Primary structure of human carcinoembryonic antigen (CEA) deduced from cDNA sequence

Vol. 142, No. 2, 1987 January

BltiCHEMlCALAND

BIOPHYSICALRESEARCH

COMMUNICATIONS Pages 511-518

30,1987

PRIMARY STRUCTURE OF HUMAN CARCINOEMBRYONIC ANTIGEN

(CEA)

DEDUCED FROM cDNA SEQUENCE Shinzo 1 Suntory

, Hiroshi

Oikawa' Institute

for

Nakazato'

Biomedical

Mishima-gun, *Tokyo Received

Metropolitan December

Research,

Osaka

Komagome

, and Goro

618,

Hospital,

.2

Kosaki

Shimamoto-cho,

Japan

Bunkyo-ku,

Tokyo,

113,

Japan

5, 1986

The cDNAs corresponainy to the mRNA encoding a polypeptide which is immunoreactive with the antisera specific to carcinoembryonic antigen (CEA) (1) are cloned. The amino acid sequences deduced from the nucleotide sequences of the cDNAs show that it is synthesized as a precursor with a signal peptide followed by 668 amino acids of the putative mature CEA peptide, whose N-terminal 24 amino acids and amino acids 286 to 295 exactly coincide with those known for N-terminal sequences of CEA (2) and NFA-1 (3), respectively. The first 108 N-terminal residues are followed by three very homologous repetitive domains of 178 residues each and then by 26 mostly hydrophobic residues which probably comprise a membrane anchor. Each repetitive domains contains 4 cysteines at precisely the same positions and as many as 28 possible N-glycosylation sites are found in the CEA peptide region agreeing with high carbohydrate content of purified CEA. o 1987 Academic press, lnc.

CEA first glycoprotein (4)

found

thelia

described with

by Gold

a molecular

in adenocarcinomas

and foetal

of

colon.

serially

monitoring

cancer

particularly

in

and documented.

one encounters e.g.

(NFA-1, the These mostly

NFA-2

serum,

But

case

(5),

because NCA-2

and NFCA)

saliva,

CEA-like on peptide

faeces

substances moieties

(9)

(6),

the the

TEX (B),

and BGP-I (lo),

and colon share

with

in addition

is

lavages

derived

digestive

have

immunoassay presence

distinct

diagnosis to

evalu-

such

binding

purposes, substances

faecal

colonic

binding

and

been widely

of CEA-like

apparently

epi-

or response for

normal

in normal of

system for

disease

cancer,

various

itmnunoreactive

60% carbohydrate

CEA immunoassay

CEA antibody to

a complex

comprising

recurrent

colonic of

(1)

180,000 the

for of

in practicing

difficulties

NCA or NCA-1

of

patients the

of

endodermally

The roles

therapy, ated

and Freedman

weight

antigens mucosa

normal sites sites

and in

individuals. which

are

suggesting

Abbreviations: CEA, carcinoembryonic antigen; NCA, nonspecific crossreacting anl;igen; TEX, tumor-extracted CEA-related antigen; NFA, normal faecal antigen; NFCA, normal faecal cross-reacting antigen; BGP-I, biliary glycoprotein-I; b, base; kb, kilobase; bp, base pair.

511

0006-291X/87 $1.50 Copyright 0 1987 by Academic Press, Inc. AN rights of reproduction in any form reserved.

Vol. 142, No. 2, 1987

the

presence

of

relationships nature

clinical In

deduced

of

closely

this

report

these

primary

nucleotide

for

study

of

the

which

as well

It

(11).

antigens the

a polypeptide the

genes

them

we describe

to CEA and the from

related

some of

use and for

mRNA encoding cific

very

between

molecular for

BlOCHEMlCALANDBIOPHYSlCALRESEARCHCOMMUNlCATtONS

is

essential

establishing

the

their

biological

cloning is

structure sequences

as precursor-product

of

of

the the

precursor

the

tumor-specific

assays

significance.

cDNAs corresponding

imnunoreactive of

to clarify

with

the for

to

antisera the

the spe-

putative

CEA

cDNAs.

