Proc. Natl. Acad. Sci. USAVol. 83, pp. 1261-1265, March 1986Biochemistry

Primary structure of the human melanoma-associated antigen p97(melanotransferrin) deduced from the mRNA sequence

(tumor-associated antigen/amino acid sequence/transferrin superfamily)

TIMOTHY M. ROSE*, GREGORY D. PLOWMAN*t, DAVID B. TEPLOWt, WILLIAM J. DREYERt,KARL ERIK HELLSTROM*t, AND JOSEPH P. BROWN*t*ONCOGEN, 3005 First Avenue, Seattle, WA 98121; tDepartment of Pathology, University of Washington Medical School, Seattle, WA 98195; and tDivisionof Biology, California Institute of Technology, Pasadena, CA 91125

Communicated by Hans Neurath, October 21, 1985

ABSTRACT p97 is a cell-surface glycoprotein that ispresent in most human melanomas but only in trace amountsin normal adult tissues. To determine the structure of thistumor-associated antigen and to identify its functional do-mains, we have purified and cloned p97 mRNA and determinedits nucleotide sequence. The mRNA encodes a 738-residueprecursor, which contains the previously determined N-termi-nal amino acid sequence of p97. After removal of a 19-residuesignal peptide, the mature p97 molecule comprises extracellu-lar domains of 342 and 352 residues and a C-terminal 25-residue stretch of predominantly uncharged and hydrophobicamino acids, which we believe acts as a membrane anchor.Each extracellular domain contains 14 cysteine residues, whichform seven intradomain disulfide bridges, and one or twopotential N-glycosylation sites. Protease digestion studies showthat the three major antigenic determinants of p97 are presenton the N-terminal domain. The domains are strikingly homol-ogous to each other (46% amino acid sequence homology) andto the corresponding domains of human serum transferrin(39% homology). Conservation of disulfide bridges and ofamino acids thought to compose the iron binding pocketssuggests that p97 is also related to transferrin in tertiarystructure and function. We propose that p97 be renamedmelanotransferrin to denote its original identification in mel-anoma cells and its evolutionary relationship to serotransferrinand lactotransferrin, the other members of the transferrinsuperfamily.

p97 is a tumor-associated antigen that was first identified inhuman melanoma by using monoclonal antibodies (1-3). Ithas been studied extensively with regard to its expression innormal and neoplastic tissues and is present in most humanmelanomas and in certain fetal tissues, but it is found only intrace amounts in normal adult tissues (4-6). p97 has beenused as a target for diagnostic imaging of melanomas inhuman clinical trials (7).p97 is a monomeric cell surface sialoglycoprotein, with an

apparent molecular weight as determined by NaDodSO4/polyacrylamide gel electrophoresis of slightly less than97,000 (4). Use of monoclonal antibodies has defined threemajor antigenic sites, which are present on a stable Mr 40,000tryptic fragment (4). Subsequent work has shown that at leasttwo other independently characterized human melanoma-associated antigens, gp95 (3) and gp87 (8), are identical top97.The N-terminal amino acid sequence of p97 is homologous

to transferrin and, like transferrin, p97 binds iron (9). Anal-ysis of somatic cell hybrids and by in situ hybridization hasshown that the p97 gene, like the genes for transferrin and the

transferrin receptor, is located on chromosomal region3q21-3q29 (10, 11). These observations suggest that p97 playsa role in iron metabolism. To determine the structure of p97and identify functional and antigenic domains, we havecloned and sequenced p97 mRNA. The availability of clonedp97 cDNA will allow us to study the regulation of theexpression of p97 and to develop animal models to study theusefulness of such tumor-associated antigens in tumor therapy.

