in normal andjimpy mutant

mice. An A to G conversion was observed in the conserved "AG" splice acceptor signal of the fifth exon in thejimpy PLP gene. This resulted in deletion of the fifth exon from the PLP and DM20 mRNAs. The polyadenylation sites of the PLP gene are shown by

arrows.

The

bold lines represent the coding regions in PLP or DM20 mRNA.

Morello et al (1986) and Hudson et al (1987) suggested that DM20mRNA is produced by alternative splicing of the PLP-mRNA precursor and that part of PLP-mRNA is deleted to form DM20-mRNA. Nave et al ( l 987b) confirmed this hypothesis by directly cloning and sequencing mouse DM20 cDNA (Figure 3). The three different sized mRNAs are also present in mouse DM20-mRNA and are probably formed by the alternate usage of the same polyadenylation sites as PLP-mRNA (Ikenaka et al 1988).

The PLP Gene Structure The genes for mouse (Ikenaka et a11988, Macklin et al 1987a) and human (Diehl et al 1986) PLP have been isolated and both have been shown to consist of seven exons (Figure 3). The exon-intron junctions of PLP are completely conserved between the mouse and human. Homology between mouse and human PLP untranslated regions (94%) and that upstream of


208

MIKOSHIBA ET AL

the major cap site for at least 100 bases (89% ) is remarkably high. This suggests some very strong functional constraints on these regions that limit the variation allowed in these sequences (Macklin et al 1987a). An interesting aspect of the exon usage is that four of the five hydrophobic domains are encoded in individual exons (Diehl et al 1986). Southern transfer analysis revealed that a single gene encodes for PLP and DM20 (Dautigny et al 1986, Milner et aI1985). The 3' end of the deleted portion in DM20-mRNA corresponds to the junction of exons 3 and 4 of PLP mRNA, whereas the 5' end of the deletion starts in the middle of exon 3 (Figure 3) (Ikenaka et al 1988, Nave et al 1987b). Therefore, alternate usage of the splice donor sites is involved in the formation of PLP and DM20-mRNA. All the polyadenylation sites are present in exon 7 (Figure 3, exon 7). Transcription of rat, mouse, human, and baboon PLP gene has been shown to start from multiple sites by several methods, including primer extension analysis, S l nuclease protection, T4 DNA polymerase primer extension, and RNase mapping (Ikenaka et al 1988, Macklin et a1 1987a, Milner et al 1985). No common sequences were found upstream of each initiation site. Possible "TATA" and "CAAT" boxes were found at posi tions - 26 and - 10, respectively, arranged in a rather odd manner. The unique feature of the sequence of the 5'-flanking region of the PLP gene is that it has four tandemly repeated 11-base-pair sequences [consensus: GGGAGGAG(A/G)AG], which share a high degree of homology (9/11) with a herpes simplex virus repeated sequence (DR2) (Ikenaka et aI 1988). This sequence is believed to be involved in the cleavage and packaging reactions of the virus (Chou & Roizman 1985, Varmuza & Smiley 1985). At present, however, the function of this sequence is not known.

Synthesis of PLP PLP is synthesized in oligodendrocytes and its synthesis increases con comitantly when myelin is actively formed. Synthesis of PLP in Schwann cells was also reported; however, it was not incorporated into the pe ripheral myelin (Puckett et a11987). PLP-mRNA is located only in the cell body of the cultured oligodendrocyte, whereas MBP-mRNA is present over the length of the slender processes as well as in the cell body. Though PLP is present in all parts of the cell in primary culture, as is the case with M BP, PLP-mRNA is located exclusively in the cell body (Shiota et al 1989). Electron-microscopic immunohistochemical study demonstrated that the location of PLP is restricted to the rough endoplasmic reticulum (rER), Golgi apparatus, and apparent Golgi vesicles (Schwob et al 1985). It is clear that PLP is synthesized in the rER in the perikaryon and transported to the myelin sheath.


