Characterization of a cloned cDNA encoding rabbit myelin P2 protein.

THEJOURNAL OF BIOLOGICAL CHEMISTRY 0 1988 by The American Society for Biochemistry and Molecular Biology, Inc.

Val. 263,No. 17, Issue of June 15,pp. 8332-8337,1988 Printed in U.S.A.

Characterization of a Cloned cDNA Encoding Rabbit Myelin P2Protein* (Received for publication, October 20, 1987)

Vinodh NarayananSQ, ErnestBarbosaS, Randall Reedll, and Gihan TennekoonSll From the Departmentsof $Neurology and (IMoleculorBiology and Genetics, The Johns Hopkins University School of Medicine, Baltimore, Marylond21205

Myelin P2 is a 14,800-Da cytosolic protein found in proteins being a relatively minor component (30%) (3). In rabbit sciatic nerves. It belongs to a family of fatty central nervous system myelin, the major proteins areproteoacid binding proteins and shows a 72% amino acid lipid protein, myelin basic protein (MBP’), and theWolfgram sequence similaritytoaP2/422,the adipocyte lipid proteins (including 2’,3’-cyclic nucleotide 3”phosphodiesterbinding protein,a 58%sequence similarity to rat heart ase). In peripheral nervous system myelin, the major proteins fatty acid binding protein, and a 40% sequence simi- are Po glycoprotein, MBP (also called PI),and Pzprotein, the In order one of interest to us. The function of these proteins is not larity tocellular retinoic acid binding protein. to isolate cDNA clones representing P2,a cDNA library known, although it has been postulated that proteolipid prowasconstructedfrom poly(A+) RNA isolated from tein, Po, and MBPserve as structuralcomponents of myelin sciatic nerves of 10-day-old rabbit pups. By use of a mixed synthetic oligonucleotide probe based on the (4,5), with Poand MBP playing an additional role in myelin rabbit Pz amino sequence, 12 cDNA clones were se- compaction (6). During the past several years, cDNA clones lected from about 25,000recombinants. Four of these representing proteolipid protein, Po, 2’,3’-cyclic nucleotide were further characterized. They contained an open 3‘-phosphodiesterase, and MBPhave been isolated and charreading frame, which when translated, agreed at 128 acterized (6-10). Encouraged by these results, we have applied out of 131 residues with the known rabbit P2 amino similar methods to the study of Pzr a 14,800-dalton cytoacid sequence. These cDNAs recognize a 1.9-kilobase plasmic basic protein. The amino acid sequences of bovine ( l l ) , human (12), and rabbit(13) Pzprotein have been determRNA present in sciatic nerve, spinal cord, and brain, but not present in liver or heart. Thelevels of P2mRNA mined, and each displays a strikingsimilarity to thesequences parallel myelin formation in sciatic nerve and spinal of proteins that bind fattyacids in adipocytes (14), liver (15), cord withmaximal amounts being detected at about 15 intestine (16), andheart (17). Postulated roles for these postnatal days. This initial study will allow character- proteins include uptake and transport of fatty acids to intraization of the P2 gene and its regulation, as well as cellular organelles, targeting fatty acids to specific metabolic further studies into the role of P2,the first metaboli- pathways, and maintenance of intracellular pools of fatty cally active myelin-specific protein tobe characterized acids. This suggests that P, may serve as a lipid carrier and at the genetic level. is probably involved in assembly, remodeling, and maintenance of myelin. Immunization of susceptible animals with Ppprotein leads to T cell-mediated destruction of peripheral Myelin sheaths thatinvest axonsof the central and periph- myelin (18). We report here the characterization of cDNA clones reperal nervous systems are elaborations of the plasma membrane of myelin-producing cells. In the central nervous sys- resenting Pzisolated from a cDNA library constructed from tem, this is done by oligodendroglia, and in the peripheral rabbit sciatic nerve RNA. In additionto thecomplete nucleonervous system, by Schwann cells (reviewed in Ref. 1).The tide sequence, we have studied the tissue distribution and morphological changes that are observed during myelination variation of Pz mRNA levels during development. This inforare preceded by biochemical changes that include synthesis mation is a starting point for studies of the P2 gene and its of lipid and protein components, and transport of these com- regulation, as well as characterization, at a molecular level, of ponents to appropriate intracellular destinations where mye- the process of myelination. lin membranes are assembled. Initially myelin is loosely EXPERIMENTAL PROCEDURES’ wrapped around axons and subsequently undergoes compaction to give the membrane its characteristic appearance (2). RESULTS Myelin is composed predominantly of lipids (70%), with Characterization of cDNA Clones-A cDNA library was * This work was supported, in part, by Grants NS21700-01A1 and PO1 NS22849-01 from the National Institutes of Health. The costs constructed in bacteriophage hgtl0 from poly(A+) RNA isoof publication of this article were defrayed in part by the payment of lated from sciatic nerves of 10-day-old New Zealand white page charges. This article must therefore be hereby marked “adver- rabbit pups. A 64-fold degenerate 26-base-longoligonucleotide tisement’’ in accordance with 18U.S.C. Section 1734 solelyto indicate this fact. The nucleotide sequence(s) reported in thispaperhas been submitted totheGenBankTM/EMBLDataBankwith accession number(s) 503744. § Recipient of a Charles A. Dana Foundation Fellowship. 11 To whom correspondence and reprint requests should be addressed Dept. of Neurology, Meyer 6-119, The Johns Hopkins Hospital, 600 N. Wolfe St., Baltimore, MD 21205.