METHODS

Polysome Immunopurification. Polysomes prepared fromSK-MEL 28 melanoma cells (12) by magnesium precipitation(13) were purified by affinity chromatography using threemonoclonal antibodies specific for p97 as described (14).p97-enriched mRNA was isolated by elution with EDTA andpurified by affinity chromatography on oligo(dT)-cellulose(Bethesda Research Laboratories).cDNA Cloning with Oligo(dT) as Primer. For first-strand

cDNA synthesis, p97-enriched mRNA and oligo(dT) (Col-laborative Research, Waltham, MA) were incubated withreverse transcriptase (Molecular Genetic Resources). Thesecond strand was synthesized by incubation with the largefragment of Escherichia coli DNA polymerase (BethesdaResearch Laboratories), and the double-stranded cDNA wasdigested with S1 nuclease (gift from D. Durnam). The cDNAwas then dC-tailed with terminal deoxynucleotidyltransferase(Bethesda Research Laboratories), hybridized with Pst I-digested dG-tailed pBR322 (Bethesda Research Laboratories)(15), and used to transform CaCl2-treated E. coli RR1.cDNA Cloning with Synthetic Primers. cDNA was prepared

as described above using SK-MEL 28 mRNA and syntheticoligonucleotide primers. The cDNA was dG-tailed, ligatedwith EcoRI-cut XgtlO (16) and an oligonucleotide (AATTC-CCCCCCCCCCC) bridge, packaged (17), and plated on E.coli C600 rK- mK+ hfl.RNA Blot Analysis. SK-MEL 28 mRNA was denatured,

electrophoresed on a 0.7% agarose/2.2 M formaldehyde geland transferred to nitrocellulose. The filters were probed witha nick-translated p973a2fl cDNA insert.

Screening of cDNA Libraries. DNA from colonies of trans-formed bacteria was bound to paper (18) and screened bydifferential hybridization with cDNA probes synthesized onp97-enriched and unenriched mRNA templates. Libraries inXgtlO were screened for p97 inserts by plaque hybridization(19) with genomic exon fragments as probes. Probes wereradiolabeled with [32P]TTP (New England Nuclear; 3200Ci/mmol; 1 Ci = 37 GBq) by nick-translation to a specificactivity of 5-10 x 101 cpm/,ug.DNA Sequence Analysis. cDNA inserts were excised and

subcloned into the plasmid vector pEMBL18+ (20) forsubsequent propagation and restriction mapping. cDNA wasalso subcloned into the M13mpl8 phage cloning vector (21)and sequenced using the dideoxy chain-termination method

1261

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Proc. NatL. Acad. Sci. USA 83 (1986)

A 1 2 B 1 2

FIG. 1. Cell-free translation_MW of p97 mRNA. Five nanograms

of mRNA was translated in thereticulocyte lysate system, andthe translation products wereanalyzed by NaDodSO4/PAGEand autoradiography. (A) Totaltranslation products (0.5 p1), 6-day exposure. (B) Translationproducts (5 ul) immunoprecipi-tated with anti-p97 serum, 1-dayexposure. Lanes: 1, p97-en-riched mRNA; 2, unenrichedmRNA.

of Sanger et al. (22). M13 clones containing large inserts weresequenced by generating deletions using DNase I (23) orexonuclease III (24) and by using synthetic 21-mer oligonu-cleotide primers.

RESULTS AND DISCUSSIONPurification of p97 mRNA. Polysomes bearing p97 nascent

chains were purified by incubation with three IgG2a mono-clonal antibodies (96.5, 118.1, 133.2) specific for distinctepitopes of p97 (2, 4, 5, 10) followed by affinity chromatog-raphy on protein A-Sepharose. In a typical experiment, 150A260 units of polysomes yielded 260 ng of p97-enrichedmRNA, 0.23% of the total mRNA. When translated inXenopus oocytes (25) and assayed for p97 as described (5,10), p97-enriched mRNA yielded 80 pg of p97/ng ofmRNA,whereas p97-unenriched mRNA yielded only 0.44 pg ofp97/ng ofmRNA, showing that p97 mRNA activity had beenenriched 180-fold. The yield of p97 mRNA activity was 42%.Translation in the reticulocyte lysate system (26) showed thatp97-enriched mRNA coded for a major polypeptide with anapparent Mr by NaDodSO4/PAGE of 84,000, which was notdetectable in the translation products of unenriched mRNA,and was immunoprecipitated by antiserum specific for p97(Fig. 1). We concluded that it was the unglycosylatedprecursor of p97.

