Journal of Neuroscience Research 25:143-151 (1990)

Rapid Communication Molecular Cloning of cDNAs That Encode the Chicken Po Protein: Evidence for Early Expression in Avians M. Barbu Institut d’Ernbryologie Cellulaire et MolCculaire, CNRS, College de France, Nogent-sur-Marne, France

As the major transbilayer glycoprotein of peripheral myelin, Po is believed to play a prominent role in the formation of this structure by Schwann cells. The amino acid sequence of the chicken Po molecule, deduced from the nucleotide sequence of cDNA clones, is reported here. Comparison with the mammalian molecule reveals an extensive overall homology, thus underlining the importance both of the cytoplasmic and of the extracellular domains of this protein in the establishment and preservation of peripheral myelin structure. Unexpectedly, an avian Po cDNA probe was found to hybridise with several mRNA species, present exclusively in peripheral nerve and in proportions that varied according to the developmental stage. The expression of these transcripts was detected significantly earlier than that of Po mRNA in mammals. Key words: peripheral myelin, amino acid sequence, Schwann cells INTRODUCTION Po, the major protein of peripheral myelin, is the subject of considerable interest, not only because of its presumed role in myelin formation but also as a result of suggestions that this molecule is among the most primitive of the immunoglobulin (lg) superfamily of proteins (Williams, 1987; Lemke et al., 1988). In mammals, this small integral membrane glycoprotein is expressed only by myelinating Schwann cells and accounts for over SO% of the protein present in mature peripheral myelin (lshaque et al., 1980). The amino acid sequence of manimalian Po has been deduced from a cloned rat cDNA (Lemke and Axel, 1985) and directly determined by sequencing the bovine molecule (Sakamoto et al., 1987). Analysis of the primary structure revealed a basic intracytoplasmic region, a single membrane-spanning domain, and an extracellular portion that is homologous to the variable region fold of Ig. 0 1990 Wiley-Liss, Inc.

The abundance of this Ig-related protein in peripheral myelin, together with comparative studies on normal mice and shiverer mutants (Kirschner and Ganser, 1980), led to the hypothesis that Po participates in the adhesion and subsequent compaction of adjacent myelin lamellae (Lemke and Axel, 1985). More specifically, this molecule has been suggested to act as a “transbilayer spacer,” ensuring by means of molecular bridges the preservation of the lamellar structure: The basic intracytoplasmic domain could interact with microtubules (Braun, 1984) or with acidic lipids (Lemke and Axel, 1985) to stabilise the major dense line of myelin, while the extracellular domain could play the same role in the case of the intraperiod line via homotypic interactions (Braun, 1984; Lemke, 1988.). Furthermore, recent results suggest that Po could also act prior to myelination, at the time when contact between Schwann cclls and appropriate axons takes place (Martini et al., 1988). If this is so, the extracellular Ig-like domain of Po could be implicated in axonal recognition. To determine which parts of the Po molecule are functionally and structurally important, i.e., have been highly conserved during evolution, we have isolated and analysed cDNAs coding for the complete chicken Po protein. Comparison of the amino acid sequence deduced from this cDNA with sequences of two mammalian species shows the three proteins to be identical in size, except for a single additional amino acid in the carboxyterminal region of chicken Po. The overall conservation of the molecule, the first nonmammalian myelin protein to be analysed in this way, is extensive, although thcrc are several important substitutions. Using a cDNA probe

Received September 10, 1989; revised September 29, 1989; acccptcd October 5 , 1989. Address reprint requesta 10 M. Barbu, Institut d’Embryologie Cellulaire et Moltculaire, CNRS, Collcgc dc Francc, 49bis. avcnuc de la Belle Gabrielle, 94736 Nogent-sur-Marne Cedex. France.

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to analyse the levels of Po mRNA during development of the chicken, we have found the existence of several niRNAs. They are present only in the peripheral nervous system, and their expression can be detected earlier than that of the Po transcript in mammals.

