Neuron,

Vol. 8, 323-334,

February,

1992, Copyright

0 1992 by Cell Press

Molecular Characterization of the Schwann Cell Myelin Protein, SMP: Structural Similarities Superfamily within the Imtknoglobulin Catherine Dulac,* Michael 6. Tropak,+ Patrizia Cameron-Curry,* Jean Rossier,* Daniel R. Marshak,§ John Roder,+ and Nicole M. Le Douarin* *Institut d’Embryologie du CNRS et du College de France 94736 Nogent-sur-Marne Cedex France +Samuel Lunenfeld Research Institute Mount Sinai Hospital Toronto Canada M5G IX5 +Laboratoire de Physiologie Nerveuse CNRS 91198 Gif-sur-Yvette Cedex France Kold Spring Harbor Laboratory Cold Spring Harbor, New York 11724

Summary The Schwann cell myelin protein (SMP), previously defined in quail and chick by a monoclonal antibody, is in vivo exclusively expressed by myelinating and nonmyelinating Schwann cells and oligodendrocytes. The isolation of the complete nucleotide sequence of SMP is reported here. The predicted polypeptide chain reveals that SMP is a transmembrane molecule of the immunoglobulin superfamily showing sequence similarities with several surface glycoproteins expressed in the nervous and immune systems. In spite of a 43.5% overall sequence identity between rat myelin-associated glycoprotein (MAC) and quail SMP, SMP does not seem to be the avian homolog of MAG, since their expression, regulation, and functions are significantly different. Unusual sequence arrangements shared by SMP, MAC, and two lymphoid antigens suggest the existence of a particular subgroup in the immunoglobulin superfamily. introduction In the peripheral nervous system, glial cells, which have been shown to be derived from the neural crest (Le Douarin, 1982), can be classified into threecategories: the Schwann cells lining the peripheral nerves, the satellite cells associated with neuronal somata in peripheral ganglia, and enteric glia. The process of peripheral glial cell differentiation is a model system in which a cascade of regulatory events directing the emergence of glial cell diversity has been partly identified (Le Douarin et al., 1991; Jessen and Mirsky, 1991). Specific signals arising from axons have been shown to be of outstanding importance in controlling the proliferation of Schwann cell precursors in nerves (Salzer and Bunge, 1980; Ratner et al., 1988; Bunge

et al., 1989) and in directing their differentiation into myelinating or nonmyelinating cells (Monuki et al., 1989, 1990; Morgan et al., 1990; Aguayo et al., 1976; Jessen et al., 1990; see lessen and Mirsky, 1991, for a review). However, recent results concerning earlier steps of glial cell differentiation challenge the view that the entire process of gliogenesis is directed by neurons (seeLeDouarin etal.,1991,forareview).Thisworkwas made possible by a newly characterized monoclonal antibody (MAb) that is specifically directed against avian glial cell membranes and that identifies a M, 75,000-80,000 antigen. This marker has been called Schwann cell myelin protein (SMP), since in addition to being a myelin constituent in both the central and the peripheral nervous systems, it is, in contrast to already identified myelin proteins, also expressed on all external membranes of Schwann cells: those surrounding the cell body of myelinating Schwann cells as well as those constituting the surface of nonmyelinating Schwann cells (Dulac et al., 1988; CameronCurry et al., 1989). In contrast, satellite cells that form a glial capsule around neuronal cell bodies in sensory and autonomic ganglia and enteric glial cells located in the neural plexuses of the gut wall do not express SMP. SMP therefore appears to allow discrimination between myelinating and nonmyelinating Schwann cells and all other peripheral glial cells. SMP is first synthesized at embryonic day 5 (E5) of quail development, 5-6 days before the onset of myelination, and remains expressed by cultured Schwann cells. Therefore, this marker has been useful for the identification of glial precursors present in the neural crest by means of clonal analysis in culture (Dupin et al., 1990). These studies have demonstrated that crest cells committed to a glial fate spontaneously synthesize SMP after several days in culture even in the absence of neurons. Moreover, recent studies of the microenvironmental conditions in which the SMP+ Schwann cell phenotype appears showed that, in contrast to the expression of myelin components directed by neurons, the initiation of SMP synthesis by peripheral glial precursors is a constitutive pathway of differentiation. As for the two categories of SMP- glial cells, i.e., satellite and enteric glial cells, their development and phenotypic stability are due to a constantly exerted inhibitory influence of ganglionic and enteric environments. When withdrawn from the ganglia or the gut wall, both SMP-glial cell types re-acquire theability to synthesize SMP (Dulac and Le Douarin, 1991; Cameron-Curry et al., unpublished data). Since the cellular distribution of SMP, togetherwith the regulatory mechanisms controlling its expression, appeared to be strikingly different from those described for other glial markers and particularly for myelin components, we have undertaken further char-

NellrOn 324

gttqgg*tCcCt*Cggqc*ttg*9*cccccccccc.ucctgcJ*9accccccc cca*tsaggg*9a9*pc*~g*~t~t~c~ccccccatcc CTG

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GTG

CM;

ACG

GTG

Cpc

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52 Am GAG 102

GGA

II Gd?i%

AU

1

2

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141 15 180 28

LLVLTVLLNGTGC A: A P CrrlTcGGTAcG TX CAT TAC ~DYPKBLRPASIG GGT TTA TGG

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219 41 256 54 297 67

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336 80

CACGAGAGTTRGCCGGTAGAGCTXG~TTAGGTGAC HIS?AGRAS?LCD CCG Act GGA cm GAT ACC PTGRDCTLNIARL AGcGAGGMl-rGGcG cl MA S L L L A C I