1 2

ai 9 0~~~U

Isolation of cDNA Clones for Human p97. p97-enrichedmRNA was used as template for oligo(dT)-primed cDNAsynthesis. The cDNA was cloned in pBR322, and the result-ing library was screened with cDNA probes. A 243-base-pairclone, p973a2fl, was identified that hybridized to p97-enriched cDNA but not detectably to unenriched cDNA and,in addition, selected p97 mRNA in hybrid-selection transla-tion experiments (data not shown). A polyadenylylationsignal (AATAA) and a poly(A) tract were present at the 3' endof the cDNA. Nick-translated p973a2f1 hybridized 100-foldmore strongly to p97-enriched mRNA than to unenrichedmelanoma mRNA and not detectably to fibroblast mRNA(data not shown). RNA blot analysis with the cloned cDNAas a probe identified an mRNA of approximately 4 kilobasesthat was present in SK-MEL 28 melanoma cells and absentfrom fibroblasts (data not shown).Attempts to obtain cDNA clones extending more than 1

kilobase from the polyadenylylation site were unsuccessful,possibly due to a region of high G+C content (>80%6) withextensive secondary structure. Genomic cloning was used tocircumvent this problem. Four overlapping genomic cloneswere isolated from libraries of X L47.1 containing size-fractionated SK-MEL 28 DNA enriched for a specific p97restriction fragment (unpublished data). Restriction frag-ments that hybridized to the 4-kilobase p97 mRNA on RNAblots were sequenced and p97 exons were identified by acomputer-assisted homology search between the predictedcoding sequences and the amino acid sequence ofhuman andchicken transferrin (11, 27, 28). Three synthetic oligonucle-otides based on p97 genomic exon sequences were used toprime cDNA synthesis on SK-MEL 28 mRNA and the cDNAwas cloned into XgtlO. Three overlapping cDNA clones(lOal, lji, 2fl) spanning 2368 nucleotides of the p97 mRNA,including the entire coding region, were identified by usingp97 exon-specific fragments as probes (Fig. 2).

Structure of p97. The p97 cDNA sequence is shown in Fig.3. An open reading frame of 2214 nucleotides extends fromthe first ATG, around which the sequence conforms with theconsensus initiation sequence determined by Kozak (29), tothe TGA at position 2215. The most 5' cDNA clone containsan additional 60 nucleotides upstream of the initiating ATG.The 3' noncoding region of p97 mRNA, which was notobtained as a cDNA clone, was identified as a single genomicexon containing 1667 nucleotides (unpublished data). Resi-dues 20-32 ofthe predicted amino acid sequence are identical

3x.m

W x

4kb

-3'

3 UT

Signal N-terminal C-terminal Anchor

3a2fl1

2fl

loal

FIG. 2. Structure of p97mRNA. The p97 cDNA clonep973a2fl was isolated from anoligo(dT)-primed p97-enrichedmelanoma cDNA library in pBR-322, whereas cDNA clones p972fl,p971jl,andp9710alwereisolatedbypriming with p97 exon-specificoligonucleotides and cloned inXgtlO. The structure of the codingand noncoding regions of themRNA are indicated as is the du-plicated domain structure ofthe p97precursor. kb, Kilobase(s).

97>78*66e

ft..5

43>0

30

20 _

0

- m

5 'UT

lii

1262 Biochemistry: Rose et al.

Biochemistry: Rose et al. Proc. Natl. Acad. Sci. USA 83 (1986)

ATC CCC CCt CCC ACC CCC CCt CTC TCC CTCNt? Arg Cly Pro Ser Cly Ala Lou Trp Lcu

CCA GAG CAC CAC AAC tCC CCC AAC ATC ACCPro ClI ClG lte Lye Cys Cly Ass Not Ser

CAC CAC tCC CtC CAC CtC ATC CCC CCC CACAsp lie Cys Vat CIs Lou Ite Ala Ala Cl.

CtC AAC CCC CtC CtC CCC CAA CtC TAC CATLou Lye Pro Vatl Vat Cly Clu Val Tyr Asp

ATT CAC ACC CTC AAA CCC CtC AAC tCC TCCIle Aop Thr Lou Lye Cly Vat Ly Ser Cys

CCC CCC CTC TCG CC ATC CCC tCC CAT CTACly Arg Leu Ser Vat Not Cly Cys Asp Vat

TAC tCt CAC tCC CTC tCt CCC CTC TCC ACCTyr Ser CIi Ser Los Cys Arg Lou Cys Arg

AGC CCC CCC TTC CCC tCC CTC CCC CAA CCCSer Cly Ala Phe Arg Cys Lou Ala CIs Cly

CTT CCC TCC TCC CCC CAC CCC CTC CTC TCALou Pro Sr Trp Cly CIs Ala Lou Lou Ser

tCC CAT CtC CCC CCC CTC CCt CCt CAC CCCCys lis Lou Ala Arg Vat Pro Ala His Ala

CCt CtC TTC ACC CAC CAC CCC ACC ACC TTCArg Lou Pe Scr lie Clu Cly Ser Ser Ph.