The abbreviations used are: MBP, myelin basic protein; FABP, fatty acid binding protein; bp, base pair; kb, kilobase pair; N, a nucleoside; HPLC, high performance liquid chromatography; SDS, sodium dodecyl sulfate; AMV, avian myeloblastosis virus. The “Experimental Procedures” are presented in miniprint at the end of this paper. Miniprint is easily read with the aid of a standard magnifying glass. Full size photocopies are included in the microfilm edition of the Journal thatis available from Waverly Press.

8332

Cloning of cDNA Encoding Rabbit Myelin Pz Protein

8333

3, TCG T I G TlT MA GAT CCC 5 ' mixture, GT-5, corresponding to amino acids 14 to 22 of the I C ~ T C C C A C C T A G M C T G T G C G G A G ~ T I C ~ C U T U fATG f i AGC M C MA TIl CTA GGC ACC TGG MG Ser Asn Lys Phe Leu Gly Thr Trp Lys 9 rabbit P, protein (5' GAA/G-AAC/T-TTC/T-GAT-GAC/T_"""" "-

TAC/T-ATG-AAA/G-GC 3') was synthesized. The oligonucleotide was chosen to correspond to a peptide fragment close to theNH, terminus of the protein, increasing the likelihood of isolating a full length clone. This oligonucleotide probe selected 12 cDNA clones from about 25,000 recombinants. Four of these, pSN2.1, pSN2.9, pSN2.8, and pSN2.2-2, were subcloned into plasmids for further analysis (Fig. L4). When used as a probe, pSN2.1, pSN2.9, and pSN2.2-2 each recognized the cDNA insert fragments in each of the 12 phage clones isolated, suggesting that they belonged to a single crosshybridizing family. The nucleotide sequences at the 5'- and 3"termini of pSN2.1, pSN2.9, pSN2.8, and pSN2.2-2 were determined. An open reading frame was found, which when translated, corresponded to the NH2 terminus of rabbit myelin P, protein. This confirmed that these clones represented P, and not some other related protein. Northern analysis (Fig. 5) suggested that the Pa mRNA was about 2 kb in length, and, therefore, that pSN2.2-2 was closeto a full length cDNA clone. When transcribed and translated in vitro, this clone yielded a 15-kDa protein (data not shown). The strategy that we adopted for nucleotide sequence determination is shown in Fig. 1B. Nested deletions were made from both ends of clone pSN2.2-2 by using exonuclease I11 and S1 nuclease. Single-stranded DNAwas rescued from transfected NM522 cells with the R408 helper phage and used for dideoxynucleotide sequencing. Combining the data from pSN2.1 and pSN2.2-2 yields the sequence for a 1863-bp Pz cDNA (Fig. 2). This cDNA encodes a short 5'-untranslated region, a 393-bp coding region, and a 1395-bp-long3'-untranslated region followed by a poly(A) tail. Translation of this nucleotide sequence agrees at 128 out of 131 residues with the published rabbit Pz amino acid sequence (13). The differences occur at positions 72 (glutamine by protein sequencing and glutamic acid from the nucleotide sequence), 83 (threonine by