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CYCGGGGGTTACAATCUTACACCTTC~GAGCACGCC LGGYNQTSlSLRA GAGCTCGATGTGTGGGCGGCG LLDVWAA H

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TTGGGGTCA~CGClTCCGACCCCCTAMGAAGATRA LGSLRIRPRIIDL GGA CGA CGT GTC GGA GGG GTC Act G R R V G t% G V T AGC CTC AGC TTC CM kf CAT GIG GGG SLS?QADVGLDVQ TAT GAG 03 CAG GTT GTG GGT CYG Y E P Q V V G L” GTGGTCGAAGGTXCGATGTTGMCXGGGTTGTGMGCC VVtGSDVKLG

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960 288 999

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1. DNA

ACG

Deduced

Amino

Acid

301 1038 314 1077 327 1116 340 1155 353 1194

cGGGccGAGAGcGMccGGcr

m

and

862 ;;: 275

ITy;cTcTTAXGAATcIcGGGCCCGACGACGGGGGGAGC LLLSNVGPDDGGS TTC AGC I S C AGG AGC H RSLQLRVAVAPRA

492 132 531 !I5 570 158

Sequences

AI~TVr.RGCKVl4A CCC GCC A.TT TAT GM CAT UC GTG ACC

366 ATC

GM

AAIYLDHVTMEMR CCC GCC AU PARPEDCGTYS GCC GAG MC ACN~HGASSTS~ ATC XC GTC I S V

CCC

GM

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GGC

GGC

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cu:

GCC

AGC

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ACC

GAG E

TM: Y

CCC P

CCC P

l7G L

GTC V

cn: L

CCC P

HVNSIPDSSLVFG CT’2 CCC ACC I. P T

CGG R

UGACGGTCTCGGACGGCCATAGG Q T V S D CCl’ CCC GGT TCC GAC

y CAT ‘PIT ACG GCG GCC ‘JITAAPPCSDGSI ACGccCATA~A~c1c~ccCccPTTAcU~GCcG TGILTLRGPLLPS crGcIcmm GcGGcAcaAAccGGcAcGuAcc LLVLCAARNRBGT ACC GCC CGG CAG H cGcl7ccAccAccccGGGGGcm TARQLR?HHPGGL GTTTGGGCCMGGTGGGTCCGGEGGTGCC~CGTG@?!

G GGG

ATG

CCC

1233

TEc

GTG

379 1272

GCC A

XC S

” TCC

R ATA

1350 416

444 1467 457 1506 470 !545 483 1564 496 1623 509 1662

GnuG~t~~n:cnccc~~~u;cccc~ccn:~c

,779

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GAG

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R

v

MC

TCA

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GGT

E

G

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CCC

GM

561 1818 574 1857

GM

GAC

$7 1896 600 1935 613 1979

ctccq,xcaccgcccc.qctg

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627 2031

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2551 2603 2655

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**

2707 2715

of SMP

Two hydrophobic regions corresponding to the signal sequence (amino acids I-17) and to the predicted transmembrane domain (506533) are underlined by a thick bar. Thinner underlining represents amino acid sequences determined by Edmann degradation of intact SMP and of proteolytic fragments. Underlining arrows beneath the sequences of the amino-terminal portion of SMP and of peptide 1 represent oligonucleotide primers used to isolate the 69 and 400 bp DNA probes. Sites of potential N-glycosylation are marked by inverted triangles. The RCD motif is boxed. The IS cysteines of the sequence are circled.

acterization of the structure of SMP. In this paper we report the complete nucleotide sequence of SMP as determined from cDNA clones. The predicted polypeptide chain suggests that SMP is a transmembrane molecule belonging to the immunoglobulin superfamily of proteins. SMP is also compared with other immunoglobulin-like glycoproteins at both the molecular and the functional levels. Results Isolation of cDNA Clones Encoding SMP An anti-SMP MAb, obtained after immunizing mice with quail nerve glycoproteins (Dulac et al., 1988), was used to immunopurify the corresponding antigen from neural tissues as described in Experimental Pro-

cedures. lmmunopurified SMP was submitted to automated, repetitive Edmann degradation in order to determine its amino-terminal sequence. Additional immunopurified material was treated with Asp-Nendopeptidase or with trypsin, and the resulting fragments were purified by high-pressure liquid chromatography (HPLC). The amino-terminal sequences of three of these purified fragments were determined. The amino-terminal sequences of the internal peptides and of intact SMP were all found in the complete sequence deduced from the cDNA clone, as shown in Figure 1. The peptide sequences were used in our strategy to obtain a SMP cDNA clone. A 69 bp probe was first isolated by polymerase chain reaction (PCR) on El5 quail spinal cord poly(A)’ RNA using two degenerate

SMP, a Schwann 325

Cell-Specific,

Ig-like

Molecule

2

Figure

2. The Expression

of SMP-like

Antigen

on CDMBSMPCTransfected

Anti-SMP immunoreactivity of CDMBSMPCtransfected analysis with the anti-SMP MAb of membrane proteins compared with immunopurified SMP (lane 3). Bar, 50 pm.