METHODS cDNfA Cloningi. Poly(A)+ RNA was prepared from total RNA extracted from 1.39 o human ma ignant colon tissue from a patient as described (16), and a cDNA library was constructed using a phage expreSsion vector hgtll (17). oly(A) RNA was prepared according Double stranded DNA corresponding to the to the RNaseH method of Watson et al. (18 P , methylated at EcoRI sites, ligated to EcoRI linkers, digested by EcoRI, and size fracxnated on a Bio-Gel A-5rcolumn. The cDNA largerxan 500-bp was ligated to the Eco_RI-cut dephosphorylated hgtll DNA (Promega Biotec, U.S.A.) and pagkaged using Gigapack (Vector Cloning Systems, U.S.A.). Approximately 3x10 clones were plated and submitted to immunological screening. One positive clone termed hKr40 was detected and purified by plaque purification. A cDNA insert was isolated by electrophoresis following cleavage with EcoRI and ligated to pBR322. Resulting plasmid, pBRCEA5 was used to transforin7.coli DHl to prepare DNA for further study. Two PvuII fragments (from 566 to 1099 and from 1100 to 1633, Fig.2) were labelled by nicktranslation with [a-s*P]dCTP and used as a hybridization probe in RNA blot analysiq shown in Fig.4 and in the screening of a cDNA library prepared from poly(A) RNA isolated from healthy part of the colon tissue of the Same patient as above. After sucrose density gradient centrifugation, poly(A) RNA of 2 to 5-kb was pooled and used for the construction of a cDNA library by the method of Okayama and Berg (19). Approximately 7,000 clones of E.coli DHl transformants thus obtained were transfered to nitrocellulose filters and screened. After hybridization with the probes described above for 16hr at 65"C, 8 positive clones, of which 4 contained longer cDNA inserts than xKr40 were obtained. After restriction enzyme mapping, plasmids, pCEA55-2 and pCEA8-11 were isolated for nucleotide sequence analysis as described in Fig.2 legend. Immunological staining of plaques transfered Immunological Methods. nitrocellulose membranes was performed accordins to the manufacturers instruction (Express-Blot Assay Kit, Bio-Rad Lab. U.S.A). The first antibodies for screening and for the confirmation of the inununoreactivities were rabbit ant-CEA antibody (DAKOPATTS, Denmark) and those described in Peroxidase labelled goat anti-rabbit IgG Fig.1 legend, respectively. antibody and rabbit anti-sheep IgG antibody are from Bio-Rad Lab. (U.S.A.) and Kirkegaard Perry Lab. Inc. (U.S.A.), respectively.

to

Blot Hybridizations. RNA was separated by electrophoresis in a 1% agaromation with formamide and transfered to nitrocellulose sheet. Hybridization with the 32P-labelled probe described above was done for 16hr at 45°C. Ten pg of DNA was digested with 50units of endonuclease for 3hr at 37°C and electrophoresed, transfered to nitrocellulose sheet (25), hybridized for 17hr at 65°C with N-terminal 201-bp CEA 80-11 insert which had been labelled by nick PstI-PvuII fragment of translation with [a-s*P P dCTP. 512

Vol.

142,

No.

BlOCHEMlCALANDBlOPHYSlCALRESEARCHCOMMUNlCATlONS

2, 1987

RESULTS AND DISCUSSIONS A cDNA library tumor

of

E.coli

a patient

and the make sure

of

antibodies

specificity the

of

including

different

which

the

As the indicated

known

for

as above. 534-bp

PvuII

Fig.4A)y clones

of

fragments the

of

different

the with

all

imnunoanti-CEA

four

xKr40

antibodies

serum carries

the

kinds.