76 CTI GTC TGC AGC G M M T TlT GAT GAT TICA% MA K T CTG GCT GTG GGG TTA GCC ACCAGA Leu Val Ser Ser Glu A m Phe Asp Asp Tyr net Lys Ala Leu G l y Val Gly Leu A h Thr Ar8

30

139 MA CTG GGA M T Tx GCC MA CCC M T GTG ATC ATC AGC MG MA u% GAT ATC ATC ACT ATA Lyr Leu Gly A m Leu Ala Lyr Pro A m Val Ile Ile Ser Lys Lyr GlyAsp 11s I1e Thr Ile

51

202 CGA ACT GM AGT ACC TIC MA M T ACT GAG ATC TCCTIC MG CTA GGC CAG GM TlT GM GM Arg Thr Glu Ser Thr Phe Lys As" Thr Glu Ile Ser Phe Lys Leu Gly Gln Glu Phm Glu

72

265 ACC ACA GCT GAC M T AGG MA ACC MG AGT ATC ATA ACC CTG G M AGA GGC GCA T I G M T CAG Thr Thr Ala Asp A m k g Lys Thr Lyr Ser & Ile Thr Leu Glu Arg Gly Ala Leu As" Gln

93

328 GTA CAG MA TGG GAT GGC MA GAG ACA ACC ATAMC AGG M G Tx GTG GAT GGG A M ATG G T I Val Cln Lys Trp & Gly Lyr Glu Thr Thr Ile Lys Arg Lys Leu Val Asp Gly LysMet Val

114

391 GTG CM TGT MC ATG MG GGC GTG GTC lYX ACC AGA ATC TAT GAG M G GTC TGA GTA A T I TGT Val Clu Cyr Lys Met Lya Gly ValVal Cys Thr Arg Ile Tyr Glu Lys Val * 131 GCT G M GTG ATA lTI U T UTIA T ATG l T G GTA GTC M T TGA C M GAC CTGAGT ATA EGCT T CAT lTG lTI GGT lTI GCT TlT G T I TCA MG GCT CAG ATA GGC MA GGC C M GGA TIA ACC C M AGT TCA GTA TIA TlT MA U T TCT CCA TGT ATA TGC ACG TCA TIA T M AGC M C TTA M T MTGAT TGA E T UTTA T ATI M C TGC C M ATI ATA ACC TAT TMA U AGT M T TGT AGA CTG T I A MA GGA M T MA M T A T I TAT TAT MA TGA T M M T MMA C CTC MA TAG ATI CAG GGA CCA GGA MG TIA AGA GGA AGG AGG ATC AGA GCA ATC TGCCTI MG TlC AGG K T KGCT T GM GGC ATI GTG GGA ACA TGG T I A AGA GGG TAT GCC TGC A T I CCA TAT CAG U T MTGA C G T I TGA G T T CCC ACT CTG CTI CCT GCT TTC TAC T M TGC ATA TCC TGG GAG GCA GCA GGT GTT GGC T U ATA CTI GGT CCC TGG MA CCA U C GGG AGT CCC AGA T I G AGT TCT GGA CTC ATG G T I TIA GCC K C CTC AGC CCC AGC TGT GGC AGG U T T I G GAG AGT G M TCA GTG GGT GGC T l ICTC TCT CTC TCT CTC TCT CTC TCT CTC TCT CTC TCT CTC TCT CCC TCT TlT CCC CTT ACT GTC TCT CTC ACC CTC CTC CCT CCC TIC CTI CTI TIC TIC CTI CCT T U TIl CTI TIl CCT GTC TCT CTC TlT CCC TCT CCC ACC CTC CCT CTCTCT CTA T I G CTI TIC CTT TCA M T GM T M MA T M ATA U T AGA GGA lTI CTA GTC TCA GCT GM AGG GGC ACC CTC TAC CCC M C T U TIC TIl MA TPG AGG GAT U T GTA TAG MG 1399 GTG MA TGT AGA TAT T M GGG TAG AGT TGA A T I GCT TGT GAT GM Tcc A X CAC CAT GTAACT TACA U TIC MG ACA TlT CCT T M TCC TAG MA G T I TIC TITI E C CCT TGC CCA 14b2 CACAGC C M 1525 A T G TCC TGC CCA GGG CCA CTC TCA CTA M C C U ACT ATA GTA GTG ATI TAG TIC CTC TCA CTT 1588 CAG ATI AGT lTG TCT G T I CAT ATA T M ATG GGA CTA TAC ATI GTI TIT CTI K T TCT G T I TIC TIT Tx ACT TM: ATC CAC ATP GTG CCA TAT AGT GGT AGT ACA 1651 CTI Tl7 MA TIC ATG 1714 TICU T TIT A T I GTA MG TAG CAT TCC ATT CTA AGG ATA TAT CAC MA ATA TGT ATA CAT TCT 1777 TCT G T I MA ACA A 1 7 GAG l T G TIl TTA G T I Tx GCT ACT ATG W T T GCT ACC M C MA 1840 P

