BIOCHEMICAL AND BIOPHYSICALRESEARCHCOMMUNICATIONS Pages 578-585

Vol. 176, No. 2, 1991 April 30, 1991

THREE NOVEL MOLECULAR FORMS OF BILIARY GLYCOPROTEIN DEDUCED FROM cDNA CLONES FROM A HUMAN LEUKOCYTE LIBRARY*

Motomu Kurokil, Fumiko Arakawal, Yoshino Matsuol, Shinzo Oikawa2, Hiroshi Nakazato2 and Yuji Matsuokal§ 1Department of Biochemistry, School of Medicine, Fukuoka University, 7-45-1 Nanakuma, Jonan-ku, Fukuoka 814-01, Japan 2Suntory Biomedicallnstitute, l-l-lWakayamadai, Shimamoto-cho, Osaka 618, Japan Received February 22, 1991

Three cDNA clones that encode three novel variants of biliary glycoprotein a (BGPa), a glycoprotein belonging to the CEA gene family, were identified in a human leukocyte cDNA library. The domain structures of the predicted proteins of the three clones W211, W233 and W239 are N-A1-B1-A2, N-A1-B1 and N-A1-B1-C, respectively; they lack the transmembrane and cytoplasmic domains that exist in the four BGP species (BGPa, BGPb, BGPc and BGPd) previously reported. Their sequences from N to B1 or to A2 are virtually identical to those of BGPa-d. Comparison with the genomic sequence for BGPa-d suggested that these three new BGP variants as well as BGPa-d are generated from the same single gene by alternative splicing of RNA. ~ 1991AcademicP . . . . . I n c .

Biliary

glycoprotein

a (BGPa),

one

of the

cross-reacting

antigens

of

carcinoembryonic antigen (CEA), is a glycoprotein originally found in normal human hepatic bile (1,2). Immunofluorescence studies (3) and the findings that the serum level of BGPa was often elevated in patients with liver or biliary tract diseases (4) suggested that the protein is a product of biliary tracts and gall bladder. A recent study using Northern blot analysis has suggested that liver cells produce BGPa (5). The complete primary sequence of BGPa has been demonstrated by cDNA cloning of a library derived from normal colon (6), revealing that the sequence of the antigen is highly similar to that of CEA. In contrast to CEA that is a cell surface protein anchored with a glycosyl-phosphatidylinositol tail (7,8), BGPa has a transmembrane (TM)

*In this paper, we use the new nomenclature for the CEA gene family members (Ref. 17). The three cDNA clones in this paper, W211, W233 and W239, will be designated as BGPg, BGPh and BGPi, respectively. §To whom correspondence should be addressed. 0006-291X/91 $1.50 Copyright © 1991 by Academic Press, Inc. All rights of reproduction in any form reserved.

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domain followed by a cytoplasmic (Cyt) domain, in addition to an N domain and three repetitive domains (A1, B1 and A2) similar to those of CEA.

On the basis of the

findings obtained by cDNA cloning, Barnett et al. (9) have indicated that BGPa and its three variants, BGPb, BGPc and BGPd, which are all predicted as integral membrane proteins with domains TM and Cyt, can be generated from a single gene by alternative splicing of RNA. Hinoda et aL (5) have also reported two BGP cDNA clones, 4-22 and 4-13, which can be derived from mRNAs produced by different splicing of the same transcript of a gene; 4-22 and 4-13 are probably equivalent to BGPc and BGPd, respectively, in coding region, whereas their 3' untranslated region (UTR) are the same as that of BGPa and BGPb. When we screened a cDNA library of human leukocytes to elucidate the primary structures of the granulocyte nonspecific cross-reacting antigens (NCAs), another group of CEA-cross-reacting antigens (10,11), we unexpectedly identified a clone encoding BGPa.

