Mammalian and avian PO protein of the peripheral nervous system myelin are different

Comp. Biochem. PhysioL Vol. 87B, No. 4, pp. 895-905, 1987 Printed in Great Britain

0305-0491/87 $3.00+ 0.00 © 1987 PergamonJournals Ltd

MAMMALIAN A N D AVIAN PO PROTEIN OF THE PERIPHERAL NERVOUS SYSTEM MYELIN ARE DIFFERENT ANDRi~A C. LEBLANC and CATHERINEMEZEi Department of Biochemistry, Dalhousie University, Halifax, Nova Scotia, Canada, B3H 4H7 (Tel: 902-424-2409) (Received 30 September 1986)) Abstract--1. The mammalian PO gene exhibits low homology to the avian PO gene and transcript. 2. The avian PO mRNA is smaller than the mammalian mRNA. 3. The primary structure of mammalian and avian PO proteins differ in their molecular weight, isoelectric point, and chymotryptic peptide pattern. 4. Similarity between the PO proteins is indicated by immuno-cross-reactivityof the anti-chicken PO IgG to mammalian PO proteins. 5. Similarities at the level of amino acid sequence could provide insight on the structure and function of the PO protein.

INTRODUCTION

tion, during the course of a long range investigation concerning the expression of the PO gene in the developing, regenerating and degenerating avian and mammalian PNS, we observed major differences between the primary translation products of the PO mRNA of the two species (LeBlanc and Mezei, 1985; LeBlanc et al., 1986). In this paper we describe the results of experiments in which the properties of the PO genome, transcript and translation product in the mammalian and avian PNS were compared. The results indicate a rather surprising divergence between the structures of this important PNS myelin protein from the two species. The results of this investigation appeared in a preliminary form (LeBlanc and Mezei, 1986).

The most plentiful and characteristic protein of the PNS myelin is the PO protein (for review see Lees and Brostoff, 1984). The protein is an excellent marker of myelination in the developing peripheral nerve: it is first detected in the Schwann cells at the onset of myelination (Trappet al., 1981; Winter et al., 1982). Thereafter the protein is deposited at a rapid rate in the PNS myelin following the pattern of active myelination (Oulton and Mezei, 1976; Uyemura et al., 1979; Wiggins et al., 1975). Various morphological, physico-chemical and recent molecular approaches indicate that the PO protein is an integral membrane protein that spans the bilayer of the compact myelin sheath (for review see Kirschner et al., 1984). Besides the single membrane spanning region, the protein contains a large extracMATERIALS AND METHODS ellular domain and probably a smaller, basic intraImmunoblotting of PO protein from rat and chicken sciatic cellular domain. It has been suggested that each of nerve and brain these domains plays an essential role in producing The preparation of protein samples, electrophoresis, and maintaining the highly ordered structure of the transfer of proteins to a nitrocellulose membrane and compact myelin sheath (Lemke and Axel, 1985). immunoblotting with rabbit anti-chick PO (IgG fraction) PO protein has been purified from the PNS of was carried out as described by Nunn and Mezei (1984) with various mammalian species (Kitamura et al., 1976; a few modifications. The anti-PO IgG fraction was used at Roomi et al., 1978; Wood and Dawson, 1973). It has a 1:10,000 dilution (0.08mg/100 ml buffer). The second a molecular weight of about 30,000, exhibiting micro- antibody used for detection was HRP-coupled goat antiheterogeneity due to post-translational glycosylation, rabbit IgG (BioRad, Quebec, Canada) at the same dilution. acylation, sulfation and phosphorylation (Agrawal et Epitope selection of anti-chick PO antibody from purified IgG al., 1983; Quarles, 1980; Singh and Spritz, 1976). A fraction report from our laboratory demonstrated that alThis procedure was based on the method of Weinberger though the amino acid compositions of chick PO et al. (1985) with the following modifications. Approxiprotein and that of the mammalian PO protein mately, 5/~g of purified chick PO protein was electroexhibit overall similarities, there are also definite phoresed on 10% (wt/vol) SDS-polyacrylamide slab gels differences. The purified protein from the chick sci- and electroblotted on a 9 x 8 cm nitrocelluose sheet as atic nerve contains a higher percentage of polar described recently (Nunn and Mezei, 1984). A portion of the amino acids and a lower proportion of hydrophobic sheet was immunostained with rabbit anti-PO antisera and amino acids (Mezei and Verpoorte, 1981). In addi- HRP-conjugated goat anti-rabbit IgG to locate the exact position of the pure 30 K PO protein band. The remaining portion of the nitrocellulose containing the pure 30 K Abbreviations: HrP, Horseradish peroxidase; PAGE, poly- antigen was then cut from the nitrocellulose sheet and acrylamide gel electrophoresis; PNS, peripheral nervous incubated overnight at room temperature with 1.2 mg of system; SDS, sodium dodecyl sulfate. chick anti-PO IgG/50 ml of TBS buffer (20 mM Tris; 0.5 M 895