primers (I and III; see Experimental Procedures) based on the aminoand carboxy-terminal portions of the sequence of peptide 1. The predicted amino acid seq uence of the 69 bp DNA probe corresponded exactly to the sequence of peptide 1. A 400 bp DNA fragment was then obtained by PCR using the nondegenerate oligonucleotide 3, which is part of the 69 bp probe, and the degenerate primer N, based on the aminoterminal sequence of SMP. The predicted amino acid sequence of the 400 bp DNA contained the sequences of the amino-terminal portion of SMP and of peptides 1 and 2. The 400 bp probe was then used to screen a sizeselected cDNA library from El5 quail spinal cord

1 -

2 -

3

4

5

28s

18s

Figure

Cells nontransfected COS-7 cells (B). (C) Western (lane 1) and nontransfected (lane 2) COS-7

blot cells,

mRNA cloned into the CDM8 vector. The primary screening of 3 x IO5 transformed bacteria resulted in many positive clones corresponding to a frequency of about 1 per 1000. The longest cDNAs were identified by Southern blot analysis from 60 of these clones, using the 400 bp probe and the oligonucleotide corresponding to the amino-terminal sequence. The restriction map of clone #4 corresponded to the longest cDNA clone that was also in the correct orientation for expression in COS-7 cells. Plasmid CDMSSMP4 was used to transfect COS-7 cells. This led to a transient anti-SMP immunoreactivity at the surface of a subpopulation of transfected COS-7 cells (Figures 2A and 2B). Analysis by Western blot of the protein content of transfected cells further demonstrated the identity of the SMP antigen and the protein encoded in plasmid CDMSSMP4 (Figure 2C). The identity of the cDNA clone was further confirmed by Northern blot and in situ hybridization. The 32P-labeled 400 bp probe selectively hybridized to one band at 3 kb present in quail brain, spinal cord, and nerve RNAs, but not in thymus or liver (Figure 3). In situ hybridization experiments showed a strong hybridization signal detected in nerve, brain, and spinal cord white matter from adult and embryonic quails. Gray matter, cartilage, meninges, and surrounding nonneural tissues showed no signal over background (Figure 4). These results are consistent with the location and developmental pattern previously obtained by immunocytochemistry using the anti-SMP MAb.

-

3. Northern

COS-7

COS-7 cells (A) and of control extracted from transfected

3

Blot Analysis

Using

the 400 bp DNA

Probe

A 3 kb transcript is detected in quail adult nerve (lane 1) and spinal cord (lane 2) and in El5 spinal cord (lane3), but not in adult liver (lane 4) and thymus (lane 5).

SMP Sequence Analysis The SMP cDNA predicts an open reading frame that encodes a 621 amino acid polypeptide. The predicted ATG start codon at nucleotide position 96 is flanked by the consensus sequence for eukaryotic translational initiation sites (Kozak, 1984). The open reading frame

Neuron 326

Table 1. Sequence Molecules

Pre-OB-CAM Chicken N-CAM Ll Rat TAG-1 MAC

Figure 4. In Situ Hybridization on a Cryostat Section of a El0 Quail Embryo with a 32P-Labeled SMP DNA Probe A strong hybridization signal is detected in white matter of the spinal cord and in spinal nerves.The regions of transcript expression match well the expression pattern of the SMP antigen as detected by immunocytochemistry. Bar, 500 pm.

G

P

hfC

B

Immunoglobulin-like

Residues of Proteins

Residues of SMP

187-320 301-506 33-106 7-133 l-626 19-140 141-240 241-326 327-414 415-509 510-540 541-626

201-331 133-331 238-313 206-323 l-620 17-139 140-239 240-325 326-413 414-509 410-540 541-621

% Identity 27.1 27.7 28.2 28.1 43.5 61.4 44.0 36.0 41.4 38.9 80.0 18.7

Sequence Alignment with Immunoglobulin-like Molecules A search of the NBRF data base showed that SMP is most similar to members of the immunoglobulin superfamily expressed in the nervous system, particularly rat myelin-associated glycoprotein (MAC), rat TAG-l, mouse Ll, OB-CAM, and chicken N-CAM (Table 1; Arquint et al., 1987; Lai et al., 1987; Salzer et al., 1987;Furleyetal.,1990;Moosetal.,1988;Cunningham et al., 1987). Thegreatestdegreeof similaritywasfound between the SMP and rat MAG amino acid sequences. Alignment of SMP and MAC sequences is shown in Figure 6. Sequences of the recently identified antigens of

h L h

hWp

C

with

2, 3,4, and 5 most closely match the definition of the immunoglobulin-likedomainsbelongingtotheC2set (Williams and Barclay, 1988; Hunkapiller and Hood, 1989). Attempts to identify alternatively spliced forms ot SMP by examining other cDNAs that hybridized with the 400 probe were unsuccessful. Moreover, PCR analysis of spinal cord and nerve transcripts purified from adult and embryonic quails revealed the absence of isoforms of the cytoplasmic domain.

ends at position 1957 and is followed by 0.74 kb of untranslated DNA. A potential polyadenylation signal was found at position 2664, followed by a poly(A) tail. The hydrophobicity profile of the translated cDNA sequence (Kyte and Doolittle, 1982) indicates two hydrophobic regions (data not shown). The first region (amino acids I-17) corresponds to the signal peptide preceding the experimentally determined amino-terminal sequence of SMP. The second hydrophobic region (amino acids 506-533), flanked by consensus basic amino acids (Sabatini et al., 1982), is predicted to form a transmembrane domain. The presumed extracellular portion of SMP contains five sites of potential N-glycosylation (amino acids 222, 314, 331, 405, and 449), matching the consensus sequence NXTIS. One RGD motif was found at residues 119-121. By manual alignment based on cysteine position, the extracellular domain of SMP was found to consist of five repeating units of about 100 amino acids each (Figure 5). These homology units fold up to form immunoglobulin-like domains, as suggested by the intercysteine spacing of about 60 amino acids and conserved residues flanking the cysteines. Domains