CGC CTT CtC CCC ATC CCC ACA CAC ACC TATClI Lou Val Pro Ite Ala Thr CGa Thr Tyr

CCC CTC CCC CCC TAC CTC CCC tCC TCt CtCArg Lou Pro Pro Tyr Lou Arg Trp Cys Vat

CTC AAC CCA CAC ATC CAC TCC CtC TCA CCCLou Lye Pro CGl Ite CIs Cys Vat Ser Ala

ACT CCC CAC CAC ATT TAC ACC CCC CCC AACSer Cly Cli Asp Ite Tyr Thr Ala Cly Lye

TAC TAC CTC CTC CCC CtC CtC ACA CCC CACTyr Tyr Vat Vat Ala Val Vol Arg Arg Asp

CCC ACC CCt CCA CCC TCC CGA CTC CCC CTCCly Ser Pro Ala Cty Trp Asp Val Pro Vat

ACC GAG TTC TtC AAT CCC ACC TCC CtC CCCer ClIi Phe Phe Ass Ala Ser Cys Val Pro

CCC CCC AAC AAC TCT CtC CCC AAC ACC CACCly Arg Ass Lys Cys Vat Cly Ass Ser Clo

CCC TTC CtC ACC CAC ACA ACC CTC TTT CACAla Phe Vat Arg lie Tbr Thr Vat Phe Asp

CTC CTC TCC CCC AAC CCC cCC CcA CCC CACLou Lou Cys Pro Ass CLy Ala Arg Ala ClI

CCC CAC ACC AAC ATC TTC ACC CTC TAT CCAPro Asp Tbr Ass Ie Ph. Tbr Val Tyr Cly

TTC CAC TCC tCC AAC TAT CAT CCC CAA CACPh. A Sepr Scr Ass Tyr Hie Cly Cln Asp

GCC TCC CTC CCC CTC CAC TAC CtC CCC CCCCly Trp Lou Cly Leu Asp Tyr Val Ala Ala

CtC CTC CCC CTC CTC CTC CCC CCC CTC CCCLeu Lou Pro Lou Lou Lou Pro Ala Lou Ala

CCC CCCCCA CTT TCC CCC CCC CCC TCT CCC

CAC CAT TCC TTC CTt TTT TCA AAA CCC ACT

TCA ACC TCT CCC CTC CC? CCC TCC ACT CTA

ATC TTC CtT CCT tTC A AAT AAA CCC AAA

GCC CAC tTC CTC CCA CCC CCA CCC ACC CCC

CTC CTC CCT CTC CCC ACC CTC CTC CCA CCCLou Lou Ala Lou Arg Thr Vat Lou Cly Cly

GAG GCC TTC CGC CAA GCG CCC ATC CAC CCCCGl Ala Phe Arg Clu Ala Cly IIo CGI Pro

GAG CCS CAC CCC ATC ACT CTC CAT CCA CCACGL Ala Asp Al& Ita Thr Lou Asp Cly Cly

CAA GAG CTC CCT ACC TCC TAT TAC CCC GCTClo Clu Val Cly Tbr Ser Tyr Tyr Ala Val

CAC ACC CCC ATC AAT CCC ACA CTC CCC TGCHis Thr Cly Ite Asa Arg Thr Vat Cly Trp

CTC AAA CCT CTC AGC CAC TAT TTT CCC CCCLou Lys Ala Val Ser Asp Tyr Ph* Cly Cly

CCT CAC ACC TCT CCC CAA CCC CTC TCT CACCly Asp Ier Ser Cly Clu Cly Vat Cy$ Asp

CCA CCC CAC CTC CC? Ttt CTC AAC CAC ACCAla Cly Asp Val Ala Ph. Vat Lys lie Ser

CAC CAC TtC GAG C?C CTC tCC CCC CAT CCTCtn Asp Phe Clu Lau Lou Cys Arg Asp CGy

CTC CTC CTC CCC CCC CAC ACA CAT CCC CCCVat Vat Val Arg Ala Asp Thr Asp Cly Cly

CAC ATC TTC ACC TCT CAC CCC TAT CCC CACClo Net Phe Ser Ser Clu Ala Tyr Cly Cl.