MATERIALS AND METHODS Isolation of RNA RNA was isolated from chicken sciatic nerve, brain, thymus, or liver by homogenisation in 6 M guanidinium-HCI followed by ultracentrifugation through a 5.7 M CsCl cushion (Liu et al., 1979). Poly (A)+ RNAs were enriched by oligo (dT) cellulose chromatography (Aviv and Leder, 1972).

Amino Acid Sequence Comparisons The amino acid sequences used for similarity comparisons with chicken Po protein were obtained from the National Biomedical Research Foundation database or were entered directly from published data. Sequence alignments were obtained using the computer programmes ALIGN-Citi 2 (Dayhoff et al., 1983) and Kanehisa-Citi 2 (Kanehisa, 1984).

Production of Monoclonal Antibody Against Chicken Po A fusion protein comprising P-galactosidase and the expression product of cDNA 12 was obtained as described by Huynh et al. (1985) and separated on SDSpolyacrylamide gel. After blotting onto nitrocellulose, the fusion protein was located by staining with polyPreparation and Screening of a cDNA Library clonal anti-Po antibody. The corresponding band was cut A peripheral nerve cDNA library (3. los recombi- out from the gel and electroeluted (Hunkapiller et al., nantsipg poly (A)+ RNA) was constructed in the lambda 1983). Immunisation (250 pg of fusion protein for two phage cloning vector g t l l (Young and Davis, 1983) with female Balbic mice) and hybridoma production were carpoly (A)+ RNA from the sciatic nerve of chicken sacri- ried out as described elsewhere (Barbu et al., 1986). ficed 4 weeks after hatching. The protocol was the same Hybridomas were selected by indirect immunofluoresas that described by Bernier et al. (1987). A polyclonal cence on frozen sections of adult chicken sciatic nerves antibody directed against rabbit Po protein (a gift from following a technique similar to that described by Dulac Dr. D. Colman) was used to screen 3. los clones of this et al. (1988). except that sections were ethanol-fixed for library (Young and Davis, 1983). This antibody had been 5 min before immunocytochemistry . Culture supernapreviously purified by adsorption on an Affigel (Bio- tants of positive hybridomas were tested on blots of pure Rad) matrix covalently coupled to chicken Po protein; sciatic nerve myelin prepared from adult chicken by conthe latter was prepared by electroelution of the 30,000 M,. ventional procedures (Norton and Poduslo, 1973). Imband after SDS-polyacrylamide gel electrophoresis of munoperoxidase reaction was performed as described purified myelin proteins. previously (Rong et al., 1987) with a peroxidase-conjuScreening resulted in the isolation of a cDNA of gated goat antimouse or antirabbit Ig (Nordic) diluted to 210 bases (clone 12). This probe was "P-labeled by 11.500. random priming to a specific activity of 5.10' c p d p g and used to rescreen the same library. Filters were hybridised at 42°C in 50% formamide, 6 x SSC, 5 x Den- RESULTS hardt's solution, 0.1% SDS, and 200 pgiml sonicated Isolation and Analysis of cDNA Clones Encoding salmon sperm DNA and washed at 42°C in 2 X SSC and Chicken Po 0.1% SDS. Two clones, B91 and F1, were isolated. Three clones (Fig. 1 ) were independently isolated from a chicken peripheral nerve expression library. An DNA Sequencing and Analysis initial screening with a purified polyclonal antibody (see cDNAs 12, B91, and F1 were subcloned in M I 3 Materials and Methods) allowed us to isolate a 2 10-base mp18 and M13 mp19 vectors and sequenced on both insert (nucleotides 298 to 508 in Fig. 2). After sequence strands (see Fig. 1 ) by the dideoxynucleotide chain ter- analysis, this clone (12) was found to contain a single mination method (Sanger et al., 1977). Oligonucleotides open reading frame coding for a polypeptide displaying (269-287) and (593-610), shown in Figure 2, and the 85% identity with part of the rat Po protein and 87% M13 universal primer were used as sequencing primers. identity with the bovine homologue (Tyr'" to Val"', Fig. 3). RNA Blot Analysis To obtain the complete sequence of chicken Po, the Northern blots were performed as previously de- same library was screened by DNA hybridisation with scribed (Bernier et al., 1987) except that, after hybridi- labeled probe 12. Two overlapping inserts, B9 1 and F1, sation, the filters were washed under conditions of high of 591 and 502 bases, respectively, were found. The restriction map of these cDNAs and the strategy emstringency (30 min at 65°C in 0.2X SSC, 1% SDS).