Cons

Similarities

D

E

OsG

Y C

N

F

Figure 5. Domain Structure of the Extracellular Region of SMP Five homology units were aligned based on the position of the cysteines. Identical amino acids in the different domains are boxed. The indicated positions of the !3 strands B, C, D, E, and F are based on the location of p strands in immunoglobulin constant domains and on the positions of alternating conserved hydrophobic residues. lmmunoglobulin domains 3 and 4 contain the consensus sequences GXXhXfXCX7W and DXGXYXC, typical of immunoglobulin-like domains belonging to the C2 set. Domains 1, 2, and 5 match the first consensus sequence. However, domain 1 does not appear to contain the second consensus site, and residues of domain 2 and 5 weakly match it. Therefore, aligned domain 1 does not appear to belong to the C2 set, and domains 2 and 5 are likely to be weak members of the C2 set. f = aliphatic amino acids: L, I, V; h = hydrophobic amino acids: L, I, V, M, Y, F; p = polar amino acids: K, R, H, D, E, Q, N, T, S; s = small amino acids: A, G, S, T, V, N, D.

SMP, a Schwann

Cell-Specific,

@-like

Molecule

327

A Smpdl Magdl Cd22dl Cd33dl

5

B

C

-~

c”---

C’

D

E

F

G

Smpd2 Hagd2 Cd22d2 Cd33d2

B Smpta Magtm

Smpcyt Haqcyt

Figure

------:GAGSPEVT -NVTESPSFSAGDNP

6. Comparison

PHACPGGDPD~P~~~RG~ERUA~KtGSGAP~EVT~TSH 8 VESE 'LGSERRLLGL EPPELDLSVSHSDLGK T LVSPEFRISGA

of Quail

SMP Domains

with

Rat MAC

521 626

and

Human

CD33

and CD22

Identical amino acids found at the same position are boxed in black when the identity concerns 3 or 4 molecules and in gray when it concerns only 2 molecules. (A) Alignment of domains 1 and 2 of SMP, rat MAC, and human CD33 and CD22. Sequences were aligned manually based on the positions of the cysteines in each domain, using the alignment program MALIGNED. Positions of the B strands in domain 1 are based in part on the assignments of Williams et al. (1989) for domain 1 of MAC. Important features to note are the presence of an extra cysteine in the amino-terminal portions of domains 1 and 2 and the absence of cysteine in strand F adjacent to the consensus sequence DsGxYx. The RCD sequence in B strand F of domain 1 of SMP and MAC is similar to the RME sequence found at the same position in CD22 and CD33. (B) shows the close similarity of the transmembrane domains of MAC and SMP. This is in contrast to the cytoplasmic domains, where only the carboxy-terminal portions display significant sequence identity.

the human lymphoid system CD22 and CD33 are also shown (Stamenkovic and Seed, 1990; Wilson et al., 1991; Simmons and Seed, 1988); these are also very similar to MAC. This alignment allowed the determination of the percentage of identical amino acids between domains of SMP and the corresponding domains of MAC, CD33, and CD22 (Table 1). It is significant that the immunoglobulin superfamily consensus sequence of B strand F (DXGXY), which is normally found in the C2 set pattern before the carboxy-terminal cysteine (see Figure 6A; Figure 5), appears after this cysteine in SMP, MAC, CD22, and CD33. As aconsequence of this unusual arrangement, Williams et al. (1989) suggested that the first domain of MAC belong to the V set of immunoglobulin-like domains. The nine anti-parallel B strands proposed by Williams to constitute the V-like fold of the first MAC domain are also represented in Figure 6. In addition, the first and the second domains of SMP, MAC, CD22, and CD33 all contain an extracysteine near the amino-terminal cysteine of the immunoglobulin domain. It has been suggested for MAC (Pedraza et al., 1990) that these 2 extra cysteines of the first and the second domains form an intramolecular disulfide bridge between domains 1 and 2. Alignment of the transmembrane domains of SMP and MAC (Figure 6B) shows that this domain is very

similar in the two proteins (80%). In contrast, the cytoplasmic regions of SMP and MAC display very little similarity (18.7%), with the exception of the 8 terminal amino acids, which are identical. The pattern YXEXbXb (b = basic residue) is also found in CD22 and CD33 (data not shown). Analysis of conservative substitutions shared between SMP and MAC is in agreement with the results reported above, i.e., the relatively low level of homology between the cytoplasmic domains of SMP and MAC (40%) and the high homology of the first immunoglobulin-like domains of the two molecules (80%). At the DNA level, we saw no significant homology between the 3’ and 5’ noncoding regions of rat MAG and quail SMP. Moreover, when the 5’ noncoding region of SMP was used to search GenBank, we did not identify MAC or any other members of the immunoglobulin superfamily. Liposomes

Assay

To determine whether SM P displays adhesive properties to axonal or glial membranes, as has been shown for the other glial cell surface immunoglobulin-like proteins MAC, Po, and Ll (Johnson et al., 1989; Sadoul et al., 1990; Filbin et al., 1990; Crumet et al., 1984), we used thepreviouslydescribed liposome bindingassay (Johnson et al., 1989). lmmunopurified SMP from