CAC CCC TCC CC CCGC CA? CAG TAC CtC CACClu Ala Trp Lou Cly Nis CGl Tyr Lou H1i

CTC TCC ACT CCC CAC ATC CAC AAC TCT CCALcu Ser Thr Pro Clu I1c ClG Lye Cys Cly

AAC TCC CCC CAA CAC TCC ATC CAC CCC ATCLys Ser Pro Clo His Cys Net Clu Arg Ie

AAC TAC CCC CtC CT CCC CCA CCC CCC GAGLye Tyr Cly Lou Val Pro Ala Ala Cly CGl

ACC TCC CAC CCC TTC ACC TTC CAT GAG CTTSer Ser Nis Ala Phe Tbr Lou Asp Clu Law

CCt CCC Ctt ATT CAC ACA CCC TtC ATC CCCCly Ala Lou Ite Clo Arg Cly Pbe Ite Arg

CTC AAC AAC CCC AAC AAC TAC CCC TCC TCCVat Ass As. Pro Lys Ass Tyr Pro Ser Ser

CAC CCC TAT TAC CCC TAC CCC CCC CCC TTCClu Arg Tyr Tyr Cly Tyr Arg Cly Ala Phe

AAC ACA AAC CCC CAC AAT tCC CAC CCC TCCAss Thr Asa Cly lie As Ser Clu Pro Trp

CTC TCC CAC TTT CCA CCC TCC AAC CTC CCAVat Ser Clo Pbs Ala Ala Cys Ass Lew Ala

CTC CTC CAC AAC CCC CAC CAC CTC TTT CCALou Lou Asp Lye Ala Cl. Asp Lou Pbe Cly

CTC CTT TTC AAC CAT CCC ACC CtC CCC CCCLeu Lcu Pbs Lye Asp Ala thr Val Arg Ala

CTC CAA CCC ATC TCC TCT CAC CAC tCC TCCLou Clu Cly Hot Ser icr Cln ClG Cy Ser

CCC CCC CTC CTC CCC CCC CCC CTC TCA CCCAts Arg Leu Lou Pro Pro Ala Lou ***

CCT CCC CAA TCC ACA ACC AAC CTC CCC A..

.(28...... ... ... bp)... ... ... ...