Further analyses suggested the existence of clones similar to but

distinct from any of BGPa-d in the same library. We report here the sequences of three cDNA clones that encode three novel BGP variants which lack domains TM and

Cyt. MATERIALS AND METHODS eDNA library construction and eDNA sequencing. Construction and screening of a cDNA library from human leukocytes were described previously (10,11). Briefly, Poly(A)+ RNA was purified from peripheral leukocytes of healthy donors. A cDNA library was constructed in ZZAP II (Stratagene) and screened with a 32P-labeled Nco I-Bgl II fragment of cDNA corresponding to the N domain of NCA (12). Positive cDNA inserts were subcloned and mapped with restriction enzymes. The inserts whose restriction enzyme sites were similar to but different from those of BGPa were sequenced by the dideoxynucleotide chain termination method (13). Construction of expression vectors and their expression on COS-1 cells. cDNAs of W211 (1,759 bp), W233 (1,789 bp) and W239 (1,631 bp) were inserted into an expression vector, pSG5 (Stratagene), to yield expression vector plasmids, pSG5W211, pSG5-W233 and pSG5-W239, respectively. COS-1 cells were transiently transfected with each vector by calcium phosphate-mediated precipitation and cultured for 2 d before use (10). Metabolic labeling, immunoprecipitation and SDS-PAGE. Transfected cells (~ 5 x 106) were metabolically labeled with 3.7 MBq [35S]methionine (TranS~5-1abel, ICN) according to the method previously described (10). For inhibition of Nglycosylation, tunicamycin (2 I~g/ml) (Sigma) was added to the culture. Labeled cells were solubilized in a lysis buffer containing 1% Nonidet P-40 (15). Immunoprecipitation from cell lysates and spent culture medium with an affinitypurified rabbit antibody against CEA (16), SDS-PAGE and fluorography were performed as described before (10,15). [14C]methylated molecular weight standards were obtained from Amersham. 579

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AND

DISCUSSION

We have previously obtained 95 cDNA clones that hybridized with a cDNA probe for the N domain of NCA from a cDNA library of human peripheral leukocytes (10,11). We identified in them a cDNA clone encoding BGPa (11), which consists of domains N, A1, B1, A2, TM and Cyt (6,9).

Furthermore, dot hybridization analysis using an

oligonucleotide probe specific for the N domain of BGPa suggested that about a quarter of the 95 clones encode BGPa or BGPa-like proteins (11).

In the present

study, we have sequenced three cDNA clones that were reactive with the BGP probe and whose recognition sites of restriction enzymes were similar to but distinct from those of BGPa. The nucleotide and deduced amino acid sequences of the three clones designated as W211, W233 and W239 were depicted in Figs. 1 and 2. Their sequences revealed that W233 (Fig. 1), W211 and W239 (Fig. 2) encode BGPa-like proteins of 287, 383 and 317 residues, respectively. The molecular masses of these peptides calculated from the sequences are 42,191 for W211, 31,542 for W233 and 34,818 for W239. All the three clones contain a sequence identical to that from the 5' UTR to that encoding the B1 domain of BGPa. However, the rest of the structure of each protein encoded was found to be different from one another and from BGPa or other known BGP variants, BGPb, BGPc and BGPd (9), the overlapping amino acid sequences being identical to those of the BGP variants.

Our three new clones do not carry

polyadenylation signal but carry part or full of Alu family sequences.

Apparently,

reverse transcription started from the oligo(dT) primer hybridized to the oligo(A) stretches in the middle or in the 3'-terminal portion of the two different Alu sequences, respectively (Figs. 1 and 2). The CEA gene family members are coded by more than 15 independent genes (17). Besides BGPa-d, isomers of pregnancy-specific 131-glycoproteins (PSG), a subfamily in the CEA family, have been shown to be generated by alternative splicing of RNA (18,19). By comparing with the sequences of cDNAs and of exon-intron boundaries of the genomic DNA for BGPa-d reported by Barnett et aL (9), we concluded that the three new BGP sequences identified here were also derived from the same gene as that for BGPa-d by splicing of the precursor for mRNA. In W233, apparently, the 5' part of the intron between the two exons for the domains B1 and A2 of BGPa is used as the exon for 3' UTR and additional one amino acid at the C-terminus of B1 domain (Figs. 1 and 3A), yielding a BGP protein composed of domains N, A1 and B1 (Fig. 3B). In W211, the splicing between the exons for domains 580