Cloning of cDNAs Encoding Chicken Po Protein

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Fig. 1. Restriction map and sequencing strategy of the chicken Po cDNAs. The relative positions of cDNAs 12, B91, and F1 are presented with respect to the schematic representation of the full-length cDNA encoding chicken Po. The thick lines

represent the translated region, and sequencing strategy is shown by arrows. The two boxes mark the position of oligonucleotides used as sequencing primers.

ployed for their sequencing are shown in Figure 1 . The sequences of the overlapping regions of B91, 12, and F1 were identical. Translation of the total nucleotide sequence (841 bases) shows an open reading frame corresponding to a protein 249 amino acids long, if one assumes the first ATG of the sequence, at nucleotide position 38, to be the initiation codon (Fig. 2). This ATG is preceded by a CCGCC sequence, which is in good agreement with the consensus sequence for eukaryotic translation initiation sites (Kozak, 1986). The assumption is further supported by the comparison of chicken and rat sequences (see below). Mezei and Verpoorte (1981) have identified isoleucine as the N-terminal amino acid of the mature chicken Po protein. This amino acid is found downstream from the first methionine, thus delimiting a 29 amino acid signal peptide. Consequently, the total length of the mature protein is 220 amino acids (calculated molecular weight is 24.7 kDa). This value is less than the apparent molecular weight (28,600) determined for the deglycosylated molecule separated on SDS-acrylamide gel (Mezei and Verpoorte, 1981). As in the mammalian species, chicken Po can be subdivided into three domains relative to the myelin membrane. A highly hydrophobic domain, containing 26 residues from TyrI2' through Ilei5', defines a single membrane-spanning region; the extracellular domain (Ile' through Arg'24) exhibits a relative hydrophobicity compared with that in the cytoplasmic region (ArgI5' through Lys2*O), which bears a high positive charge (Fig.

2). One potential N-glycosylation site, Asn"3-Gly-Thr"" is found in the extracellular domain.

Comparison of Avian Po With Its Mammalian Homologue Mature chicken Po protein shows an overall homology of 78% and 77% with the rat and bovine moleculer, respectively (Fig. 3). Identity ranges from around 80% in extracellular and cytoplasmic domains to only 58% in the transmembrane region. The signal peptides of the primary translation products in chicken and rat are of equal length, but display only 41 % identity. Interestingly, the five amino acids upstream to the N-terminal isolcucine of the mature protein (Ser-' to Ala-I) are exactly conserved between chicken and rat. The amino acid composition of chicken Po is notably different from that of the mammalian molecule. In the extracellular domain, hydrophobic amino acids are less abundant in the avian (42 compared with 46 in the rat). The opposite situation is observed in the cytoplasmic region, with 26 hydrophobic residues in avian compared with only 18 in rat. The comparison of the hydropathic index of chicken and rat Po protein (data not shown) reveals that conserved stretches of hydrophobic amino acids between the two species are essentially localised to the transmembrane domain, four positions in the extracellular domain (9-28,46-52, 81-88, and 110118) and one in the intracytoplasmic region ( 1 88-196). Concerning the charge of the mature molecule, pH, val-

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ATC CAC GTG TAC ACA CCG CGG GAG GTG TAC GGC ACC GTG GGC TCC CAC GTC ACC CTC TCG Ile H i s Val Tyr Thr Pro Arg Glu Val Tyr Gly Thr Val Gly Ser H i s Val Thr Leu Ser