454 517 580 b43 706 769 832 895 958 1021 1084 1147 1210 1273 1336

TIC CTI CTG U T MA TGA CTG TAT GCT GTA G T I TGG ACT GAG ACC TCT GTG CTI

FIG. 2. Nucleotide sequence and corresponding P, amino acid sequence. The sequence of the sense strand of the P, cDNA clones, a composite of pSN2.1 and pSN2.2-2, 1863 bases long, and the translated amino acid sequence, are shown. The nucleotide number is shown to the left of each line, and, in the coding region, the amino acid number is indicated to the right of the corresponding line. Stop codons are indicated by (*) under the TAG or TGA sequence. Differences between this and the published rabbit P, amino acid sequence occur at residues 72, 83, and 98, and are underlined. The position of the mixed synthetic oligonucleotide, GT-5, used to screen the cDNA library, is indicated (- - - -) above positions 88-113. The sequence of the complementary oligonucleotide, GT-7, used for primer extension is shown above positions 49-66. Possible polyadenylation signals and a long CT repeat in the 3"untranslated region are underlined.

protein sequencing and isoleucine from the nucleotide sequence), and 98(asparagine by protein sequencing and aspartic acid from the nucleotide sequence). Sequence data from psN 2.9 pSN2.8 and pSN2.9 (Fig. 1B) agree at these positions with the data obtained from pSN2.1 and pSN2.2-2, implying that Pa28 these three amino acid differences were not an artifact of pSN 2.2-2 ; cloning. The putative translation initiation ATG at position 5' 3' 46 is preceded by a stop codon at position 13 in the cDNA, suggesting that no amino-terminal peptide is removed after (B) I P2 tDNA translation. A polyadenylation signal consensus sequence AA-2.1 4 TAAA is found 174 bases away fromthe poly(A) tail, although c . ( -2.9 a nearly identical heptanucleotide AATGAAA is found within 10-15 bases of this site. This same motif also occurs internally in the 3'-untranslated region of the cDNA (at positions 725, 757, and between 1304 and 1319). These internal sequences do not appear to serve as polyadenylation signals, as only a single size mRNA species is detected on Northern blots (Fig. " 5). This is distinct from the situation for myelin proteolipid c-.l protein, where two mRNAspecies, 3.2 kb and 1.6 kb in length, 4 " result from polyadenylation at two distinctsites (7). The FIG. 1. cDNA clones and sequencing strategy. A, diagram segment from positions 1126 to 1167 (CT repeat) undoubtedly indicating the relative lengths and position of the coding region renders a unique conformation to the DNA in this region, (shaded) of the cDNA clones pSN2.1, pSN2.9, pSN2.8, and pSN2.2- although the importance of such a structure is not known. 