NC3JKJll 328

Figure

7. Liposome

Binding

Analysis

The assay was performed with liposomes containing rat MAC (A), human glycophorin (D-C), or SMP (B, C, H, and I) on E9 quail DRG cells after 3 days in culture (A, F-l) and on hybridoma cells producing anti-SMP MAb (B-E). Rat MAC liposomes bind to the cell surface of cultured quail neurons, whereas SMP and glycophorin liposomes do not. The binding of SMP liposomes to the surface of the hybridoma cells allows verification that the extracellular portion of SMP is found at the surface of the liposome vesicles. Bars, 50 urn (A, F-l); 25 urn (B-E).

adult quail brain was incorporated into fluoresceinfilled liposomes. Rat MAG and human glycophorin were in parallel incorporated into similar liposomes as positive and negative controls, respectively. SMP liposomes were observed to bind to the surface of hybridoma cells producing the anti-SMP MAb (Figure 7). This result demonstrated that the SMP ex-

ternal region is on the outside surface of the liposomes and is folded properly, within the limits of the recognition of the antibody, since the anti-SMP MAb recognized an extracellular and conformational epitope (Dulac et al., 1988). Liposomes were incubated with 15-day-old cultures of fetal rat dorsal root ganglion (DRG) cells and with

SMP, a Schwann 329

Cell-Specffic,

Ig-lfke

Molecule

3

Figure

8. Western

Blot Analysis

The SDS-polyacrylamide Western blot technique was applied to SMP (lane 1) and MAC (lanes 2 and 3) immunopurified from adult chicken spinal cord. Chicken MAC is identified with a MAb recognizing rat MAG and cross-reacting in avian species (lane 3, arrow), whereas the anti-SMP MAb clearly distinguishes chicken SMP (lane I), but does not react with chicken MAC (lane 2, arrow).

3-day-old ganglion

cultures of El5 cells. As shown

quail DRG in Figure

and sympathetic 7, rat MAC lipo-

somes bound strongly to neurites and neuronal cell bodies in all three types of cultures tested. This adhesion was totally inhibited by preincubating MAC liposomes with Fab fragments or ascitic fluid of the antiMAC MAb 513, but was unaffected by preincubation with anti-SMP MAb. However, no binding to any DRG or sympathetic cell was ever observed with SMP or glycophorin liposomes (Figure 7).

Different lmmunoreactivity Chicken MAC

of Chicken

SMP and

Chicken MAC equivalent was immunopurified with a MAb recognizing rat MAC (Poltorak et al., 1987). Western blot analysis (Figure 8) showed that the antiSMP MAb recognizes immunopurified chicken SMP, whereas no reactivity was ever detected against the immunopurified chicken MAC. Similar results were obtained when three different batches of chicken MAC were tested, the immunoreaction being performed with two MAbs directed against distinct epitopes of the SMP protein core (data not shown).

Discussion SMP is a surface glycoprotein specifically expressed by oligodendrocytes in the CNS and by Schwann cells enveloping myelinated and nonmyelinated peripheral nerve fibers (Dulac et al., 1988; Cameron-Curry et al., 1989). In this study we have predicted the amino acid sequence of SMP after isolation of the corresponding cDNA. The identity between the SMP antigen and the cDNA-encoded protein was validated by several criteria. First, amino acid microsequences determined by Edmann degradation were included in the predicted SMP open reading frame. Second, transfected COS-7 cells expressed anti-SMP-immunoreactive material at their surface. Finally, the tissue specificity of SMP mRNA expression coincided well with the distribution of the anti-SMP immunoreactivity.

The amino acid sequence of SMP predicts an integral membrane protein. The first 20 codons of the open reading frame encode a signal peptide preceding the amino terminus of the mature protein. Cysteine-containing repeats found in the extracellular part of the molecule exhibit a sequence pattern characteristic of the C2 set of immunoglobulin-like domains (Williams and Barclay, 1988), with typical consensus residues flanking 2 cysteines spaced by 50-60 amino acids. Moreover, SMP shares significant sequence homology with several molecules of the C2 subgroup of the immunoglobulin superfamily, such as Ll, TAG-l, N-CAM, and OB-CAM (Furley et al., 1990; Moos et al., 1988; Cunningham et al., 1987; Schofield et al., 1989). The most important similarity has been found between SMP and a myelin constituent, MAC, whose sequence has been identified in rat, mouse, and human (Arquint et al., 1987; Lai et al., 1987; Salzer et al., 1987; Spagnol et al., 1989; Fujita et al., 1989). The overall sequence identity is 43.5% and appears to be heterogeneously distributed along thewhole molecule, ranging from 61% and 75% for the first immunoglobulin-like domain and the transmembrane domain, to 36%-42% for the four other immunoglobulin-like domains, to only 18.5% for the cytoplasmic part. The latter shares only 8 consecutive identical residues between SMP and MAC at the carboxy-terminal end of the molecule. The fact that the molecules of the immunoglobulin superfamily share striking sequence homology has been interpreted on the assumption that they are derived from common ancestors. Therefore, the 43.5% overall sequence quail SMP can

identity found between rat MAG and be considered in one of the following

ways: either SMP is the quail SMP and MAG are encoded common, recent evolutionary

homolog by distinct ancestor.