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Fig. 3. Comparison of the amino acid sequences of chicken (middle), rat (lower; Lemke and Axel, I985), and bovine (upper; Sakamoto et al., 1987) Po proteins. Mature protein is comprised between Ile' and L ~ S ~ ' " A~ ~ ~ )at. position 212 gap has been introduced in the mammalian sequences in order to

align the avian one. The dashes denote identity with the chicken sequence; conservative and nonconservative substitutions (Kanehisa, 1984) are indicated in Roman and italic type, respectively.

ues of 10.06 and 9.83 were calculated for avian and rat Po, respectively. However, the strong positive charge ( + 16) of the cytoplasmic domain is precisely maintained. In contrast, the extracellular portion of the protein contains three additional basic residues in the chicken. Examination of the amino acid sequences of the chicken and mammalian proteins brings to light the following points: 1) The majority of the amino acid substitutions that have occurred in Po during the evolution of avians and mammals concern the N-terminal part of the molecule (residues 1 to 41) and a short stretch of the cytoplasmic domain (Leu17' to ProIx7): These regions also exhibit the lowest conservation score between bovine and rat Po. 2) In addition to the seven major amino acid substitutions observed in the N-terminal region, two others concern adjacent sites within the extracellular domain, at positions 77 and 78, where arginine residues are

replaced by serine and tryptophan in the rat, although arginine is retained at position 78 in the bovine sequence.