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ANDREA C. LEBLANC and CATHERINE MEZEI

NaC1, pH 7.5) containing 1% (wt/vol) gelatin. The nitrocellulose strip was then washed three times with 20ml quantities of TBS and blotted dry. The epitope-selected anti-chick PO antibody was eluted from the nitrocellulose strip three times at room temperature with 5 ml of 5 mM glycine-HCl (pH 2.3), containing 0.5M NaC1 and 1% (wt/vol) gelatin. The combined eluates were immediately neutralized with Tris-HC1 pH 7,4 to a final concentration of 50 mM and diluted with TBS containing 1% (wt/vol) gelatin to a final volume of 50 ml. The epitope-selected antibodies were used as first antibodies to immunostain the electroblotted samples shown in Fig. lB. Removal o f the N-asparagine linked high mannose and corn plex carbohydrates from the glycoproteins o f the sciatic nerve

The method was essentially according to Elder and Alexander (1982), with some modifications. Sciatic nerves from 19-day-old chick embryos, and 21-23-day-old rats were dissected as described previously (Nunn and Mezei, 1984) and homogenized in 10 vol. of 0.1 M Tris buffer, pH 7.5. To the homogenate was added 15 vol. of 0.1 M sodium phosphate buffer, pH 6.1 containing 0.05 M EDTA, 1% (vol./vol.) Nonidet-P40, 0.1% (wt/vol.) SDS and 1% (vol./vol.) 2-mercaptoethanol to obtain a protein concentration of approximately 1/~g/~l. Twelve /~1 of the above diluted nerve homogenate was then treated with 2#1 of Endo F solution (0.125q).5 units//zl, New England Nuclear, Boston, MA) and I #1 of phenylmethylsulfonyl fluoride (0.2 #g/#l) and incubated for 18 hr at 37°C. The reaction

was stopped by adding 15 #1 of 0.25 M Tris buffer, pH 8.6, containing 1.92M glycine, 1% (wt/vol.) SDS and 2% (vol./vol.) 2-merceptoethanol to the reaction mixture and boiling for 2 min. Control samples were treated as above but in the absence of Endo F solution. Solutions containing approximately 3-5/zg of total protein were analyzed by SDS polyacrylamide slab-gel electrophoresis and immunoblotting. Isolation o f poly(A +) R N A from sciatic nerve or brain

Total cellular RNA was isolated from sciatic nerves of approximately 10-20 dozen chicken embryo (Gallus domesticus~olden Comet strain, Cook's Hatchery, Truro, N.S.) or 20-40 rats (Rattus--Sprague-Dawley; 21-23 day old). Brain RNA was prepared from approximately 1 g of tissue. The RNA was extracted from the tissue by the guanidine/hot phenol method described in Maniatis et al. (1982) with the following modifications. The tissue was immediately dropped into liquid nitrogen upon dissection. The tissue, immersed in liquid nitrogen, was then ground to a fine powder using a mortar and pestle. The powder was transferred to a 5 M solution of guanidine thiocyanate at 5 ml/g weight of frozen tissue and homogenized in a Duall glass homogenizer (Kontex Scientific, Vineland, N J). The rest of the procedure was carried out as described in Maniatis et al. (1982). After an overnight ethanol precipitation at -20°C, the RNA was recovered by centrifugation at 5800g, washed as previously described (LeBlanc and Mezei, 1985) and the poly(A ÷) RNA separated by two

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Fig. 1. Immunoblots of total proteins from sciatic nerve extracts of rats and chicks. Proteins were extracted, fractionated by 10% SDS-PAGE and electroblotted as described recently (Nunn and Mezei, 1984). The nitrocellulose sheets were immunostained with anti-chick PO IgG (A). In (B), immunostaining was carried out with epitope-selected anti-chick Po IgG as described in Materials and Methods. The following materials were immunoblotted in (A) and (B). Lane 1, 6/~g of protein from sciatic nerve of 19-day-old chick embryo; lane 2, same as lane 1 but after 0.5 units of endoglycosidase F treatment as described in Materials and Methods. Lane 3, 30/~g protein from brain of 19-day-old chick embryo; lane 4, 35 ~g protein from brain of 21-day-old rat; lane 5, I0/tg protein from sciatic nerve of 21-day-old rat; lane 6, same as lane 5 but after 0.5 units of endoglycosidase F treatment as described in Materials and Methods. Lanes 7 and 8, 130 ng of purified chick PO protein without or with endoglycosidase F treatment as described for lanes 1, 2, 5 and 6 above.