of rat MAG, or genes with a

Owing to the considerable differences in the expression patterns and functional properties of SMP and MAG, the first proposition can be ruled out. This view is further substantiated by the fact that there are several examples of presumed avian and mammalian homologs belonging to the immunoglobulin superfamily that are very highly conserved, with more than 70% sequence identity. For example, mouse F3 and avian Flllcontactin are 78% similar, mouse N-CAM and chicken N-CAM are 81% similar, and chicken and bovine PO are 75% identical. In addition, since the sequence of MAG showed a very high degree of conservation, with 95% and 98% identical amino acids between rat MAG and human and mouse MAG, respectively (Fujita et al., 1989; Spagnol et al., 1989), it may also be expected that mammalian MAC and avian MAG are equally similar. In this view, SMP and MAG (with 43% sequence identity) would be different, although phylogenetically related, molecules. This kind of relationship has already been described for chicken contactin/FlI/mouse F3 and rat TAG-l, which are clearly distinct molecules and are also 50% identical. In the same way, the unexpected low level of se-

Neuron 330

quence identity (40%) found between chicken NgCAM and mouse Ll sequences has led to a reevaluation of the hypothesis that they are homologous counterparts (Burgoon et al., 1991). One can therefore put forward the hypothesis that avian SMP and mammalian MAG are encoded by distinct genes subjected to different developmental controls. Preliminary results showed the existence of two distinct proteins in chicken (see Results), as well as two distinct transcripts (unpublished data). During development, MAG synthesis has been shown to start when the myelination process is initiated (Martini and Schachner, 1986), whereas SMP appears at the surface of Schwann cells 5-6 days before the onset of myelin formation. Moreover, SMP is expressed in vivo by both myelinating and nonmyelinating Schwann cells, whereas MAC is synthesized only by myelinating Schwann cells. SMP is constitutively synthesized by cultured Schwann cells, and it is spontaneously expressed on the surface of a neural crest-derived cell subpopulation after 8 days in culture. In contrast, direct contact between axonal and Schwann cell membranes is required for MAC synthesis, as well as for the expression of all the recognized myelin proteins (Brockes et al., 1981; Mirsky et al., 1980; Gupta et al., 1990). Therefore, MAC appears as a typical myelinassociated component, whereas SMP is an early and a constitutive glial cell marker that does not appear to be specifically associated with myelination. The idea that MAG and SMP are distinct molecules is further supported by functional studies. Data have already suggested that MAG plays an important role in the specific adhesion between myelinating cells and axons (Johnson et al., 1989; Sadoul et al., 1990; Poltorak et al., 1987). We have shown here that rat MAG liposomes adhere to quail neurons, indicating that MAG and the MAG receptor are likelyto be highly conserved among birds and mammals. However, under the same conditions, SMP liposomes do not display the same properties. The absence of binding of SMP liposomes to neurites does not constitute the absolute proof that SMP is not a cell adhesion molecule, but it does demonstrate that SMP and MAG are functionally distinct. Molecules of the immunoglobulin superfamily play a role in a great variety of signaling processes, such as antigen recognition, membrane adhesion, and signal transduction, and serve as receptors for growth factors and cytokines (Williams and Barclay, 1988). Moreover, some molecules containing certain structural units have been shown to bind to several different ligands (Diamond et al., 1991). In particular, binding sites for heparin have been experimentally demonstrated in N-CAM and MAG (Cole and Glaser, 1986; Fahrig et al., 1987; Cole et al., 1986). These binding sites might be important during the process of glial cell development, since it has been shown that heparan sulfate proteoglycans and some other components of the extracellular matrix play a crucial role in controlling glial cell proliferation and differentiation

(Ratner et al., 1988; Bunge et al., 1989). Therefore it is possible that SMP may have functions other than cellcell adhesion. The carbohydrate moiety of some immunoglobulin-like molecules has also been implicated in their functional properties (Ktinemund et al., 1988; Riopelle et al., 1986; Rutishauser et al., 1988; Diamond et al., 1991). It is therefore interesting to note that among the five N-glycosylation consensus sites found in the extracellular portion of the SMP sequence, one of the carbohydrate determinant has already been identified by MAb 4B3 (Cameron-Curry et al., 1991). This epitope appears to be strictly specific to glial cell membranesand ispresentonasubpopulationof SMP as well as on other unrelated molecules. The transmembrane domains of SMP and MAG are strikingly similar, which may attest to their structural and functional importance in the Schwann cell membrane. It is particularly interesting that the cysteine which has been demonstrated to be palmitylated in MAG (Pedraza et al., 1990) is conserved in SMP. A similarly positioned cysteine is also found in CD22 (Stamenkovic and Seed, 1990). No potential tyrosine, serine, or threonine phosphorylation sites or actin-binding sites were found in the cytoplasmic domain of SMP. Although overall there is no significant homology between the sequences of the SMP and MAG cytoplasmic domains, the 8 carboxy-terminal amino acids are identical. Furthermore,asimilarsequenceconsistingofthepattern YXERIKXHIQIK is also found at the terminal ends of CD22 and CD33 and of CEA (Stamenkovic and Seed, 1990; Wilson et al., 1991; Simmons and Seed, 1988; Barnettetal., 1989)Thismayrepresentasofaruniden tified consensus sequence whose functional significance remains to be determined. Membrane interactions have been shown to perturb the Cal+ level and inositol phosphate metabolism (Schuch et al., 1989), and developmentally regulated phosphorylation has been demonstrated on some isoforms of MAC and N-CAM (Sorkin et al., 1984; Edwards et al., 1988). Moreover, cytoskeleton binding sites have been demonstrated in one N-CAM isoform (Pollerberg et al., 1987) and in contactin (Ranscht, 1988). In contrast to several other members of the immunoglobulin superfamily, such as MAC and N-CAM, an alternative cytoplasmic domain of SMP does not exist. An RGD tripeptide was found in the sequence of the first immunoglobulin-like domain. This motif has been implicated in interactions between integrins and extracellular matrix glycoproteins (Ruoslahti and Pierschbacher, 1986,1987), and it has been shown to play a role during the myelination process (Cardwell and Rome, 1988). However, it is not absolutely clear whether the RGD motif is always implicated in adhesion phenomena. For example, biochemical (Pedraza et al., 1990) and modeling studies (Williams et al., 1989) on MAG have demonstrated that the RGD sequence is sequestered within the interior of the protein and is therefore unavailable for interactions. Moreover,