2. B , sequencing strategy. Indicated at the top is the P, cDNA We compared the amino acid sequences of rabbit Pzprotein (composite of pSN2.1 and pSN2.2-2) with the coding region shaded. with that of aPz/422, rat heart, liver, and intestinal fattyacid Numbered arrows represent the sequence data obtained from the 5'and 3'-ends of clones pSN2.1 and pSN2.9, and one entire strand of binding proteins (FABPs), and cellular retinoic acid binding pSN2.8. The remaining arrows represent sequence information of protein. Alignment of the amino acid sequences of these overlapping segments of pSN2.2-2 spanning the entire cDNA and proteins, comparing each to P, protein, is shown in Fig. 3. P, including both strands. and aPz/422 are identical at 95 amino acid residues, yielding

(A)PsN 2.1

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Cloning of cDNA Encoding Rabbit Myelin Protein P2

SNKFLGTWKLVSSENFDDVMKALGVGLATRKLGNLA

KPNVllSKKG

HCDAfVGTWKtVSSbNFDDYMKEVGVGFKTRKVAGMA KPNMIISVNG TEKNF.VGTWKLVDSKNFDDYHKSCGVGF~T~OVASHT K P T T l l E K N G

P2

a b 1422 HeartFAEP

HPW FAGTWKMRSSENFDELLKAT;GVNAMLRTVAVAKASKPMVEIRODG C W W MNFSGKVOVOSOENFEPFHKAMGLPEDLIOKGKDI K G V S E I V H E G UverFAEP N L K L T l T O E G InlestineFABP HAFDGTWKVYRNENYEKFMEKMGINVVKRK~GAHD

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P2 mRNA

5' " " "

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3' GT-7

5' D I I T I R T E S T F K N T E I S F K L G O E F E E T T A D N R K T K S I I T L E R GALNO D L V T I R S E S T F K N T E I S FKLGVEkDElfADDRKVKSfITLDG GALVO DTIVGKTHSTFKNTEISNCOLGVEDDRKVKSVVTADDRKVKSVVTLDG GKLVH

3' pSN2.1

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D O F Y I K T S T T V R T T E I N FKVGEGFEEETVDGRKCRSLPTWENENKIHC Cw\Bp KKVKLTITVGSKVIHNE FTLGEECELETMTGEKVKAVVKMEGDNKMVT UverFAEP N K F T V K E S S N F R N I D V V F E L G V D F A V S L A D G T E L T G T L T M E G NKLVG lnles11neFAEP

VOK VOK VOK

WDGKETTIKRKLVDGKHVVECKHKGVVCTRIVEKV

WDGKSTTIKRURDGDKLVVECVHKGVTSTRVYERA

WDG E T T L T R E L S D G KLLTI L T H G N V V S T R T Y E K E A TOTLLEGDGPKTVWTRELANDELILTFGADDVVCTRIYVRE TFK GIKSVTEFNGDTITNTMTLGDIVYKRVSKRI KFK R V D N G K E L l A V R E l S G H E L l O T Y T V E G V E A K R l F K K E

G

A

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FIG. 3. Alignment of FABP amino acid sequences. Alignment of the amino acid sequences of rabbit P, protein with that of aPZ/ 422, rat heartFABP, rat liver FABP, rat intestine FABP,and cellular retinoic acid binding protein (CRABP)is shown. Amino acid identity is indicated by shoding. The degree of sequence identity is 72% between P, and aP2/422, 58% between P, and heart FABP, 40% between P, and cellular retinoic acid binding protein, 29% between P, and liver FABP, and 21% between P, and intestinal FABP.