Fig. 2 . DNA and deduced amino acid sequences of B91, 12, and FI . Numbers at the end of each line correspond to amino acids of the mature protein. The putative membrane-spanning domain (TyrI2' to 11~150)is boxed, The N-terminal amino acid (Ile') of the mature protein, the two cysteine residues (Cys'l and Cys9') delimiting the Ig-like extracellular domain, and the potential N-glycosylation site are underlined.

~~~~~~~i~~ of chicken p0 m~~~ When incubated with total or poly (A)+ RNA from different tissues, probe 12 hybridised only with mRNA present in sciatic nerve (Fig. 5A,B). No signal was found in brain or liver, even after overexposure (Fig. SA), and Po mRNA was not found in the thymus, unlike mRNA

Specificity of Monoclonal Antibody 112-2 lmmunisation of two mice with the chimaeric protein resulting from the fusion of p-galactosidase with the expression product of probe 12 allowed us to isolate a clone of positive hybridoma cells. Tested on sections of adult chicken sciatic nerve (see Materials and Methods), the supernatant (112-2) of cultures of thcse cells displayed a strong immunoreactivity with myelin (data not shown). To determine which protein was recognised by this antibody, the crude supernatant was tested by Western blot analysis on purified peripheral myclin. As shown in Figure 4, 112-2 reacted specifically with a protein of 30,000 M,. On the same blot, the affinity-purified polyclonal anti-Po (i.e., the antibody initially used to screen the library) recognised a molecular species of similar electrophoretic mobility.

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-0.56 Fig. 4. Western blot of pure myelin prepared from the sciatic nerves of 4-week-old chickens. Equal amounts of protein (50 ygilane) were subjected to electrophoresis on a 12% SDSpolyacrylamide gel, blotted onto nitrocellulose, and immunorevealed by a goat antimouse Ig (lane I ) , purified polyclonal anti-Po diluted 1/50 (lane 2), and 112-2 crude supernatant (lane 3), followed by the appropriate peroxidase-conjugated second antibody. Molecular weights of calibration standards (Sigma) are indicated on the right.

encoding another myelin protein, CNPase (Bernier et al., 1987). The strongest signal with probe 12 was observed with the 1900-base mRNA species: This agrees with results obtained in the rat (Lemke and Axel, 1985). In addition, RNAs of different sizes (900, 1,350, 3,800, and 6,500 bases) were also found to hybridise with the probe, even after washing the blot under highly stringent conditions (see Materials and Methods). When labeled cDNA 12 was incubated with the same quantity of total RNA from sciatic nerves removed at various embryonic stages or from the 15-day-postnatal chicken, a very early expression of Po mRNA was revealed: Under conditions of overexposure (Fig. 5 A ) a signal could already be seen in embryos of 11 days (El I ) , the youngest stage tested in these experiments. This expression is earlier than that observed in the rat (Lemke and Axel, 1985) and that detected in chicken by another technique (Leblanc and Mezei, 1985). Interestingly, only 1,900- and 3,800base-long mRNA species were found at this stage of

Fig. 5 . Northern blot of 20 yg of total RNA (from liver and sciatic nerve) and 10 pg of poly (A) ' RNA (from total brain and thymus) of chicken. Liver, brain, and thymus were taken from adult chickens (4 weeks old); sciatic nerves were dissected from embryos (El 1 through E19) and from young, posthatching (PlS) chickens. All samples were subjected to electrophoresis through the same 1.5% agarose-formaldehyde gel, blotted onto Gene Screen (NEN), and probed with 32P-labeled chicken Po cDNA (insert 12). After the last wash (0.2 x SSC, 1 % SDS, 30 min at 65"C), the blot was exposed, using Amersham MP film with an intensifying screen, for 12 hr (B) and again for 3 days (A) to reveal faintly positive bands. Size markers are shown on the right (calibration in kb).

development, the latter form being more abundant. Finally, this Northern analysis revealed that the maximal expression of Po mRNA occurs at E17, when all the different size classes of mRNA are represented. At E l 9 the expression has already decreased, and at PI5 only the 6,500-, 3,800-, and 1,900-base species are still detectable.

DISCUSSION Because of its particular transbilayer position in the Schwann cell membrane, it has been suggested that the Po molecule plays an important role in adhesion of peripheral myelin lamellae (for reviews, see Braun, 1984; Lemke, 1988). The basic cytoplasmic domain was pro-

Cloning of cDNAs Encoding Chicken Po Protein

posed, via heterotypic interaction, to mediate the compaction of the major dense line of myelin (Braun, 1984; Lemke and Axel, 1985); in contrast, the cohesion of the intraperiod line was suggested to correspond to a homotypic interaction between the extracellular domains of two Po molecules facing each other (Lemke, 1988). The analysis of the primary structure of chicken Po reveals an extensive overall homology with the mammalian molecule and consequently underlines the importance of both the cytoplasmic and the extracellular domains of this molecule in the formation and preservation of peripheral myelin structure. The cytoplasmic region of Po has been proposed to play the same role as MBP in stabilising the ma$or dense line of myelin, since, in the shiverer mouse (MBP deficient) the lack of adhesion of cytoplasmic surfaces of the glial cell bilayer is observed in the central but not in the peripheral nervous system (Kirschner and Ganser, 1980; Ganser and Kirschner, 1980). Interestingly, both proteins share a high basic charge that is particularly well conserved between the bird and the mammal in the cytoplasmic domain of Po. These positively charged amino acids could interact with acidic lipids of the apposed membrane (Lemke and Axel, 1985). The abundance of Po glycoprotein and the