SMP, a Schwann 331

Cell-Specific,

Ig-like

Molecule

several molecules such as contactin and its homologs Fll and F3, or even I-CAM, that bind to the integrin LFAI (Simmons et al., 1988) do not possess an RCD sequence.BecausethefirstdomainsofSMPandMAG are so similar, it is unlikely that the RGD motif plays a classic role in adhesion for either molecule. Although RGD in SMP and MAG is unlikely to interact with an integrin, it most likely plays an important structural role, as does a similar sequence, RME, found in CD22 and CD33 (Williams et al., 1989). It has been proposed that the first domain of MAC belongs to the V set of immunoglobulin-like domains and that the disulfide bond which normally exists between the two p sheets is in fact found between the two p strands B and E of the same p sheet. The conserved residues found in the sequencesof MAG, SMP, CD22, and CD33 suggest a similar folding. In addition to this similarity, all four molecules-SMP, MAG, CD22, and CD33-contain the extra cysteines found near the amino-terminal cysteine of the first and the second immunoglobulin domains, which were shown in MAC to form an intramolecular disulfide bond. CD22, CD33, SMP, and MAG are the only known members of the immunoglobulin superfamily that contain the cysteines forming these two unusual disulfide bridges. Therefore, these four members may form a distinct subgroup of the immunoglobulin superfamily. The arrangement of the first two domains of these molecules may represent a novel structural motif with important developmental and functional consequences. Experimental

Procedures

Protein Purification and Microsequencing TheSMPantigen was immunopurified using batch immunochromatography. Adult quail spinal cords or sciatic nerves and brachial plexuses were homogenized in phosphate-buffered saline (PBS) plus 0.5% Nonidet P-40 (NP-40). Tissue homogenates were first applied to Sepharose 4B (Pharmacia) coupled with normal mouse IgG (Nordic) in order to reduce the nonspecific adsorption to the Sepharose and then transferred to Sepharose 48 coupled with anti-SMP IgG. After extensive washing of the Sepharose with PBS, 0.5% NP-40, the bound antigen was eluted with 0.2 M glycine at pH 2.5 and then with 6 M guanidine at pH 3. The eluate was neutralized, dialyzed, and lyophilized. After SDSpolyacrylamide gel electrophoresis under nonreducing conditions, the major protein band of M, 75,000-80,000 was electroeluted. lmmunopurified SMP was treated with Asp-Nendopeptidase or trypsin (Boehringer), and the fragments were separated on a C8 column by HPLC. The native protein as well as three HPLC-purified peptides were subjected to automated, repetitive Edman degradation. Thefollowingsequenceswereobtained:amino-terminal protein sequence, APWAAWMPPKMAAL; peptide 1, DWAAPHLEVPHELVAGSEAEIL; peptide 2: DLGCYNQYSFSEHA; peptide 3, DLRPQQVXW. RNA Purification and Northern and Southern Blot Analyses Quail tissues were quickly frozen in liquid nitrogen after their dissection. RNA purification was performed according to Chomc?zynski and Sacchi (1987). Poly(A)+ RNAwas isolated byoligo(dT) chromatography (Pharmacia). For Northern analysis, 1 kg of mRNA or S-IO pg of total RNA was separated on 6% formaldehyde, 1% agarose gelsand then transferred toa nylon membrane (GeneScreen, New England Nuclear). For Southern blot analysis,