tissue

for

RNA blot

cDNA

mode of

transcription

A

the

as shown

from

with

xKr40

the

poly(A)+ had of

(lane

two

5 in

screening,

have

RNA

same patient

mixture

hybridization

in

that

analysis the

cDNA insert

which

to

using

hybridization

hybridized

and pCEA80-11,

two

cDNA inserts

longer

(Fig.2). determined

reading

by the

frame

The cDNA in 500-b

removed

a colony

the

of AATAAA sequence about

determined corresponding

was prepared

colon

for

isolated open

a long

stretch

cDNA library

from

pCEA55-2 were

reveal two

sequence

RNAs which

same probe

plasmids,

downstream

cDNA insert

isolated

sequences

poly(A)

To

non-immune

that

the

was chosen,

existence

in xKr40

least

not

N-terminal

normal

The nucleotide (Fig.2)

but

indicating

of

of

Okayama-Berg

apparently

Using

that

21-b

absence

The tissue

abundant

than

sequence

the

from

shown

purification.

CEA.

CEA (2),

islolated

antibody

we conducted

antibody

product

in the

first

on E.coli in Fig.1,

on

stained

as the

by plaque

shown

colon

DNA and plated

antibody

hKr40

As is

adsorbed

encoded

nucleotide

Fig.2

imnunoreaction by plating

sources.

xKr40

encodes

the

formed

NCA- or NCA-2-

recognized

was purified

from

was positively

anti-CEA

xKr40 of

plaques

RNA extracted xgtll

A plaque

a rabbit

termed

poly(A)+

dephosphorylated

legend.

using

phage

the

staining

in Fig.1

assay

cloned

using

and EcoRI-cut

as described

inununoscreening

at

was constructed

or

followed

clone

that

processing

B

the

clone

suggesting of

the

three

the

C

primary

has a

existence

of

transcript.

D

The

E

2

Fig.1.

Reactivity

Plaques

formed

of

xKr40

by 1; xKr40

product

or 2; hgtll

to

antibodies

plated

of

and fixed to nitrocellulose membrane which is themnto reacted with anti-CEA antibodies of different animals straining as described in METHODS. A, rabbit (A115, sheep, NCA-'2 adsorbed (640733-1, Miles, U.S.A.); C, England); 0, sheep, NCA adsorbed (AHP 105B, Serotec); serum.

513

different

on E.coli

of

stretch

pCEA55-2 the

clones

region

has a poly(A)

of

down stream

of

by 3'-non-coding

pCEA80-11

whereas

further

analyses

sources.

Y1090 were transfered

pieces, and and sources before DAKOPATTS, Denmark); B, sheep (AHP 105A, Serotec, E, sheep, non-imune

Vol. 142, No. 2, 1987

A

8lOCHEMlCALAND8lOPHYSlCALRESEARCHCOMMUNlCATlONS

0

566

1oou

1500

2000

2520

3000

3500

I

I

I

I

I

I

I

I

Pv

WPV

I

so

I

Ps/Pvnc

II

1

I

PSlPv I

s

II

I

Pv

II

AKr 40

pCfA 55-2

--pm

80-11

o-

:

Fig.2. Sequencing strategy of cloned cDNA (A), and primary structures of cDNA and the CEA (B). In A, schematic structure of the cDNA depicting protein-coding (open boxes) and non-coding regions (I) is shown at the top. The restriction endonuclease sites only relevant to the present work are shown. Three cDNA inserts are shown by thick horizontal bars. The direction and extent of sequence determinations are shown by horizontal arrows under each clone used. H , signal peptide; 0, N-terminal 108-residue sequence; a, m,m, domain I, II, III; hydrophobic C-terminus. Restriction endonuclease and 5'-terminal nucleoti Y e'generated by cleavage are: Pv, PVUII (187, 566, 1100, 1634 & 2043) ; Ps, PstI (565, 1099 & 1633) ; S, Sac1 (m & 1375); B, BamHI (873); Hc, HincII (1193). In B, nucleotide resid= are numbered inhe 5' to 3' direction, beginning with the first residue of the insert in pCEA80-11, the number of the nucleotide residue at the right end of each line is given. The deduced amino acid sequence is shown below the nucleotide sequence, and amino acid residues are numbered so that N-terminal sequence coincides with that reported for CEA. Residues of the signal peptide are indicated by negative numbers. 0, start of pCEA55-2 insert; o, start and end of XKr40 insert; *, translational stop; 1, start of poly(A) in pCEA80-11. Truncated Alu family sequence is underlined and the direct repeats boxed. N-tetmm 24 amino acids identical to those of CEA are boxed; $I, the residue is alanine for NCA-1. Amino acids identical to those of N-terminus of NFA-1 are underlined. Starts and ends of domain I, II and III are shown above the nucleotide sequence. C-terminal hydrophobic region is shown by broken underline. Nucleotide sequencing of XKr40 insert was done l), a 2,400-bp EcoRI restriction fra ment of the insert was by two methods. recloned into M13mp18 and M13mpl??%id sequenced (20) 9 -); 2), appropriate restriction fragments shown in the figure were recloned into M13mp18 or M13mpl9, and sequenced by the chain termination method (21) (k--d), pCEA55-2 and pCEA80-11 were analysed by l), chain termination method on appropriate restriction fragments recloned into pUC9 (22) using general and reverse primers ( W ); 2), chain termination method using two synthetic oligodeoxyribonucleotide primers 5'-GTTTCACATTTGTAGCT-3' and 5'-ATGTGGTCGCTCCAGAC-3' ( W). analysis noted

of

this

about

sequence

the

(12)

family

consensus

region

of

the

region

is

left

3'-non-coding flanked left

region

by a pair

sequence half

(12), of

for

the

future is

of direct

study. that

514

feature

to be

contains an Alu family Compared to the Alu repeats.

71 nucleotides dimer.

Another

it

are

deleted

from

5'-terminal

Vol. 142, No. 2, 1987

BIOCHEMICAL

AND BIOPHYSICAL

RESEARCH COMMUNICATIONS

GOCAliAOGel~~Cl~RCT~CRCl~CCGCCUCUClGCC S’..... cTaGccctlccccAcAGATGGlGcAlccccl

91

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-1

CRTTTGGCMAGCTGGlACMAGGTGMAGAGlGGAl L~PhrG~l~k~TrpTy~l~Gl~l~A~V~lA~p

211 40 331

451

571

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TATGGCCCGGATGCCCCCACCATATnCCCCTCTAAAcAcATCTTAcAGATUGGGGMM TCTGAACCTCTCCTGCWGWC CTCTMCCCACCTGCACAOTACTCTTGGTTTGTCAAT 811 TyrGlyProAspAlaProThrIlekrProLcuAsnfhrkrTyrArg~~lyG1uArnL~uAsnL~ukrC~HIsA1~Al~k~~ProPrdl~GlnTyrScrTrpPhrVs1Asn 210 220 240 GGW\CTTTCCAG~TC~CC~GCTC~TAlCCC~T~CTGT~TMTffil~TCCTATACGT~CUTMCT~~CT~CT~Tffi~C~CAGT~CG GlyThrPhcGlnG1nScrThffilnGluL~uPhcll~prdsnIl~ThrV~1AsnAsn~~~ly~rTyrThrCyrGlnAl#1rAsn~f~AspThffilyLauAs~rgThrThrV~lThr OuSn I1 ACGATCACAGTCTATGCA r CCACCCMACCCtTCATtACCAGCAACMCTCtMCCtCGTOOC ThrIlsThrValTyrAlrGluProPraLysPw~Il~Thr~rAsnAsnSerAsn~~V~lGl~s~luAs~l~V~lAl~L~u~~~GluP~luIl~GlnAsnThrThrTyr 290