Avian and mammalian PO protein successive cycles of oligo-dT-cellulose chromatography, according to the method of Maniatis et al. (1982). The poly(A ÷) RNA was kept at - 7 0 ° C in 95% ethanol; it was washed twice before use with 70% ethanol containing 5 mM EGTA, lyophilized and dissolved in sterile H20. In vitro translation and immunoprecipitation o f PO protein In vitro translation was performed as described previously (LeBlanc and Mezei, 1985) using 35S-methionine ( ~ I000 Ci/mmole, DuPont Canada Inc., Quebec, Canada). Immunoprecipitation of the PO protein was done according to the method of Frail and Braun (1984) using 10#1 of rabbit anti-chick PO antibody containing 16 p g IgG/p 1 for each 25-50 #1 of translation mixture. Protein-A sepharose was used to isolate the immune complex from the translation mixture. The total translation and immunoprecipitated products were separated by electrophoresis on a 10% polyacrylamide gel as described previously (Nunn and Mezei, 1984). The gels were processed for fluorography (Bonner and Laskey, 1974) exposed for 2-10 days on Kodak XAR-5 film. Processing o f in vitro translation products

Processing of in vitro translation products was done according to the method of Jackson and Blobel (I 977). Dog pancreas microsomal membranes (Dupont Canada Inc., Quebec, Canada) were added to the in vitro translation mixture at a concentration of 0.5 #1 membranes/25/tl mixture. The mixture was incubated for 1 hr at 37°C. SDS was added to give a final concentration of 4% (w/v) and the mixture boiled 2 min. After cooling the samples on ice, the immunoprecipitation was carried out as described above. Two-dimensional gel electrophoresis of i n vitro translated PO protein

Immunoprecipitated PO protein was dialyzed against 0.1% Triton X-100 for 24 hr. The protein sample contained PO protein from a 50~1 translation reaction, l mM Tris-HC1 pH 8.0, 8 M urea and 30 mM dithiothreitol as described by Maguire et al. (1984). The protein samples were electrophoresed in the first dimension on a prefocussed 10% tube gel containing 8 M urea and 1.2% ampholytes pH 5-8 (Pharmacia, Quebec, Canada). Prefocussing was done at 100 V for 30 min. The protein samples were overlaid with 100 #1 of 5% sucrose and 1% ampholytes in 0.02 M NaOH. The anode solution was 0.01 M phosphoric acid and the cathode solution was 0.02 M NaOH. The samples were electrophoresed for 18 hr at 200 V. After electrophoresis, the gels were removed from the tubes and put into a solution of 0.1 M sodium phosphate (pH 7.0), 2% SDS and 1 mM dithiothreitol for 30-60 min. The gels were run in the second dimension on a vertical 10% SDS-polyacrylamide gel prepared as previously described (Nunn and Mezei, 1984). The tube gel was set into place with 0.7% agarose and electrophoresis carried out at 60 mA for 2 hr. The gel was then fixed in 25% isopropanol, 10% acetic acid for 1 hr, processed for fluorography, dried and exposed on Kodak Xomat-S film for 2~, days. Limited proteolysis o f in vitro translated PO from rat and chicken

PO protein immunoprecipitated from a 50/~1 translation mixture directed with exogenous chick or rat sciatic nerve poly(A ÷) RNA was electrophoresed in a 10% SDSpolyacrylamide gel as previously described. After electrophoresis, the gel lanes were cut into 1 cm pieces. The [35S-meth~on~ne]-iabeled immunoprecipitated PO protein was located with a Geiger counter (Technical Associates, Canoga Park, California) and the gel piece crushed into small pieces with a glass rod. Approximately 200 #1 of 0. 1% Triton x-100 containing 0.2% sodium azide was added to the gel mixture and left at room temperature for 24 hr. Proteolytic digestion was carried out in the crushed gel

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following the method of Cleveland (1983) with the following modifications. The chymotrypsin digestion was carried out at 37°C for 3 hr in 0.15 M Tris, pH 6.8, 0.12% SDS, 1 mM EDTA, 0.05 M 2-mercaptoethanol, 0.002% Triton X-100 with 0.125 units of enzyme (~-chymotrypsin from bovine pancreas: type II, Sigma, St. Louis, MO). The reaction was stopped by adding 0.02% bromophenol blue/7.5% glycerol and boiled for 2 min. The gel particles were spun down in a microfuge for 10 min and the supernatant applied to a 15% SDS-polyacrylamide running gel with a 3% (w/v) stacking gel. Electrophoresis was carried out at 40 mA until samples were into the running gel and 80 mA until completion. The gel was then treated for fluorography and autoradiography as described above. Preparation o f DNA probe for Northern and Southern