DNAwastransferredonanylon membraneafteracidicdepurination followed by denaturation according to Sambrook et al. (7989). Oligonucleotide probes were radiolabeled usmg T4 polynucleotide kinase (Boehringer) in the presence of [yJ2P]ATP. Hybridization was performed at 42OC in 6x SSC, 5x Denhardt’s solution, 0.05% NaPi, 0.2% SDS, 100 vglml salmon sperm DNA. DNA probes were radiolabeled using the Random Prime DNA labeling kit (Boehringer) in the presence of [a-SZP]dCTP and hybridized at 42OC in 50% formamide, 6x SSC, 5x Denhardt’s solution, 0.2% SDS, 100 pg/ml salmon sperm DNA. cDNA Synthesis, PCR, and cDNA Library Screening Oligonucleotides I (5’CTAACCTTGA(CT)CT(ATCG)TGGGClGC(ATCC)CC), III (5’~CTGAAlTCA(GA)CTAG)ATCTC)TClGC(lC)TC), and 3 (5’.CTCAAlTCAGGATCTCGGCTrCTGACC) were synthesized based on the sequence of the peptide 1. Oligonucleotide N (5’~CTAAGCTlTCCATGCC(ATCG)CC(ATCG)AA(GA)ATGG) was synthesized based on the amino-terminal sequence of SMP. Single-stranded cDNA was synthesized with murine leukemia virus reverse transcriptase (Bethesda Research Laboratories) usin’g poly(A)+ RNA purified from adult quail spinal cord and oligo(dT) (12-18) as primer. One microgram of cDNA template was denatured at 94OC for 1 min and annealed with synthetic oligonucleotide at 42°C for 2 min. DNA was then synthesized with Taq polymerase (Perkin Elmer Cetus) for 3 min at 72’C. This cycle was repeated 30 times to amplify the DNA segment between the oligonucleotide primers. A size-selected (>2 kb) cDNA library was made by In Vitrogen (La Jolla, CA) from 15-day-old postnatal quail spinal cord mRNA cloned into the CDM8 vector. DNA Sequencing and Sequence Comparison Suitable restriction fragments of the CDMLI-SMP4 plasmid were subcloned into BlueScript KS-. Double-stranded DNA was purified by alkaline lysis and polyethylene glycol precipitation (Sambrook etal., 1989rand sequenced on both strands by thedideoxy chain termination method (Sangeret al., 1977) using a Sequenase kit (United States Biochemical Corp., Cleveland, OH). Asearchofthe NBRFdata basewasdonefor potential polypeptide sequence similarities. Liposome Binding Assay SMP was purified from quail myelin prepared according to NortonandPoduslo(1973) byimmunoaffinitychromatographyusing MAb 513, which was raised against chicken brain glycoproteins (Poltorak et al., 1987) and recognizes SMP. After washing the antibody column with Tris-buffered saline (TBS) containing 0.5% NP-40, the detergent was changed to IO mg/ml P-octylglucoside (BMC) in TBS. The bound antigen was eluted from the column with 100 mM diethylamine (Sigma) containing P-octylglucoside (IO mg/ml). The eluate was collected in tubes containing 1 M Tris (pH 6S)and subsequentlyconcentrated. The bufferwaschanged to TBS containing 10 mglml B-octylglucoside using Centriprep concentrator tubes (AMICON). MAC was immunopurified according to the procedures of Poltorak et al. (1987), except that the starting material was myelin prepared from rat brains. Purified glycophorin, used as a negative control for liposome binding was purchased from Sigma and resuspended in TBS containing 10 mgiml P-octylglucoside. MAG, SMP, and glycophorin (approximately 100 pg each) were incorporated into liposomes as described in Johnson et al. (1989). Rat DRG cells were dissected from I- to 2-day-old SpragueDawley rat pup and maintained in vitro in Dulbecco’s modified Eagle’s medium (DMEM), 10% fetal calf serum (FCS) supplemented with 100 rig/ml nerve growth factor (Bioproducts) as described (Seilheimer and Schachner, 1988). Cultures were grown for 2 weeks in vitro before being used for liposome binding. DRGs and sympathetic ganglia were dissected from E9 quail embryos and cultured for 2-3 days in DMEM, 10% FCS. For the liposome binding test, 50 PI of liposome solution containing 10% FCS was incubated for 30 min with the monolayer

NWU3ll 332

cultures at room temperature. Cultures were washed with 20 mM HEPES (pH 7.2), 0.15 M NaCl and mounted in PBS, 90% glycerol, 100 mg/ml phenylenediamine dihydrochloride (Sigma). MAC liposome binding was inhibited with anti-MAC MAb 513 by preincubating the liposomeswith a 1110 dilution of the ascitic fluid. SMP Expression Approximately IO5 COS-7 ceils were transfected with 2 ug of SMP cDNA clone CDM8-SMP4 using the DEAE-dextran method (Lopata et al., 1984). Three days later, COS-7 cells from one 100 mm plate were scraped off, washed in PBS, and solubilized in 200 PI of buffer containing 50 mM Tris (pH 7.4), 150 mM NaCI, 0.5% NP-40, and 2 mM phenylmethylsulfonyl fluoride. One fifth of the sample was run on an 8% SDS-polyacrylamide gel and subsequently transferred to nitrocellulose. Following blocking with 5% dry milk in TBS with 0.05% Tween-20, the blots were incubated first with the anti-SMP MAb and subsequently with goat anti-mouse IgG alkaline phosphatase (BioRad). Blots were finallydeveloped with 5-bromo4chloro-3-indoyl phosphateand Nitro Blue Tetrazolium (BioRad) as alkaline phosphatase subst rate. For surface staining, COS-7 transfectants were trypsinized 2 days after transfection, plated onto 12 mm glass coverslip, and stained the following day with anti-SMP MAb and fluoresceincoupled goat anti-mouse IgC before cell fixation with PBS, 4% formaldehyde. Acknowledgments The authors wish to thank Dr. A. Williams and D. Hall for critical readingofthemanuscript.Theyacknowledgetheskillfultechnical assistance of L. Addade, J. P. Lecaer, and A. Billaut and the help of Y. Rantier and B. Henry for preparation of the illustrations. This work has been supported by Centre National de la Recherche Scientifique, Association pour la Recherche sur la Sclerose En Plaques, and Association Fransaise contre les Myopathies and Ligue Nationale Francaise contre le Cancer. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked “advertiseme&’ in accordance with 18 USC Section 1734 solely to indicate this fact. Received

September

19, 1991; revised

December

3, 1991.

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