a 72% identity. P2 and rat heart FABP are identical at 77 residues, resulting in a 58% identity, and PI and cellular retinoic acid binding protein are identical at 54 residues, yielding a 40% identity. There is less similarity between PI and the liver and intestinal FABPs (21% and 29%, respectively). Inherent in the method of cDNA library construction, where RNase H and DNA polymerase I are used forsynthesis of the second cDNA strand, is the loss of some nucleotide sequence information at the 5"terminus. To determine approximately how much of the P2mRNA was not represented in the cDNA sequence of pSN2.1 and pSN2.2-2, primer extension experiments were carried out. An 18-baseoligonucleotide primer complementary to positions 49-66 of the mRNA strand (Fig. 2) was end-labeled and annealed to template poly(A+) RNA isolated from rabbit spinal cord, a tissue in which largeamounts of P2mRNA weredetected by Northern analysis. The DNA-RNA hybridization was done in 80% formamide at 37 "C. Unlabeled excess deoxyribonucleotides and reverse transcriptase were added forenzymatic extension of the primer (Fig. 4A). Labeled oligonucleotide, primer extended product, and sequencing reaction products with pSN2.1 as template and GT-7 as primer, were separated on an 8%sequencing gel. The autoradiogram (Fig. 4B) indicates that the P2 mRNA is approximately 54 bases longer at the 5"terminus than is represented in the cDNA clones that we have isolated, making it about 1.9 kb in length. This fact will be of importance during analysis of genomic clones and the identification of the transcription initiation site. Tissue Specific ExppressionlDevelopmental Profile-On RNA blots, the cDNA clones pSN2.1, pSN2.9,and pSN2.2-2 each recognize a single mRNA species about 2 kb in length. P2 mRNA is detected in RNA isolated from sciatic nerve, spinal cord, and brain, but not in RNA isolated from liver or heart (Fig. 5).These tissues are known to contain other fatty acid binding proteins. The intensity of the signal is greatest in sciatic nerve, less in spinal cord, and even less in brain, consistent with the caudal to rostral gradient of P2 protein levels demonstrated by immunocytochemistry (19). To study the developmental profile of P2mRNA levels,we extracted RNA from rabbit sciatic nerves and spinal cord at

D

FIG. 4. Primer extension analysis. A, diagram indicating site of hybridization of the anti-sense 18-mer, GT-7, to the Pz mRNA and pSN2.1. Dashed line indicates the primer extended cDNA strand. B, autoradiogram of primer extended product, labeled oligonucleotide, and sequencing reactions of pSN2.1 done with GT-7 as primer. The position of the end of pSN2.1 insert and thebeginning of the vector sequence, marked by the EcoRI recognition site (GAATTC) is indicated to the right. The band corresponding to the primer extended product is indicated (b) and lies about 54 bases away from the end of pSN2.1.

various ages (5, 10,15, and 20 days). Northern blots were prepared from these RNAs and probed with nick-translated pSN2.2-2. The autoradiogram (Fig. 6A) showsthat the variation in P2 mRNA is most striking in the spinal cord, with a peak signal at 15 days. To ensure that thevariation in signal intensity was not due to unequal amounts of total RNA in each lane, these same filters were rehybridized with a ribosomal RNA probe, XLRlOla (20). Autoradiography showeda fairly even intensity of bands corresponding to 28 S rRNA, implying that the amount ofRNA per lane wasroughly constant. We have converted the qualitative data of the autoradiogram into a graph by densitometric scanning. The resulting graph is shown in Fig. 6B,indicating that P2mRNA levels in relation to total RNA reached a peak at 15 days in both sciatic nerve and spinal cord. Analysis of mRNA levels

Cloning of cDNA Encoding Rabbit Myelin Protein P2 1

2

3

4

DISCUSSION

5

hc. 5. Tissue specific expression of Pa mRNA. Northern blot of total RNA (IO pgllane) from liver ( l a n e I ) , heart ( l a n e 2), brain ( l a n e 3), spinal cord ( l a n e 4 ) , and sciatic nerves ( l a n e 5) of 10-dayold rabbit pups, probed with 32P-labeledP2cDNA (pSN2.2-2 insert). The probe recognizes an approximately 2-kb message in sciatic nerve and spinal cord. A faint signal is present in the brain, easily seen on longer exposures, whereas none appears in liver or heart.