209

Both PLP- and MBP-mRNAs were detected first in the pons and medulla oblongata, then in the cerebellum, and later in the cerebrum. Thus myelin-specific gene expression proceeds generally in the caudo cranial direction at the mRNA level (Kristensson et al 1986, Milner et al 1985, Shiota et al 1989, Trapp et al 1987).


Expression of the PLP Gene in X-Linked Mutant Mice

Jimpy

The X-linked recessive mutant mouse, jimpy, is characterized by abnormal myelin formation in the CNS, but not in the PNS, and is considered to be an animal model for human Pelizaeus-Merzbacher disease (Hogan & Greenfield 1984). A characteristic feature of the jimpy mouse is severe degeneration of the oligodendrocytes. A 74 bp deletion in jimpy PLP-mRNA was detected by Nave et al (1986) (Figure 3). This deleted sequence was identical to that of the fifth exon of PLP-mRNA, and thus the fifth exon of the PLP gene was not utilized injimpy (Figure 3) (Moriguchi et aI 1987). An A to G conversion at the conserved " AG" residues of the 3'-splice site was found (Macklin et al 1987b, Nave et aI 1987a), which apparently resulted in the fifth exon deletion of PLP-mRNA (Figure 3). Protein encoded by this mRNA would have an altered C-terminal sequence owing to the reading frame shift, and it is expected to be extraordinarily rich in cysteine residues (8/ 36). It must be determined whether this unusual PLP exerts a toxic effect on the oligodendrocytes or the absence of normal PLP is fatal. Recently, the nature of mutations in other X-linked dysmyelinating disorders, including human Pelizaeus-Merzbacher disease and rat myelin-deficient, has been reported (Boison & Stoffel 1989, Gencic et al 1989, Hudson et al 1989). All of them have been revealed to involve a point mutation in the coding region, which resulted in a single amino acid substitution [A -+ C; Thr -+ Pro (Boison & Stoffe1 1989), C -+ T;Pro -+ Ser (Gencic et aI 1989), T -+ C;Trp -+ Arg (Hudson et al 1989)]. These disorders are also accom panied by severe degeneration of the oligodendrocytes, and, therefore, the lack of normal PLP synthesis seems to be the major cause of these X-linked disorders. This abnormality was shown in studies of chimeric mice (Beraducci et al 1981) to be intrinsic to the oligodendrocyte itself. It was later shown, however, that normal astrocyte-conditioned medium enables jimpy oligo dendrocytes to survive and express properties of normal oligodendrocytes (Bartlett et al 1988). Because the oligodendrocyte degenerates before it expresses PLP, it is possible that PLP or a related protein is synthesized in non-oligodendrocyte cells and perhaps it is necessary for oligodendro cytes to maturate.