931 280 1051 320 1171

1291

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CTCTCCTGCCATOCAGCCTCTMCCeAtCIOCACACtArrtCTC L~uS~rCysHlrAl~AlrkrAsnPro~~l~GlnTyrSrrTrpL~uIl~spGly~;~Il~lffilnHlsTh~lffiluLw~~~~rAsnIl~ThffiluLysAsnS~rClyL~u

-

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III

100

TATACCTGCCAGGCCMTAACTCAGCcAGTGGcUCAGWGACTAcAGTcMGAUA TCACAGTCTCTGCGGMCTGCCCAAGCCcTccATcT CUGCAAWCTCCMACCCGTGGAG TyrThrCyrGlnAl~AsnAsnkrAl~krOlyH1skrA~ThrThrV~lLyrThr~~ThrV~lkrAlrCluLwProL~Pro~;;f1ekrkrAsnAsnkrLyrProV~1Glu 450 480

1531

1651

1771

1891

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2251 2371 2491 2611 2731 2651 2929

Fig.

Z%Continued.

515

Vol. 142, No. 2, 1987

The long including

open

the

amino

reading

(13)

which core

signal

sequences

and is

followed

mature

form

generally (14).

CEA peptide

identical

to

those

and TEX (8) the

(Fig.1). at

latters.

reported

for

21st

residue

Second,

it

comprises

0.15%

agreement

with

CEA from

quantity

of

methionine

NFA-2

(3).

acids

of a fragment

Denmark)

treated

when

protease

I (Wako, experimental

Japan)

call

the

178 residues

72.5,

and 61.8%,

found

in this

region,

at

of

II

somewhat

The domain form -*i.e 11,

positions

product

is

a membrane

cells multiple

and 1.6-kb

to size

the

using

blot

of reflects

protein

Accordingly,

we

are

of

RNA, i.e.

major

partly

the 516

fact

PvuII species

are

same positions

as

10 amino

acids

is

this

the

degrada-

that

the

region

acids

(Fig.3).

which

seem to sites, sequence,

The peptide

CEA (Fig.2). that

residues

2 in N-terminal short

3.5-kb

67.4,

N-glycosylation

analysis the

73.0,

context

amino

Pro),

32P-labelled

homology

(3)

in

respectively.

C-terminal

repeats

sequence

the

in this

28 possible

except

homologous is

NFA-1

I and III

hybridization

domains

and

14 amino

N-terminal

hydrophobic

III,

the

as a probe

repetitive

species

species,

the

for

NFA-1

and Achromobacter

acid

at

that

to note

and

a definite

12 cysteine

to the

domain

residue II

except

4A depicts

and tissues

correspond

I,

and

DAKOPATTS,

II and III

4 residues

interesting

(X is any

amino All

identical

by 26 mostly

domain

but

in CEA (2),

by three

the

& Fig.3).

There

i.e.

of methionine (15)

49 to 62 in the

followed

295 suggests

region.

and hydrophilic

are

the

for

CEA.

having

from

followed

Asn-X-Thr/Ser

Figure

is

(7)

658,

content

elsewhere).

and I and I,

domain

exactly

of NCA-1

position

U.S.A.)

positions

II and III, III

diverges

anchor

7 and 8 in the

acidic

acids I,

a sequence

It

the

the

and alanine

(X556,

(Sigma,

case the

are

to N-terminal

be published

286 through

CEA.

III

at

acids

small

found

this is

to

and Laferte

identical

trypsin

(Fig.2

each of

is

in

those at

to have

many

likely

former

The low

any,

peptide

respectively

of tion

will

II and III,

The finding

domain

found

108 amino

others. NFA-1

is

named domain

I and II,

between

of

details

from

a CEA preparation

668-residue

The N-terminal of

if

from

most

in addition

the

a

end of

24 amino

by Krantz

sequen,ce

the

sequence

which

for

reported

little,

at

reasons

698 signal

and contains

signal

residues. are

encoding

a truncated

one methionine

reported

a mixture

the

acids,

and differ

only

acid

nucleotides

found

valine

total

released with

is

which

amino

(Fig.28, tentatively

of that

2095

N-terminal

CEA (2)

which

NCA-1

Finally,

the

portion

proteins

following

contains

is

distinguish

the First,

methionine in good

end of

668 amino

for

N-terminal

is often

the

of

its

secretory

which

marks

RESEARCH COMMUNICATIONS

seem to comprise

precedes

also

at

contains acids

Alanine

(14)

itmnunoreactivities

truncated

30 amino

by a protein

of

AND BIOPHYSICAL

methionine

The first

hydrophobic

frame

initiative

acids.