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W233 5'-GGAAAACAGCAGAGGTGACAGAGCAGCCGTGCTCGAAGCGTTCC

44 119

TGGAGCCCAAGCTCTCCTCCACAGGTGAAGACAGGGCCAGCAGGAGACACCATGGGGCACCTCTCAGCCCCACTT

[_~ G H L S A P L LEADER

-30

CACAGAGTGCGTGTACCCTGGCAGGGGCTTCTGCTCACAGCCTCACTTCTAACCTTCTGG~CCCGCCCACCACT

194 W Q G L L L T A S L L T F W N P P T T -20 -I0 GCCCAGCTCACTACTGAATCCATGCCATTCAATGTTGCAGAGGGGAAGGAGGTTCTTCTCCTTGTCCACAATCTG 269 H

R

V

R

V

P

AIQLTTESMPFNVAEGKEVLLLVHNL -i ~-~DOMAIN-N I0 20 CCCCAGCAACTTTTTGGCTACAGCTGGTACAAAGGGGAAAGAGTGGATGGCAACCGTCAAATTGTAGGATATGCA P Q Q L F G Y S W Y K G E R V D G N R Q I V G Y A 30 40 ATAGG~CTCAACAAGCTACCCCAGGGCCCGCAAACAGCGGTCGAGAGACAATATACCCC~TGCATCCCTGCTG I G T Q Q A T P G P A N S G R E T I Y P N A S L L 50 60 7O ATCCAGAACGTCACCCAGAATGACACAGGATTCTACACCCTACAAGTCATAAAGTCAGATCTTGTG~TGAAGAA I Q N V T Q N D T G F Y T L Q V I K S D L V N E E 80 90 GCAACTGGACAGTTCCATGTATACCCGGAGCTGCCCAAGCCCTCCATCTCCAGC~CAACTCCAACCCTGTGGAG

344

419

494

569

ATGQFHVYP[_~LPKPSISSNNSNPVE 100 DOMAIN-AI 120 GACAAGGATGCTGTGGCCTTCACCTGTGAACCTGAGACTCAGGACAC~CCTACCTGTGGTGGATA~CAATCAG D K D A V A F T C E P E T Q D T T Y L W W I N N Q 130 140 AGCCTCCCGGTCAGTCCCAGGCTGCAGCTGTCCAATGGCAACAGGACCCTCACTCTACTCAGTGTCACAAGG~T S L P V S P R L Q L S N G N R T L T L L S V T R N 150 160 170 GACACAGGACCCTATGAGTGTGA~TACAGAACCCAGTGAGTGCGAACCGCAGTGACCCAGTCACCTTGAATGTC D T G P Y E C E I Q N P V S A N R S D P V T L N 9 180 190 ACCTATGGCCCGGACACCCCCACCATTTCCCCTTCAGACACCTATTACCGTCCAGGGGCA~CCTCAGCCTCTCC T I Y G P D T P T I S P S D T Y Y R P G A N L S L S

200L~DoMAIN-B1

210

644

719

794

869

220

TGCTATGCAGCCTCTAACCCACCTGCACAGTACTCCTGGCTTATCAATGGAACATTCCAGCAAAGCACACAAGAG 944 C Y A A S N P P A Q Y S W L I N G T F Q Q S T Q E 230 240 CTCTTTATCCCTAACATCACTGTGAATAATAGTGGATCCTATACCTGCCACGCC~TAACTCAGTCACTGGCTGC 1019 L F I P N I T V N N S G S Y T C H A N N S V T G C 250 260 270 AACAGGACCACAGTCAAGACGATCATAGTCACTGGTAAGTAATTCCTGGAGCATCAACACTAAGATCTGGGGTAC 1094