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MIKOSHIBA ET AL

MYELIN-ASSOCIATED GLYCOPROTEIN


Structure

of MAG

MAG, a heavily glycosylated protein (> 30% carbohydrate) with a molec ular mass of 100 kD (Quarles et al 198 3), constitutes approximately 1% of total myelin protein. Its peptide backbone consists of two polypeptide chains (67 kD and 72 kD in size), as revealed by in vitro translation of brain RNA (Frail & Braun 1984). The two isofonns of MAG were shown to be encoded by two MAG-mRNAs generated by alternative splicing of exon 12 (Lai et a1 1987a, Salzer et aI1987). An in-frame termination codon, UGA, within the 45 nucleotides derived from exon 12, creates a shorter coding region in MAG-mRNA than would be present in its absence. Thus, MAG-mRNA with and without exon 12 encodes 67 kD MAG (S-MAG) and 72 kD MAG (L-MAG), respectively. Exon 2, which is part of the 5' untranslated region, is also alternatively used; however, it does not affect the structure of the protein encoded (Lai et al 1987a). The complete amino acid sequence deduced from the cDNA sequence revealed that MAG has a long extracellular domain, which can be divided into five subdomains with varying degrees of homology to each other. Each domain also has significant identity with the immunoglobulin domain, and, therefore, MAG is a member of the immunoglobulin gene superfamily (Sutcliffe et al 1983, Salzer et al 1987, Lai et al 1987b). It is most closely related to N-CAM, L l , and neural glycoprotein contactin (Milner et al 1990). An "RGD" (Arg-Gly-Asp) sequence is present in the first domain, which is known to play a critical role in attachment to the integrin family (Rouslahti & Piersbacher 1987). MAG does not bind to laminin or fibronectin, however, and its binding to collagen is RGD-independent (Fahrig et aI1987). Thus, whether the "RGD" sequence has any biological significance is still obscure. Eight consensus sites for N-linked glycosylation are found in the N terminal extracellular domain (Lai et aI1987a). The carbohydrate moiety contains the L2/HNK-I epitope, which is shared by a number of cell adhesion molecules, such as N-CAM and L l (Kruse et al 1984), and is believed to be itself a ligand in adhesion. The structure of the C-terminal cytoplasmic domain differs between the two isoforms: 53-amino acid residues of the 92 C-terminal residues of L MAG are replaced by a different sequence of 9 amino acids in S-MAG (Lai et a11987a, Salzer et aI1987). L-MAG contains several potential sites for serine and threonine phosphorylation, and it also contains a tyrosine residue that is surrounded by a sequence highly homologous to the major



211

autophosphorylation site of the epidermal growth factor receptor within this domain (Arquint et a11987, Salzer et a11987, Lai et aI1987a). Edwards et al ( 1988, 1989) demonstrated that only one of the MAG isoforms (L MAG) is phosphorylated both in vivo and in vitro, mainly at serine residues and to a lesser extent at threonine and tyrosine residues. Human MAG eDNA has also been cloned and the amino acid sequence of human MAG deduced (Sato et al 1989, Spagnol et al 1989). The overall homology between rat and human L-MAGs is very high ( 89% in nucleotide sequence and 95% in amino acid sequence), thus suggesting close interdependence between its structure and function. All the general features described above for the rat MAG are conserved in human MAG, except for the presence of another consensus N-glycosylation site (Sato et a1 1989, Spagnol et aI1989).

Gene Organization of MAG The rat MAG gene consists of 13 exons and encompasses about 16 kb (Lai et al 1987a). The genetic locus for MAG has been assigned to chro mosome 7 of the mouse (Barton et al 1987, D'Eustachio et al 1988) and chromosome 19 ( I9q12 -+ q I3.2) of the human (Barton et a11987, Schonk et al 1989, Spagnol et al 1989). The mouse MAG gene is located close to the quiverer (qv) locus (Yoon & Les 1957), a spontaneous mutation characterized in the homozygous state by locomotor instability, quivering, priapism, paralysis, and death at < 5 months; however, no gross alteration in the gene structure or the expression of MAG protein or mRNA has been identified in quiverer mutant mice (Barton et al 1987, D'Eustachio et al 1988).

Regulation of MAG Gene Expression The synthesis of MAG is tightly coupled with myelin synthesis (Quarles 1984). Preference in the expression of the two isoforms of MAG appears to be regulated during postnatal development in the eNS. The mRNA lacking exon 12 (encoding L-MAG) is expressed more abundantly during early development and reaches its highest concentration at approximately three weeks after birth, the peak of myelinogenesis. Thereafter, its level declines to the adult level by day 62. The mRNA containing exon 12 (encoding S-MAG) is a minor form early in development, but it becomes increasingly abundant and is the predominant form in the adult brain (Frail & Braun 1984, Lai et al 1987a, Tropak et al 1988). The mRNA encoding L-MAG is characteristically induced during the remyelinating stage (Fujita et al 1990a). In the PNS, mRNA encoding S-MAG is pre dominant throughout development, reaching peak levels at days 6-10,