TTT CTC CCC TCA CAA CTC TCT TTC TCt CTC

ACT CAC CCT CCA TTC TCA CCT CCC ACC ACC

CAA CCC ACC ACA TCC CCA CCC TTC CAC CCT

ACC CCC CCC CCA CCC ACC CCC CAC CCC CCC

ATC CAC CtC CCC TCC TCC CCC ACC SCC CACNot Clu Vat Arg Trp Cys Ala thr Ser Asp

TCC CTC CTC TCC CtC CCC CCC ACC tCC CCCSer Lcu Lau Cys Vat Arg Cly Tbr Ser Ala

CCC ATC TAT GAG CCC CCA AAC GAG CAC CCCAla Ile Tyr Clu ALa Cly Lys Cli Hie Cty

CCT CTC CTC ACC ACC ACC TCC CAT CTC ACCAla Val Val Avg Arg Ser Scr Hie Val Thr

AAC CTC CCC CC CCC TAC CTC CtC GAG ACCAss Val Pro Vat Cly Tyr Lcu Va Cliu Ser

ACC TCC CTC CCC CCC CCA CCA CAC ACC ACTSer Cys Val Pro Cly Ala Cly Cli Tbr Ser

AAC ACC CCC CTC CAC ACA TAC TAC CAC TACLy Ser Pro Ls ClIi Arg Tyr Tyr Asp Tyr

ACC CTA CTC CAC AAC ACC CAT CCC AAC ACCThr Vat Lou Cli Ass Tbr Asp Cly Lye Tbr

ACC CCC CCC CAT CTC ACC CAC TCC ACC CACSer Arg Ala Asp Vat Thr Cli Trp Arg CIs

CTC ATC TTC CCC CTC CTC AAC CAA CCC CACLci Ile Phe Arg Lou Lou Ass Clu Cly Clo

AAC CAT CTA CTC TtC AAA CAC TCT ACC tCCLye Asp Lsu Low Phe Lye Asp Ser Tbr Ser

CCC ATC AAC CCt CTC CTC TCT CAC CCC AACAla Not Lye Cly Lou Lou Cys Asp Pro Ass

CAC ATC CCC CtC CCC TtC CCC CCC CAC CCCAsp Not Ala Val Ala Phe Arg Arg ClG Arg

CAC CCt CAC CAC CTC CAC CCt CTC ACC CTACl. Ala lCli Cl Vat Asp Ala Val Thr Lou

CAC TAT CCC CCC CAA CAC ACC ACC AAC TCClie Tyr Ala Pro ClI Asp Scr Ser Ass Ser

CCC CCC AAC CCC TCC TCC CAC GCC CCt tTCArg Cly Lye Arg Ser Cys Sle Ala Cly Pbe

CCC AAC CAC TCt CAC CTC CTC ACA CCA CTCPro Lys Asp Cys Asp Val Lou Thr Ala Val

CtC tCt CCA CTC TCC GTC CCC CAC CAC CACLos Cys Ala Los Cys Vat Cly Asp ClI Clo

ACC TCC CC CTC CAC AAT CC CCt CAC CTTArg Cys Lou Val ClI Ass Ala Cly Asp Vat

CCt CCt CAC CTC ACC TCA GAG CAC TAT CAAAla Ala Cl Lou Arg Ser ClI Asp Tyr Clu

CAC ATA CCA CCC CAC CCC CtC ATC CTC CCCGIn Ie Pro Pro lie Ala Vat Not Vat Arg

CAC CAC CAC AAT AAC AAC CCC TTC AAA ATCAsp Asp lie Ass Lys Ass Cly Phe Lye Not

CtC CCT CTC CCA CAC AAA ACC ACC TAC CCCVat Pro Vat Cly Clu Lye Thr Tbr Tyr Arg

CCC CCA CC CCC CCC CC CCC CCC CCC CCCCly Ala Ala Ala Pro Ala Pro Cly Ala Pro

CCC CCC CCC CCC CCC ACA CCt CCC ATC CCC

* .. ... ... *... ... ... .*.. ... ..*. ...*

*. .. .. .. .. .*** *** *.. ..... ... ...

CCt AAC TCT CCC CtA CCC TCC CC? CCC CAT

CCC CCC CTC CTC TCT CAC TCC TAA TCA AAC

CAT AAA AAA AAA

FIG. 3. Nucleotide sequence of the human p97 precursor cDNA and the deduced amino acid sequence. The N-terminal amino acid residuesdetermined previously by protein sequencing, which are identical to those predicted from the nucleotide sequence, are underlined. The potentialglycosylation sites at amino acids 38, 135, and 515 (open bars) and the membrane anchor region at the C terminus (solid bar) are indicated. Onepolyadenylylation signal AATAAA was detected at position 3785, 47 base pairs upstream of a polyadenylylated tract.

to the known N-terminal amino acid sequence of p97 (9),proving the identity of the cloned cDNA. Furthermore, thepredicted molecular weight of the precursor is 80,196, in goodagreement with the observed molecular weight of the in vitrotranslation product.The amino acid sequence of the p97 precursor comprises

four structural domains. Since residue 20 of the precursor

sequence corresponds to the N terminus of mature p97,amino acids 1-19 constitute a signal peptide, a conclusionthat is supported by its length and hydrophobic nature.Amino acids 20-361 and 362-713 comprise homologousdomains of 342 and 352 amino acids. Potential N-glycosyl-ation sites occur at positions 38 and 135 in the N-terminaldomain and position 515 in the C-terminal domain. Finally,