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The rat PO cDNA clone pSN63c was kindly donated by Drs R. Axel and G. Lemke (Columbia University, N.Y.). The 1.85kb insert was in a pUC8 vector. DNA was prepared from a larger scale plasmid preparation by the Triton lysis method (Shepard and Polisky, 1979) and purified by centrifugation in a cesium chloride gradient (Maniatis et al., 1982). The 1.85 kb fragment containing coding and non-coding regions of PO was obtained from Eco RI digestion of the plasmid and separated on a 1% low-melting agarose gel (LMP agarose, ultra pure, BRL, Gaithersburg, MD). The 1.85 kb fragment was extracted from the gel by the method described in Maniatis et al. (1982). The coding region of the probe was obtained by Pst I digestion of the plasmid. The 604 bp fragment was separated and extracted from a 1% low melting agarose gel as described above. Nick translation Nick translation (Rigby et al., 1977) of the DNA probes was carried out by the method of Kelly et al. (1970) as modified by Fuscoe et al. (1983). The reaction was carried out in 10 mM tris pH 7.5, 5 mM MgCI 2, 1 mg/mL BSA, 30pM each of dATP, dGTP, dTTP (Pharmacia, Quebec, Canada) and 100#Ci of [ct-32P] dCTP (2000-3000Ci/ mmole, Dupont Canada Inc., Quebec), 0.1 ng DNase I (Sigma, St. Louis, MO) and 20 units of DNA polymerase I E. coli minimal nuclease, (PL biochemicals, Milwaukee, WI) for 1 #g of DNA/50 ~1 reaction mixture. Total incorporation of labeled nucleotide was measured by acid precipitation (Maniatis et al., 1982). Purification of the labeled DNA from unreacted nucleotides was done on Sephadex G-50 column (Pharmacia, Quebec, Canada) in STE buffer (10 mM Tris, I00 mM NaC1, 1 mM EDTA, pH8.0). This procedure consistently gave 0.5 to 1.0 x l0 s cpm/#g of DNA. Northern analysis

Northern analysis was performed by denaturing the RNA using the methyl mercury hydroxide method (Chandler et al., 1979). Each lane of a 1% agarose gel contained 4/tg of poly(A ÷) RNA. Electrophoresis was carried out at 3-4V/cm. The RNA was transferred onto Zeta Probe (BioRad Lab., Richmond, CA) according to the method described in the ZetaProbe Blotting manual. Hybridization to the nick-translated pSN63c rat PO specific cDNA probe was carried out according to the method of Thomas (1983). The hybridization was done using 1.0 × 106cpm of probe per ml of hybridization mixture (100/~l/cm2 of blot area). Dot blots

Dot blots were prepared by the method of Cheley and Anderson (1984) using poly(A ÷) RNA prepared from rat and chicken sciatic nerves. Each sample was serially diluted 2-fold. After baking for 2 hr at 80°C under vacuum, prehybridization and hybridization to the 1.85 kb PO clone were carried out according to the method described by Thomas (1983).

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Southern blots DNA was extracted from chick and rat liver following the method of Pellicer et al. (1978). The Chinese hamster cell (Cricetus griseus) DNA was a kind gift of Dr. R. Fenwick (Dept. of Biochemistry, Dalhousie University). Human (Homo sapiens) DNA was obtained from peripheral blood leukocytes which were prepared according to R. Fenwick (personal communication). The peripheral blood leukocytes were prepared from EDTA anticoagulated blood. The blood was centrifuged at 1000 g for 20 min. The plasma was removed and the white cells collected. The contaminating red cells were hemolysed in 0.60% ammonium chloride (w/v), 0.8 mM Tris-C1 pH 7.6 at 37°C for 8 min. The white cells were then centrifuged at 1000g for 20 min and used for the extraction of human DNA by the method of Pellicer et aL (1978). Restriction enzyme treatment of 10 #g of DNA with Eco RI restriction enzyme (Boehringer Mannheim, Quebec, Canada) was done according to the manufacturer's recommendations. The digested DNA was run on a 1% agarose gel in 1 x TAE (I0 mM Tris, 5 mM sodium acetate, 0.5 mM EDTA pH 7.4) and transferred to a nitrocellulose membrane by the method of Southern (1975) as modified by Fuscoe et al. (1983). The genomic blot was baked for 2 h r at 80°C under vacuum, prehybridized and hybridized as described by Fuscoe et al. (1983). The blot was hybridized to 7.5 x 105 cpm/ml (I00 #l/cm 2 of blot area) of nick translated 1.85 kb fragment of the pSN63c rat PO cDNA clone (0.5-1.0 x 108cpm//ag of DNA). After hybridization, the

blot was washed 3 × 40 min at 65°C in 2 × SSC, 0.1% SDS and 2 x 30 min at 50°C in 0.1 x SSC, 0.1% SDS. The blot was exposed for 3 days on Kodak X-Omat-5 film.

RESULTS I m m u n o r e a c t i v i t y of the I g G fraction of the antichick P O antisera with the chick a n d rat P O protein is s h o w n in Fig. IA, lane 1 a n d 5. The anti-chick P O a n t i b o d y reacts with the P O protein o f the rat or chicken after t r e a t m e n t of the sciatic nerve extracts with endoglycosidase F (Fig. 1A, lanes 2 a n d 6). Epitope selected a n t i b o d y also detects rat P O protein before a n d after endo F t r e a t m e n t a l t h o u g h the intensity o f i m m u n o s t a i n i n g was lower as c o m p a r e d with chicken P O (compare Fig. 1B, lanes 1, 2 a n d 5, 6). These results indicate h o m o l o g y between the chicken a n d the rat PO. However, the molecular weights o f the two proteins are different. The i m m u noblots indicate t h a t the molecular weight o f the chick P O protein is approximately 2000 higher t h a n t h a t of the r a t P O even after endo F t r e a t m e n t (Fig. I A a n d B, lanes 2, 6 a n d 8). The polyclonal a n t i b o d y also recognizes some protein b a n d s higher t h a n the P O b a n d from the nerve a n d b r a i n extracts. Epitope