NRTTVKTIIVT~K*** 280 DOMAIN-BI~287 AAGCTTTCTGGTTTTCA~TAGGAGCAGAGAAGAAATTTTCTTTTGCAGCCTGTATCCAACAGGCACAAACAAGT CCAAATTCTCCCCTGAACCCTCTCAATTCATCTGTGCAGACTCTCTTCCCTTTGTTTTTCTGATTTCTCACAGCT GACCTTAGGTCCAGCCTGGAATGTGGGGAGGGGGTTCTCTCAGCCCCAGAAAGCCCCGTGTAGCAGGAGGGGCTT CACAGAGGGGGA~GCAG~AGGGTCCTCAAGGTCAATTTGCTTCTGTCACTAACATGTCCCTTTCTGTAACTTCT TGGCCTTCTTTTACCTATTCCATGAGATATAAGGAATATGTGAGGTTTTAAAACAGACTCACAATAGTTTTCCCT AAATGAGAGAAGGAAATGCCCTTCATCAGGGATGAGCAGCTCAGACTCTGCTCCCTGCTCTACTCCGGCTTGCCC GGTGATTCGTCTGCCCTGACCCATGTGGGGTAGGACGCAGGTGTGTGCAGAAGGTGTCCAGGTGGCCTGTCATGA ATCCAGCTAAATCAAGATGGCAGTCAATGGCTGGGCGCTGTGGTTCATGCCTGTGATCCCAGTACTTTGGAAGGC CGAGGTGAGAGGATCACCTGAGGTCAGGAGTTCGAGACCAGCCTGACCAACATGGCAAAACTCCATCTCTACTAA AAATACAAAA~AAAA~AA-3'

1169 1244 1319 1394 1469 1544 1619 1694 1769 1789

Fiq. 1. Nucleotide and deduced amino acid sequences of the cDNA clone W233. Nucleotides and amino acids are numbered on the right and under the sequences, respectively. Arrows and asterisks indicate boundaries of domains and the stop codon, respectively. An underline denotes the Alu sequence in 3' UTR.

581

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280 W239

R

T

T

V

K

T

I

I

V

T

5 '- A G G A C C A C A G T C A A G A C G A T C A T A G T C A C T G A G C T A A G T C C A G T A G T A G C A A A G C C C C A A A T C A A A G C C A G C W211

R

T

T

V

K

T

I

280

W211

I

V

T

EHL

DOMAIN-B1

S

~

P

V

V

A

K

P

Q

I

K

A

DOMAIN-A2

AAGACCACAGTCACAGGAGATAAGGACTCTGTGAACCTGACCTGCTCCACAAATGACACTGGAATCTCCATCCGT K T T V T G D K D S V N L T C S T N D T G I S I R 300

310 DOMAIN-B1

~

W239

P

V

(1138)

320

DOMAIN-C

EIIs

(1063)

S

300

L

G

E

D

E

A

V

P

G

Q

H

H

P

Q

H

TGGTTCTTCAA2~.ACC~GTCTCCCGTCCTCGGAGAGGATGAAGCTGTCCCAGGGC~CACCACCCTCAGCAT 1090 A W211

W

F

F

K

N

Q

S

L

P

S

S

E

R

M

K

330 K

P

C

Q

E

G

S

Q

G

N

T

T

L

S

317 G

C

W

D

V

L

V ***

AACCCTGTCAAGAGGGAGGATGCTGGGACGTATTGGTGTGAGGTCTTCAACCCAATCAGTAAGAACCAAAGCGAC W211

1165

(1288)

1240

(1363)

GGTGTGCAAAATGGATAAAACTCACAGGAGGCAGAATATCAATGAAGAGACCATTATAGCAAACAGAATTGCAAA 1315 G T G G T T A A G A G C T C A G C T C A G G C C G G G C A C A G T G G C T C A C G C C T G T G A T C C C A G C A G T T T G G G A G G C C A A G G C G G 1390 G C G G A T C A C G A G G G C A C ~ G A G A T C G A G G C C A T C C T G G C T A A T A T G G T G A A A C C C C G T G T C T A C T A C ~ A A A T A C A A A A 1465

(1513)

N

P

V

K

R

E

D

A

G

350

W211

(1213)

I

340

310 W239

L

T

Y

W

C

E

360

V

F

N

P

I

S

K

N

Q

S

D

370

CCCATCATGCTGAACGTAAACTGTAAGTGACTCCTCACCCCTTCCTATATGTCCCTCTAGGATTACTCTGTCAAT P I M L N V N IC K *** DOMAIN-A2 ~ 383

(1438) (1588)