fact that the length of its extracellular domain is appropriate for maintenance of the intraperiod line are in good agreement with the hypothesis of a self-adhesion mechanism (Lemke et al., 1988). The substantial hydrophobicity of the extracellular domain of Po was suggested to account for the auto-adhesive properties of this molecule (Lemke and Axel, 1985; Lemke et al., 1988). However, the sequence of the avian extracellular domain does not possess exactly the same hydrophobicity profile as the corresponding sequence in the mammalian; hydrophobic sequences are limited to four discrete regions in the chicken. As in the case of mammalian Po (Lai et al., 1987; Lemke et al., 1988), part ofthe extracellular domain of the chicken protein exhibits a significant homology with Ig; the degree of amino acid sequence identity, the spacing between disulphide-bonded cysteine residues (underlined in Fig. 2), and the fact that the sequences around the second cysteine residue are more V- than C-like clearly place the chicken Po extracellular domain among variable-like as opposed to constant-like Ig domains (Williams and Barclay, 1988). Homology scores with rent members of the Ig family obtained by computer-assisted analysis (see Materials and Methods) indicate that the extracellular domain of chicken Po is most similar to the Ig heavy chain variable region ( 1 2.1 alignment score; Williams and Barclay, 1988), the first variable domain of the human T-cell glycoprotein T4 (alignment score 10.5; Clark et al., 1987), and the first domain

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of the antigen OX-2 (alignment score 10.2; Clark et al., 1985). It would be interesting to determine which part(s) of the Ig domain are exposed on the surface of the molecule. This could feasibly be done by taking advantage of the fact that Po is homologous to the Ig variable region to simulate the secondary and tertiary structure of this molecule (Amzel and Poljak, 1979; Becker and Reeke, 1985; Bjorkman et al., 1987). If the function of Po as an adhesive transbilayer spacer can most probably account for the cohesion of peripheral myelin lamellae, it remains to be determined whether the form of Po present on the surface of myelinating Schwann cells is identical to that found in compacted myelin. At these different stages, the space between lamellae is not the same (Kirschner et al., 1984). From a recent study in mammals (Trapp, 1988), it has been proposed that differential expression of Po and myelin-associated glycoprotein (MAG) underlies this phenomenon. MAG, first expressed, would act as a spacer when myelin is uncompacted. Subsequent disappearancc of MAG would allow a much closer apposition o f the myelin layers by self-adhesion of Po molecules. Another possible explanation for the different degrees of separation in immature and compacted myelin lamellac is thc existence of various isoforms of the protein moiety of Po at different developmental stages. However, although this is a well-documented occurrence in the case of several Ig-related adhesion molecules (see Salzer and Colman, 1989, for references), such a phenomenon has never been demonstrated for Po. Consequently, it is particularly intriguin,0 to note that the analysis of the expression of Po mRNA in the chicken reveals RNA species of different sizes at different stages of maturation of the Schwann cell. Thus, only 1,900- and 3,800-base mRNAs were found in the youngest embryos examined (El 1 and E13), whereas five forms were detected in actively myelinating Schwann cells ( E l 5 to E19). The smallest mRNA species (900 and 1,350 bases) were no longer detectable in young adult chickens. Whether these different chicken Po mRNAs correspond to different molecular forms of mature Po and, if so, whether they are present in myelin remain to be determined. In this context, it is interesting to note that high-molecular-weight proteins are indeed recognised in nerve by an anti-Po antibody during early devclopnient ol' chicken embryos (Nunn et al., 1987; M . Barbu, personal observations). A final interesting point arising from the analysis of Po RNA expression in the chicken concerns the strikingly early stage (El 1 ) at which mRNA was observed. The first turns of myelin observed in sciatic nerve of the chicken by electron microscopy were detected at El 3 (Uyemura et al., 1979). Thus chicken Po mRNA cxpression occurs at least 2 days before myelination begins.

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This result is in agreement with that of Martini et al. (1988), who found Po protein already present on the surface of rat Schwann cells when they associated with axons on a 1:l basis. The fact that mRNAs are already present in E l 1 stage chicken embryos suggests that Po may be functionally involved before myelination, possibly in the ensheathment of axons by Schwann cells. Further studies on the localisation of Po protein and mRNA during development of peripheral nerve will be necessary to determine the stage at which expression first occurs in the chicken.

ACKNOWLEDGMENTS The study described here was carried out in the laboratories of Drs. David Sabatini and Nicole Le Douarin, whose support I gratefully acknowledge. I particularly thank Drs. David Colman and Lise Bernier for their help and encouragement at the beginning of this work; Dr. J. Smith for critical reading of the manuscript; and F. Lapointe for help with DNA sequencing. I am indebted to E. Bourson, C . Rimy, B. Henri, and Y. Rantier for typing and illustrating this article. Financial support was obtained from the Centre National de la Recherche Scientifique, the Institut National de la SantC et de la Recherche MCdicale, the Fondation pour la Recherche MCdicale, the Ligue Nationale Franpise contre le Cancer, and March of Dimes Basic Research Grant NO. 1-811.

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