Fig. 2. SDS~PAGE electrophoresis pattern of in vitro translation products of poly(A ~) RNA from chicken and rat PNS and CNS. Poly(A ÷) RNA was extracted, translated in a reticulocyte cell-free system, the translation products fractionated and analyzed as described in Materials and Methods. (A) lanes I and 2, translation products of poly(A ÷) RNA from sciatic nerves of 19-day-old chick embryos and 21-day-old rats respectively precipitated with anti-chick PO IgG. (B) translation products of poly(A ÷) RNA from the following tissues precipitated with anti-chick PO IgG. Lane 1 and 2, from 19-day-old chick embryo brain, without or in the presence of 90-fold excess purified chick PO-protein; lane 3, from 21-day old rat brain; lane 4, from 19-day-old chick sciatic nerve; lanes 5 and 6, from 21-day-old rat sciatic nerve in the presence or absence of 90-fold excess of purified chick PO protein. (C) lane 1--4, total translation products of poly(A ÷) RNA from sciatic nerve of 19-day-old chick embryo, brain of 19-day-old chick embryo, sciatic nerve of 21-day-old rat and brain of 21-day-old rat respectively. The arrows and numbers indicate the position and molecular weights (Kda) of Pharmacia low molecular weights standards on the gels.

Avian and mammalian PO protein selection of the anti-chick PO antibody against purified chick PO demonstrates the specificity of the immuno-crossactivity to a Mr = 41,000-42,000 protein in both rat and chicken sciatic nerve (Fig. IB, lanes 2, 5 and 6). This reaction is very weak with the M r = 41,000 protein of rat sciatic nerve. Although faint on the reproduction, it is definitely discernible on the original immunoblot. No protein band in the PO or Mr = 41,000-42,000 region is immunocrossreactive with either rat or chicken brain proteins (Fig. IA, B, lanes 3 and 4). A number of lower bands appeared on the epitope selected immunoblot. These may be attributed to breakdown products of the PO protein. Molecular weight differences between the rat and chick PO proteins are also detected by the in vitro translation experiments (Fig. 2). The quality of poly(A ÷) RNA and the efficiency of the in vitro translation system is illustrated in lanes 1-4 of Fig. 2C, indicating a wide range of total translation products. In vitro translation in a reticulocyte lysate system of exogenous sciatic nerve poly(A ÷) RNA from the two species indicates that the major specific immunoprecipitated translation product of rat poly(A ÷) RNA has a molecular weight of about 31,500 in contrast to the 28,300 primary translation product of the chick message (Fig. 2A, lanes 1 and 2). Two types of control experiments were carried out to demonstrate that the above reactions were specific. Immunoprecipitation of the rat in vitro translated PO, in the presence of an excess of purified chick PO protein greatly decreased the intensity of the Mr = 31,500 band (Fig. 2B, lane 5). The same experiment with the chick in vitro translated PO demonstrated 100% competition by an excess of purified chick PO protein (LeBlanc and Mezei, 1985). Furthermore, immunoprecipitation from total translation product of rat brain did not yield a Mr = 31,500 band (Fig. 2B, lane 3). However, similar experiments with m R N A from 19-day-old chick brain also results in the appearance of an approximately Mr = 28,000 protein band crossreacting with the polyclonal anti-chick PO antibody (Fig. 2B, lane 1). This polypeptide cannot be competed out by an excess of purified chick PO protein (Fig. 2B, lane 2). Immunoprecipitation with anti-chick PO IgG of this in vitro translation product of exogenous chick brain poly(A ÷) RNA was reproducible. In addition, the intensity of the immunoprecipitated band increases significantly during brain development (results not shown). Isoelectric focussing followed by SDS-PAGE in the second dimension of the immunoprecipitated in vitro translation products of PO mRNAs from rat and chick sciatic nerve demonstrates a difference in isoelectric points between the two proteins. The in vitro synthesized chick PO protein has an approximate isoelectric point of 6.0 as contrasted to the value of 7.0 for the rat polypeptide (compare Fig. 3A and B). The rat immunopreeipitated PO migrates as a smear. The position of the PO protein is indicated by a small arrow (Fig. 3B). The heterogeneity of the protein band in the second dimension may be due to ampholyte-protein complexes which form during the first dimension. Limited proteolysis by chymotrypsin also indicates that the overall peptide pattern of the