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3

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2

3

4

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FIG. 6. Developmental profile of Pa mRNA levels. Northern blots of rabbit sciatic nerve RNA ( A ) and spinal cord RNA ( B ) (8 pgllane) isolated a t 5, 10,15, and 20 days of age (lanes 1, 2, 3, and 4, respectively), probed with the P2 cDNA. These same filters were rehybridized with a ribosomal RNA probe for normalization. The positions of the P2 mRNA band andthe 28 S rRNA band are indicated. The photograph is a composite of two autoradiograms. C and D, graphic representation of densitometric scans of the bands seen in the above autoradiogram. The area under the curve of densitometric scan was taken as a measure of the intensity of the band. The area under the curve of the P, band was divided by that of the corresponding 28 S rRNA band, and the ratio arbitrarily scaled to equal 1.0 a t 5 days. Maximal P, expression is apparent a t about 15 days.

in tissue sections by in situ hybridization (currently in progress) will determine if this variation in P2 mRNA level is because of an increase in Schwann cell number or due to increased transcription in each cell.

P,, a basic protein of peripheral nerve distinct from myelin basic protein, was first isolated from bovine nerve roots (21) and biochemically characterized in terms of its amino acid composition (22), amino acid sequence (11-13), and immunological properties. Pphas been implicated in the production of a demyelinating disease, experimental allergic neuritis, in susceptible animals (18). We have described here the isolation and characterization of cDNA clones encoding rabbit Pz protein. The nucleotide sequence data and the primer extension experiment imply that the Pz mRNA is about 1.9 kb in length, with all but the first 55 bases identified here. The ATG codon at position 46 is followed immediately by the AGC codon forserine, the first amino acidresiduein the mature Pz polypeptide, and is preceded by an in-frame stop codon at position 13. Hence, this ATG is unambiguously identified as the initiation site for translation. The sequence flanking this ATG in the P2 cDNA (5’ ACG-ATG-A 3’) is different from the “optimal” sequence forinitiation in eukaryotes, 5’ ACC-ATG-C 3‘(23). It does,however,conform tothepattern 5’ A/G NNATG-N 3’ found in 95% of eukaryotic initiation sites (24). These observations imply that no amino-terminal peptide is cleaved off after translation. Processing at the NH,-terminus is limited to co-translational removal of the initiator methionine by methionine aminopeptidase, followed by acetylation of the NHz-terminal serine residue by N-acetyltransferase (25). Translation of the coding regionof the P2cDNA nucleotide sequence differs at 3 positions (residues 72, 83, and 98) from the amino acid sequence of rabbit P, protein, as determined by peptide sequencing methods (13). The sequence data from clones pSN2.9 and pSN2.8 confirm that the nucleotide sequenceis correct at these locations, suggesting that the errors in the published Pz sequence are due to limitations in peptide sequencing methods. No regions of Pa protein are similar to peptide fragments that have been associated with tyrosine kinase activity, but asingle tyrosine phosphorylation site (amino acid 19) exists (26). Unlike the other FABPs, the PzmRNA has an extremely long 3”untranslated region. This feature is common to all the other myelin-specific proteins for which cDNA clones have been isolated. The function of this region is unknown. Comparison of the NH2-terminal amino acid sequence of P,, cellular retinol binding protein and cellular retinoic acid binding protein led to suggestions that these belong to afamily of homologous proteins (27, 28). Motivated by this relationship, Uyemura et al. (29) demonstrated that Pzhas a high affinity for oleic acid,retinoic acid, and retinol. The P, amino acid sequence bears a striking similarity to that of aP2/422 (14) and to the ratheart FABP (17). There is less similarity to other proteins in this family, including rat liver and intestinal FABPs, and cellular retinoic acid binding protein (15, 16,30). We have presented here sequence comparisons of all these proteins to P,. The similarity in sequenceand in possible metabolic function ( i e . lipid binding) support the idea that these proteins are indeedhomologous (i.e. evolved from a common ancestral gene). Further support for this idea comes from structural studies. Analysis of circular dichroism spectra indicates that thetertiary structureof P, protein is predominantly @-pleatedsheet (31). A detailed analysis using empirical algorithms (including the method of Chou and Fasman) has provided a general model for the tertiary structureof P,. The picture that emerges is one of a @-barrelconsisting of anti-parallel @-strandssurrounding a hydrophobic core (32). This same motif (a criss-crossed network of antiparallel @strands surrounding a core, whichbinds a small, hydrophobic