1263

9030

1060

27090

360120

4501 SO

540ISO

630210

720240

810270

900300

990330

Iu0360

1170390

1260420

13SO450

1440's015 30S10

Ih20S40

1710S70

1800600

1890o30

1980660

2') 70690

2160720

22SO7 38

2 106

3086

3768

3840

Proc. Natl. Acad. Sci. USA 83 (1986)

we believe that amino acids 714-738, a region of predomi-nantly uncharged and hydrophobic residues, anchor p97 inthe cell membrane (30) and may extend into the cytoplasm.The domain structure of p97 is supported by protease

digestion experiments. Digestion of p97 with trypsin, papain(4), or thrombin produced a glycosylated antigenic fragmentof Mr 40,000. The fragment was purified from a thrombindigest of p97 that had been metabolically labeled with[35S]methionine or [35S]cysteine and sequenced as described(9). Cysteine residues were identified at positions 7 and 17,and methionine residues were identified at positions 2 and 20.Identical results were obtained with intact p97 and are incomplete agreement with the N-terminal sequence of p97predicted from the cDNA sequence. We conclude that the Mr40,000 protease-resistant fragment corresponds to the N-terminal domain of p97. We have been unable to isolate theC-terminal domain of p97, possibly because it is proteasesensitive.Homology of p97 with Transferrin. A search of the amino

acid sequence library of the Protein Identification Resource(release 5.0) (31) as described by Wilbur and Lipman (32)showed that p97 is strikingly homologous to three membersof the transferrin superfamily; human serum transferrin,human lactotransferrin, and chicken transferrin (37%-39%homology; Fig. 4). Since human and chicken transferrin show50% homology to each other, p97 must have diverged fromserum transferrin more than 300 million years ago, when themammalian and avian lineages diverged (34). Conservation ofcysteine residues within the domains of p97 and between thetransferrin family members is notable. p97 has 14 cysteineresidues located in homologous positions in each domain.Human transferrin contains all of these cysteines in homol-ogous positions in both domains, while human lactotransfer-rin and chicken transferrin lack only two of these cysteineresidues (in their C-terminal domains). Unlike p97, theseproteins contain 4-7 additional cysteines in their C-terminaldomains, which have no corresponding member in the

N-terminal domain. Human transferrin also contains 2 extracysteines unique to its N-terminal domain. The positions ofmost of the disulfides in human serum transferrin,lactotransferrin, and chicken transferrin have been deter-mined directly (27, 33, 35-38), and one can thus predict thepositions of seven disulfide bonds in each domain of p97.The amino acid homology between domains of p97 (46%-

achieved by insertion of 7 gaps of 9 residues) is more strikingthan that seen in human transferrin (43%-16 gaps, 45residues) or chicken transferrin (35%-12 gaps, 49 residues).Given the extensive sequence homology between p97 andtransferrin and the apparently similar folding patterns, basedon the conservation of cysteines, we believe that if thepresent low-resolution x-ray structure of transferrin (39) canbe refined it may be possible to deduce the three-dimensionalstructure of p97.Function of p97. Its membership in the transferrin

superfamily, its ability to bind iron (9), and its commonchromosomal localization with transferrin and the transferrinreceptor (10, 11) all support a role for p97 in iron transport.The iron binding pocket of transferrin is thought to containtwo or three tyrosines, one or two histidines and a singlebicarbonate-binding arginine (33). Conservation of theseamino acids in p97 support its proposed role in iron metab-olism (Fig. 4). Since p97 is a membrane-bound transferrin-like molecule and has no homology with the transferrinreceptor (40), its role in cellular iron metabolism may differfrom that provided by circulating serum transferrin and thecellular transferrin receptor. Expression of the cloned p97cDNA in eukaryotic cells will allow experimental testing ofits functional properties. Because of its strong homology withmembers of the transferrin superfamily and because it wasfirst isolated from melanoma cells, we propose renaming p97melanotransferrin.