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Fig. 3. Isoelectric focussing, followed by SDS-PAGE in the second dimension of the immunoprecipitated in vitro translation products of poly(A +) RNAs from chicken (A) and rat (B) sciatic nerve. For details see Materials and Methods. The direction of migration for the first dimension and second dimension are indicated by the arrows. The pH gradient in the first dimension is from 8.0 to 5.0. The small arrow in (A) and (B) indicate the position of PO protein. chick and rat PO protein immunoprecipitated from in vitro translation, are different. Only two major hydrolysis products exhibit similar electrophoretic mobilities (Fig. 4, lanes 1 and 2). The difference between the properties of the in vitro translation products of rat and chicken PO message is further demonstrated by the in vitro translation of the poly(A ÷) RNAs of the two species in the presence of dog pancreas microsomal membranes (Fig. 5, lanes 2 and 4). Partial processing of the primary translation products causes the appearance of a second lower molecular weight band in both species. In addition, processing of the rat translation product indicates the appearance of a third band with an apparent molecular weight of approximately 24,700. However, complete conversion of the higher molecular weight pri-

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ANDREAC. LEBLANCand CATHERINEMEZEI is smaller and the intensity of the signal is much lower than with the rat PO message (Fig. 6B, lane 1). These results thus indicate a low degree of homology between the rat and chick PO message. The low level of interaction of the chick PO mRNA and the rat PO cDNA probe was further confirmed by quantitative dot blot analysis. Figure 7, lane B indicates that the rat cDNA probe could detect picomole of the homologous message from the young rat sciatic nerve preparations, whereas very low amounts were present in sciatic nerve extracts of chicks (Fig. 7, lane A). Finally, Southern blot analysis of rat, chicken, human and hamster DNA demonstrated again the lack of hybridization of the rat PO cDNA probe to the chicken genome (Fig. 8, lane 2). In contrast, clear-cut hybridization was achieved with the homologous (rat), and the related, but heterologous DNAs of other mammalian species (compare lanes 1-4, Fig. 8).

Fig. 4. Effect of limited chymotrypsin treatment on the in vitro synthesized PO protein from chick and rat sciatic nerve. SDS-PAGE purified PO proteins from the reticulocyte cell-free system were subjected to limited proteolysis by ehymotrypsin, analyzed by 15% (w/v) SDS-PAGE and detected by autoradiography as described in Materials and Methods. Lane 1, chymotrypsin treated rat PO protein; lane 2, chymotrypsin treated chick PO protein. Arrows and numbers indicate the position and size (Kda) of Pharmacia low molecular weight standards.

mary translation products of the PO messages could not be achieved even in the presence of saturating amounts of dog microsomal membranes (results not shown). Northern blot analysis of total poly(A +) RNA from sciatic nerves of the two species illustrates the presence of an RNA band from rat sciatic nerve at the 18S level (Fig. 6A, lane 3) which specifically hybridizes to the radioactive 1,85 kb rat PO cDNA probe. No such mRNA species could be detected in poly(A +) RNA preparations from either 19-day-old chick embryonic nerve or that of post-hatch birds (Fig. 6A, lanes 1 and 2). However, an RNA band can be detected by hybridization of the 604 bp coding region of the rat PO clone to chick sciatic nerve poly(A +) RNA (Fig. 6B, lanes 3). This RNA species

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Fig. 5. Effect of microsomal membranes on the SDS-PAGE pattern of immunoprecipitated in vitro translation products of poly(A+) RNA from the sciatic nerve of rat and chicken. The translation product from rat sciatic nerve (lanes 1 and 2), and chick sciatic nerve (lanes 3 and 4) were immunoprecipitated and analyzed as described in Materials and Methods. The in vitro translation in samples of lanes 1 and 3 was carried out in the absence of microsomal membranes, whereas those in lanes 2 and 4 were performed in the presence of dog microsomal membranes.

Avian and mammalian PO protein

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Fig. 6. Northern-blot analysis of chick and rat poly(A+) RNA from the sciatic nerve. Hybridization was carried out with 32p-labelled rat Po-cDNA probe as described in Materials and Methods. The amount of fractionated poly(A+) RNA in each lane was 4 # g. (A) lanes 1-3 are poly(A+) RNA from sciatic nerves of 19-day-old chick embryos, 8-day-old chick and 21-day-old rats respectively hybridized to the full 1.85 kb fragment of the rat cDNA clone. (B) lanes 1 3 are poly(A+) RNA from 21-day-old rat sciatic nerve, 21-day-old rat brain and sciatic nerve of 19-day-old chick embryo respectively hybridized to the 604 bp coding region of the rat cDNA clone. The arrows and numbers indicate the positions and size (S value) of rRNA markers on the agarose gels. The results of Northern blot, dot blot and Southern blot analysis therefore clearly indicate a considerable degree of dissimilarity between the structures of rat and chicken PO genes. DISCUSSION Comparative studies are very useful in the elucidation of domains in the major myelin proteins, which are essential for their function (Blaurock, 1985; Tai et al., 1986; Waehneldt et al., 1986). Our investigations provide experimental evidence for major structural differences between the mammalian and avian PO protein. The results of immunoblotting confirm our previous studies and demonstrate that in situ, the PO protein of chick sciatic nerve exists in a slightly higher molecular weight form than the rat PO protein (Mezei and Verpoorte, 1981; Nunn and Mezei, 1984). The apparent difference in molecular weight is not necessarily accurate since glycosylated proteins do not migrate according to their molecular weight in SDS-PAGE. However, even after the removal of the oligosaccharide moiety by endo F, the molecular weight differences persist between the two proteins (Fig. 1A or 1B, lane 2 and 6). The difference in the migration of the two proteins after endo F treatment