peptide

BIOCHEMICAL

is

rather

sequence. of

RNA from

fragments

human

which

The presence and minor

two cDNA clones

of

4.2-,

2.9-

which

have

Vol.

142,

No.

BIOCHEMICAL

2, 1987

I ‘:E& ; F&I&S N N SbP V E 0 K I SNNS PVEOmoAVA 1:: ELPKPSISSNNSKPVEOKOAVAFTCEPEA I

AND

0 A V “6’

BIOPHYSICAL

RESEARCH

: E P ‘TV-$ TCEPEIQNTTYLWYVNN NTTYLWWVNG

COMMUNICATIONS

109-148 287-326 465-504

QSLPVSPRLQLSNGNRTLTL QSLPVSPRLQLSN~NRTLTL

1:: 189-228 367406 545x84

II III

229-268 407-446 585-624

I 1:: I 1:: %-ii amino last boxed.

Comparison of amino acid sequences of homologous repetitive domains The three homologous domains are aligned for comparison of the CEA. The numbers of amino acid residues at the first and acid sequences. Identical residues between domains are ends of each line are given. * indicates cysteine residues. A

8

12345

1

2

3

23.1-

28S-

I

9.4u.r)

6.6-

t

01

18S4.4-

2.32.0-

Fig.4. Autoradiograms of blot hybridization analysis of RNA from human tissues+(A), and of restriction fragments of human genomic DNA (6). In A, poly(A) RNA equivalent to 15Oug of total RNA was analysed as described in RNA was prepared from; lane 1, colonic METHODS. cancer tissue from patient A; lane 2, human gastric carcinoma cell line (RAT0 III) producing CEA; lane 3, human hepatoma maintained in nude mice (total RNA, 15ug); lane 4, colonic cancer tissue from patient 6; lane 5, healthy colonic mucosa of patient A; In DNA digested with restriction endonuclease was analysed B , human lymphocyte as described in METHODS. Restriction endonuclease used was; lane 1, BamHI; lane2, EcoRI; lane 3, HindIII. Molecular weight markers are DNA digezd with run throuxthe same gel. -Him11 517

Vol. 142,No.

2, 1987

different

3'

termini

relationships stage. in

of

the

Another

the

interpret

of

these

hybridization CEA-related

data

at

multiple

region copies

CEA-related similar

this

of

genes. genes

CEA and CEA-like

are

to

a gene The expected

antigens

that

differ for

we do not

from

the

not

clear

the

RNAs expressed

cancerous It

at

this

and healthy

is

know whether sequence

The

too

early

to

the

or recognizes

other

as well. hybridization

many fragments

is

of

including

CEA encoding

cDNA (Fig.46), latter

amounts

significantly.

by blot

homologous

library RNA are

the

sources

sequences

gives the

individual

is

stage,

DNA analysed of

Okayama-Berg

and the

specific

digestion

5'-terminal

the

different

encoding

Human lymphocyte endonuclease

from

same patient

is

antigen

(Fig.2).

of

the

probe

cloned

to be noted

and cells

tissues

COMMUNICATIONS

two clones

feature

tissues

colonic

are

BIOCHEMICALANOBIOPHYSICALRESEARCH

which

hybridize

indicating to

more

the likely,

biochemical

after the

putative for and

restriction with

presence

short of

either

CEA cDNA or multiple the

presence

immunochemical

of

several data

of

(11). REFERENCES

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