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MIKOSHIBA ET AL

whereas mRNA coding for L-MAG is a very minor species (Tropak et al 1988). A defect in this alternative splicing machinery has been found in the quaking mutant mice (Frail & Braun 198 5), whose myelin synthesis is impaired. Only the mRNA containing exon 12 is present in the eNS of the quaking mouse (Fujita et al 1988). In the human, mRNA coding for L-MAG seems to represent the major population even in the adult brain (Sato et al 1989). Alternative splicing of exon 2 seems to be regulated independently of the splicing of exon 12 (Lai et al 1987a, Tropak et al 1988). This is supported by the finding of a new MAG-mRNA without either exon 2 or 12 (Fujita et al 1989).

Function of MAG rmmunocytochemical studies have also demonstrated that MAG is enriched in periaxonal regions of both eNS and PNS (Sternberger et al 1979, Trapp et aI1989). In vitro experiments in which liposomes containing either purified MAG or recombinant MAG were used have clearly shown that both isoforms of MAG are capable of binding to axons and oligo dendrocytes, and the antibody reacting specifically with the extracellular domain of MAG inhibits adhesion (Johnson et al 1989, Poltorak et al 1987). Therefore, MAG is considered to be involved in the recognition and adhesion of oligodendrocytic processes (in the eNS) or Schwann cells (in the PNS) to the neural axons in forming myelin. Other functions of MAG have also been considered. MAG was shown to be present in multivesicular bodies (MVBs), together with periaxonal membranes, in seven-day-old rat eNS. These MAG-enriched MVBs were found in oligodendrocyte perinuclear regions, in processes extending to myelin internodes, and along the myelin internode in outer tongue pro cesses and paranodal loops. They were not found in oligodendrocytes from adult animals or in myelinating Schwann cells, where S-MAG represents the major species. Furthermore, the MAG-enriched MVBs reacted with antibodies specific for L-MAG (Trapp et al 1989). All these data support the proposal that the L-MAG is involved in receptor-mediated endocytosis of components from the periaxonal space of axolemma during the active stages of myelination. MAG was also found to possess neurite outgrowth function (Johnson et al 1989). ACKNOWLEDGMENTS

We are grateful to Drs. Jeff Porter and Kensuke Nakahira for help in preparation of the manuscript, and Drs. Robert Milner and Shuzo Sato for giving us copies of their unpublished manuscripts.


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Central nervous system myelin: structure, function, and pathology.

Atomic resolution view into the structure-function relationships of the human myelin peripheral membrane protein P2.

Posttranscriptional events in the expression of myelin protein genes.

The secondary structure of myelin basic protein extracted by deoxycholate.

The structure and function of protein modules.

Protein structure and function at low temperatures.

The RecA protein: structure and function.

Myelin structure transformed by dimethylsulfoxide.

Retinoic acid-regulated expression of proteolipid protein and myelin-associated glycoprotein genes in C6 glioma cells.

Triiodothyronine has diverse and multiple stimulating effects on expression of the major myelin protein genes.

Protein folding: Structure-function analysis of KDM2A.

Myelin basic protein microheterogeneity in subfractions of rat brain myelin.

Enzymic methylation of myelin basic protein in myelin.

Genetic approaches to protein structure and function: point mutations as modifiers of protein function.

Structure of mouse myelin-associated glycoprotein gene.

Developmental expression of major myelin protein genes in the CNS of X-linked hypomyelinating mutant rumpshaker.

Mammalian protein glycosylation--structure versus function.

Structure and function of G protein coupled receptors.

Layers of structure and function in protein aggregation.

Structure and function of serotonin G protein-coupled receptors.

Structure and Function of the PriC DNA Replication Restart Protein.

Structure and function of the Epstein-Barr virus BZLF1 protein.

Fundamental Characteristics of AAA+ Protein Family Structure and Function.

Solubility as a function of protein structure and solvent components.