seems to indicate that they undergo different types of post-translational modification besides Nglycosylation. It is known that the mammalian PO contains covalently-linked phosphate, sulphate and fatty acid residues as well as the oligosaccharide moiety (Agrawal et al., 1983; Quarles, 1980; Singh and Spritz, 1976). Analysis of the primary translation products from the two species demonstrate a difference in the lengths of the polypeptide chains of the PO proteins. In vitro translation in a reticulocyte lysate system of exogenous poly(A +) RNAs from the mammalian and avian sciatic nerve demonstrates that the primary translation product of the rat PO m R N A has a higher molecular weight than that of the avain immunoprecipitated product (Fig. 2A). The in vitro synthesized chicken PO has a molecular weight of about 28,000 and that of the rat PO, 31,500. Lemke and Axel 0985) also reported a similar value for the translation product of the rat PO message. These investigators deduced the complete amino acid sequence of the rat PO protein from the nucleotide sequence of the cloned PO cDNA, and demonstrated the presence of a 28 amino acid N-terminal leader sequence. They speculated that this sequence probably aids the insertion of the primary translation product into the lumen of the endoplasmic reticulum

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ANDREA C. LEBLANC and CATHERINE MEZEI

be due to a variety of post-translational modifications. Further evidence for a structural difference between the PO proteins of the two species is provided by the processing of the in vitro translation products (Fig. 5). Dog pancreas microsomal membranes have the ability to provide both signal peptidase activity and core glycosylation of the primary translation product. The immunoprecipitated translation products synthesized in the presence of microsomal membranes appeared as double radioactive bands, indicating partial processing of the PO polypeptide. However, the partially processed doublets of chick PO still have lower molecular weights than those of the rat PO proteins. As calculated from the rat PO c D N A sequence, removal of the signal peptide would give a peptide approximately M r = 29,000. The lower band obtained from in vitro processing also migrates corresponding to a

Fig. 7. Autoradiogram of dot-blots of poly(A+) RNA from chick and rat sciatic nerve, hybridized with 32p-labelledrat PO cDNA probe. One in two serial dilutions of 2#g poly(A÷) RNA from sciatic nerves of 19-day-old chick embryos (A), and those of 21-day-old rat (B) were immobolized on nitrocellulose membranes and analyzed as described in Materials and Methods. membrane. It is most likely that this sequence is cleaved by the signalase located in the membrane during core-glycosylation or soon afterwards. The small size of the chicken PO protein might indicate it contains an internal signal sequence, which is retained in the polypeptide after insertion into the lumen of endoplasmic reticulum and finally into the plasma membrane of the Schwann cell. Precedents for this latter hypothesis have been observed to occur in other systems (Wickner and Lodish, 1985). The primary translation product of the chick PO message, synthesized in the absence of microsomal membranes has a slightly lower molecular weight than the protein extracted from the sciatic nerve of the birds (compare Fig. 2A, lane 1 and Fig. IA, lane 1). The reverse is true for the rat in vitro and in vivo product (Fig. 2A, lane 2 and Fig. IA, lane 5). The difference in molecular weight between in vivo and in vitro product could

Fig. 8. Southern-blot analysis of DNA from various species with the rat PO eDNA probe. DNA (lane 1, Chinese hamster; lane 2, chicken; lane 3, human; lane 4, rat) was extracted, digested with Eco RI, fraetionated, blotted on Zeta probe, hybridized to the 1.85 kb rat PO clone and the filter exposed to Kodak X-OMAT-S film for 3 days at -70°C. Each lane contained about 10pg of Eeo RIdigested DNA. The arrows and numbers indicate the positions of marker fragments (kb).