Obtaining a full-length cDNA for p97 has allowed a detailedanalysis of the primary structure of a human membrane-bound tumor-associated antigen. p97 should therefore serve

P97 C P S A L W L L L A R T V - - C C K EKV a w C A T SD P Q K C N N S E A F R I A C I Q - - L L a 56Tf L A V A L V C A V G L C A V P D K T V R W C A V S E H A T K Q S r R D H N K S V I P S D c V A K 60

p97 G T A D NCV Q LI AAIQAD A I G A I E C K E H - C|L K P V V C KV D Q E V C - SY Y A V A V VI 113Tf K AWT L I A A N V A L V D Y L A P N N|L K P V V A F G S K E D PQ1F2Y A V A V V 120

p97 i a SRN V T I D TYK V S C H TIG I N T V C V F V G CL SO R L S V N C C D V L K A VISOD YFC G S C V 173Tf K K D[ C F Q M N Q R K S C H T C L C S A NI I CL Y C D L P E P R K P L E - -KA A IN FF S C|A 173

P97 P1C AC Kill STYSKS L C1RL RC D S S C E C VICD K S P L E RY DY AFR CL A K C A C D V A F V K H S T|V L 233If AHC D C D F P - Q[ CQIJ P EJFC C - - - - - JjS T L N- - Q C IS C A FIKjL K D C A C D V A F V K N S T I F 230

p97 NT DCT L P SV G Q A L L S Q D F LLCRC SRA DTW R QC LARV ANA VVA D T D C C - 292Tf NL A NJ- - - - - - - - A D R D Q Y K L L CL ON T K PF D Y K DC N LQ V S N TV V AS N C C K DD[J 282

p97 FR LL L F - S K C S S F s S E A Y C Q K D L L F K D S T S E L V P I A T - Q T Y IA W L11 L H 350Tf w LLNQ A[ HGC K D K S K E L P H --CK D LLL K D SIA H C F L K V P P RM DA KKY[TY V T 340

p97 AKGKCIL - - -- -0 PN R L P P Y L Rw V T P I Q C D N A A F RI Q R L K P E QC V SAK S P Q N 405Tf A I RNNJ ECICG P E A T D E C K P V K V AL R RLN D E V S N S V- C K C V S A T TE D 395

P97 C E R IQ A I Q V V T S E DIC T A OK K I L V F A A C N A P E- - - - - S S N S VV A V VR D 460Tf CI A K N N C E A A S[D[J F V I A CKGC- VF- V L A N N K S N C E D T P E A C F A V V K K S 454

p97 So H A F L DELR R A C F7S P D V V CAI Q XQ F I R P K DCD V L T A V S F F N A SC V 520Tf A- D L W K C T A V R T N I SL Y N K I - - - - NR5R0- - D S G A 505

p97 V N N P K N Y P L C V CD Q R CV SQ R C i C A F R c L V N A D V A F v iT V D 50Tf -- S K R D S L c s - L L I P K G C C A F R C L V l D V A r | Q T Q 559

p97 [1N17111 V AIAE 51RSZD TE L LCTP N7AR'A 1SQF1AFNWQ I [P[ N Vi PT0 I F TIY 0 640Tf [ CKLUJDKN A K N N I KLD T ZL L CIL DIT[K PI I YELNJLJ R AfIJN A V|V T[K[U- K I A CWH K 61S

p97 L D K A DL F CD R N K N O-- K N FDS N Y N C Q L L FK A VR A V P V OE K TTR C W L D 698Tf ILL R Q Q F C S N V T D C SO N C L F S - - E T K IF RD C L A K LID R N

IK

Y 6 7 5

p97 A AL I C K S S Q Q CS A A A P A P C A P L L L L L P A L A A i L L P P A L 738Tf KVC N L RIK - - T SS L L E A C T F R RL 698

FIG. 4. Comparison of the predicted amino acid sequence of the p97 precursor and that of human serotransferrin (11,30). Conserved residuesare boxed. Tyrosine, histidine, and arginine residues implicated in iron binding of transferrin are indicated (33).

1264 Biochemistry: Rose et al.

Proc. Natl. Acad. Sci. USA 83 (1986) 1265

as a useful model for this class of proteins and it will be ofinterest to see which other tumor-associated differentiationantigens have structural or functional homology with knownproteins.

We are grateful to Drs. Stanley McKnight, Richard Gelinas,Gregory Barsh, Steven Henikoff, and David Lee for their advice andencouragement and to Toni Monsey, Cynthia Green, and Jan Conitzfor their excellent technical assistance. This investigation wassupported by National Cancer Institute Grants CA 34777 and 38415and by National Institutes of Health Grant GM 07266.

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