Cloning of cDNA Encoding Rabbit Myelin P2Protein

8336

molecule) has been discovered in the structures of plasma retinol binding protein (33), @-lactoglobulin(34), and insect bilin-binding proteins (35, 36). The pattern of expression of the Pz gene has been studied by Northern analysis. More Pz mRNA is present in sciatic nerve and in spinal cord than in the brain,and none is detected in liver and heart, tissuesknown to contain distinct, but similar, FABPs. This caudal to rostral gradient of P, mRNA in nervous tissue recapitulates that observed in protein levelsby immunocytochemistry (19). The presence of different FABPs in various tissues and gradients within a single tissue (e.g. the central nervous system) may reflect variations in fatty acid metabolism (37, 38). It has been postulated that in enterocytes (cells lining the small intestine), which containboth liver andintestinalFABP, one protein might serve in re-esterification of absorbed fatty acids to form triacylglycerol, while the other protein might direct transport of fatty acids to mitochondria for @-oxidation(16). Within the centralnervous system, there are regional variations inlipid metabolism. The observed gradient in P, protein abundance may reflect such regional variations in metabolism, or may be due to the presence of another FABP in regions (such as thecerebral cortex) where P, is less abundant (39). The synthesis of very long chain fatty acids is unique to the nervous system, in particular, to myelin-producing cells (40, 41). The rate of synthesis of very long chain fatty acids in spinalcord is twice that in the cerebral cortex: reminiscent of the regional variation in P, mRNA levels. Fatty acids in the developing brain undergo elongation, rather than oxidation in mitochondria or peroxisomes (42).We have found that P, mRNA levels measured during development parallel the formation of myelin, a temporal profile similar to that of microsomal enzymes involved in fatty acid elongation (41). This raises the possibility that P, protein may be involved directly in fatty acid elongation, or perhaps in transport of very long chain fatty acids to myelin. The present research facilitates the application of molecular biologic methods to the study of Pz structure and function. It also permits direct study of the P, gene and its regulatory elements. Acknowledgments-Wearegrateful to Dr. B. Sollner-Webb for providing the XLRlOla cDNA, to Dr. M. Daniel Lane for providing the aPJ422 cDNA, and to Dr. B. Weiss for providingthe exonuclease 111. We thank Dr. Brian Largent for providing us with his method for the primer extension experiments, and to D. Jones for help with the computer alignment of the amino acid sequence data. We also thank Dr. Pamela Talalay for help in the preparation of this manuscript. REFERENCES 1. Morrell, P., ed (1984) Myelin, Plenum Publishing Corp., New York 2. Peters, A., Palay, S. L., and Webster, H.deF. (1976) The Fine Structure of the Nervous System: The Neurons and Supporting Cells, pp. 190-212, W.

B. Saunders Co., Philadelphia

3. Agrawal, H. C., and Hartman, B. K. (1980) in Proteins of the Nervous System (Bradshaw. R. A., and Schneider, D. M., e&) pp. 145-169, Raven

Press, New York

J. M. Bourre, unpublished observations.

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Cloning of cDNA Encoding Rabbit Myelin Pz Protein

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