Avian and mammalian PO protein Me = 29,000; therefore we can assume that this band is the result of signal peptidase activity on the primary product. The upper band could also represent PO protein which is core glycosylated in addition to having the signal peptide cleaved. Further experiments of in vitro processing followed by endoglycosidase H cleavage on the glycosylated product should provide an answer. Isoelectric focussing experiments confirmed a difference not only in size but also in the isoelectric point between the PO proteins of the two species (Fig. 3A and B). The chick PO primary in vitro translation product had an apparent isoelectric pH of 6.0 as compared to the value of approximately 7.0 for the rat PO. Isoelectric focussing, followed by SDS-gel electrophoresis in the second dimension, of radiolabelled rat sciatic nerve proteins or myelin proteins was reported by Linington and Waehneldt (1983). These investigators commented that unlike the basic proteins which migrated as distinct spots under these conditions, the PO protein appeared as a broad band on the autoradiogram, with a distinctly more acidic range of pI. Our isoelectric focussing experiments confirm a previous report from our laboratory, in which we compared the amino acid composition of purified chick PO protein with that from mammals. This comparison revealed some definite species differences. Particularly, the ratio of the acidic amino acids glutamate and aspartate (amino acid analysis includes glutamine and asparagine as well) to the basic amino acids lysine and arginine is highest in the avian PO protein [compare Table 1 from Mezei and Verpoorte (1981) to results of Lees and Brostoff (1984), Lemke and Axel (1985) and Uchida et al. (1986).] This comparison predicts the polypeptide backbone of chick PO protein is relatively more acidic than that of the mammalian polypeptide. Further evidence for a difference in amino acid sequence between the rat and chick PO proteins was obtained in the limited proteolysis experiments of the immunoprecipitated in vitro translation products (Fig. 4). This study showed the overall peptide maps of the two proteins were different, although some of the major hydrolysis products migrated to the same position on the gel. The difference between the PO genes of chick and rat is confirmed by dot blot, Northern and Southern analysis (Figs 6--8). The Southern blot demonstrates homology of the 1.85 kb rat PO eDNA clone with Chinese hamster and human DNA but no signal is detected in the chicken DNA. Northern analysis also failed to show hybridization of the 1.85 kb rat PO clone to young and old chick poly(A ÷) RNA. However, the 604bp Pst I fragment which consists mainly of the coding region of the rat PO gene did hybridize to chick poly(A ÷) RNA. The signal is less intense than that obtained for the rat although the same amount of poly(A ÷) RNA from rapidly myelinating animals was transferred to the blot. This indicates that there exists a low degree of homology in the coding region of the rat and chick PO genes. The chicken PO m R N A is also smaller than the rat PO mRNA which is in agreement with the in vitro translation results. A number of investigators have reported the existence of substantial differences in amino acid composition and structure of CNS and PNS myelin

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proteins, particularly between fishes and tetrapods (Tai et al., 1986; Waehneldt et al., 1986). The difference between the structure of the major PNS myelin glycoprotein of mammals and birds is, however, rather unexpected. A phylogenetic tree based upon differences in the amino acid sequences of cytochrome c in a variety of organisms indicates that birds and mammals are more closely related on the evolutionary scale than fishes and mammals (Fitch and Markowitz, 1970). Furthermore, the overall physico-chemical properties of the PNS myelin in mammals and birds have been well conserved during evolution (Sedzik et al., 1985). In addition, the fact that antibodies to chick PO protein can bind to PO proteins from a variety of mammals (Nunn and Mezei, 1984), and can immunoprecipitate in vitro translated rat PO protein also indicates that certain antigenic determinants in the proteins are very similar (Figs 2-5). On the other hand, Tai and co-workers (1986) reported the shark basic protein has some antigenic determinants in common with the proteins from higher vertebrates although their structure is different. Immunoprecipitation of the in vitro translation products of chick brain poly(A +) RNA yielded a Mr = 29,000 protein. This was rather unexpected since no such protein is detected by immunoblotting chick brain protein extracts. Addition of a large excess of purified chick PO failed to eliminate the immunoprecipitation reaction. This observation was completely reproducible. Furthermore, a developmental profile indicated that the message for this protein seemed to increase from 17 embryonic days to 1 day post-hatch (results not shown). Also, no such product was immunoprecipitated from in vitro translation of rat brain poly(A ÷) RNA. In an attempt to explain these apparently contradictory observations we suggest a posttranslational modification in vivo might mask the antigenic determinants detected by the polyclonal antibody. The presence of an antibody in the polyclonal antiserum which reacts with high affinity to an in vitro translated brain protein cannot be ruled out. It must be pointed out that higher molecular weight proteins were detected by immunoblotting. These could be in vivo processed forms of the in vitro translation products. It seems plausible that there are common antigenic determinants in a variety of membrane-associated proteins. In this respect, it has been shown that the Pz protein of PNS myelin possesses close structural relationships to rat liver Z-protein or a protein important for adipocyte differentiation (Bernlohr et al., 1985; Cook et al., 1985; Takahashi et al., 1982). Furthermore, C-terminal subsequences of JC, BK and SV40T antigens are similar to sequences in myelin basic protein (Stoner, 1985). It appears that a variety of proteins have related structures which might be involved in similar function. The conserved regions in the mammalian and avian PO proteins could provide insights on the structure and function of PO proteins. This would be achieved only after the elucidation of the complete amino acid sequence of the chick PO protein. Currently, we are immunoscreening a chicken sciatic nerve 2gtll eDNA library and have obtained positive clones which are being characterized.

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ANDREA C. LEBLANCand CATHERINEMEZEI

Acknowledgements--The technical assistance of N. N. McGrath and skilful typing of L Laskey is gratefully acknowledge. This work was supported by the Medical Research Council of Canada.

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c.B.P, s7/4B~-Q

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