A A A A A T T A G C C G G G C A T G G T G G C G G G C G C C T C ~ T G G T C C C A G C T A C T C G G C ~ A G G C T G A G G C C ~ G G A G A A T G G C G T G A 1540

(1663)

ACCTGGGAGGCGGAGCTTTCAGTGAGCCGAGATGGTGCCACTGCACTCCAGTCTGGGCAACAGGGCAAGACTCTG

1615

(1738)

T C T C A A A A A A A A A A A A {AAAAA) -3 '

1631

(1759)

Fig. 2. Nucleotide and deduced amino acid sequences of the cDNA clones W211 and W239. The nucleotide sequences from 5' UTR to amino acid 275 of both clones are identical to those of W233 (Fig. 1) and BGPa (6,9) and omitted from the figure. Nucleotide numbers parenthesized are for W211. A broken underline indicates the nucleotide sequence deleted in W239. A boxed AG shows the new splicing site yielding the C domain of W239. The other features are as indicated in Fig. 1.

B1 and A2 takes place in accordance with the GT-AG rule as in BGPa (9), whereas the splicing between the exons for domains A2 and TM does not (Fig. 3A). This mode of splicing yields a new BGP protein which consists of domains N, A1, B1 and A2 (Fig. 3B) and lacks domains TM and Cyt but has two extra residues at the C-terminus of A2 domain (Fig. 2). In W239, the original 3' splicing site of the intron between domains B1 and A2 of BGPa is not used, instead the nucleotides AG, 132 bp downstream (Fig. 2, boxed), serves as a new splicing site, resulting in a short C-terminal domain of 31 residues whose amino acid sequence

is entirely different from that of the

corresponding portion in A2 domain. The 3' UTR of W239 is identical to that of W211 except for the 64 residues following the C domain (Fig. 2); in W211, these residues code for C-terminal portion of A2 domain.

The C-terminal portions of these three

proteins are rich in hydrophilic amino acids, suggesting that, unlike the BGPa-d which are transmembrane proteins, they are secretory proteins lacking membrane binding portions. 582

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A W233

-TAGTCACTGGTAA~-~ B1

***

A2

W211

-TAGTCACT~taag-- -gaca+ G--AGAG--TGA--CTGTAA

W239

-TAGTCACT+taag---gacagag--a~~--CTGTAAGTGA--I

BGPa

TM B1 A2 ~A C taag-- --gaca+T-TAGTCACT~taag---gaca+G--AGAG--x~--~

BI

B

W233

a

I AIlBtl

W211

N

J A1

I

BllA2

W239

N

I

I

B1 ICl

BGPa

a

I M

I alIA2

BGPb

N

]

I

BGPc

N N

I A1 I I A2 IrMIC yt I A1 I B1 ItalCyt

BGPd

A1

A1

]

Italcytl

B1 ITMlCytl

Fig. 3. A) Possible splicing modes of W211, W233 and W239 predicted from the splicing sites of BGPa. The genomic sequence and splicing mode of BGPa are from Barnett et aL (9). Thick boxes indicate the exons for domains B1, A2, C or TM, and thin boxes indicate the exons for 3' UTR; unboxed regions are introns. Dashes represent omitted or unknown sequences. Asterisks show the stop codons. B) Schematic comparison of the domain structures of BGP proteins. BGPa-d are from Barnett et al. (9). Thus, these three molecular forms of BGP were suggested to be generated from the gene for BGPa by alternative splicing of RNA.

As several cell surface proteins,

including major histocompatibility complex antigens (20) and N-CAM (21), have their soluble isoforms generated by alternative splicing mechanisms, these three BGP species seem to be soluble counterparts of BGPa-d. It should be noted here that, comparison with the donor sequences at the exonintron junctions, B1-A2 and A2-TM (9), with the C-terminal coding sequences of W233 and W211, respectively, strongly suggests that the introns following the domains B1 and A2 of BGPa (9) are utilized as exons (Fig. 3A) which terminate the proteins with the hydrophilic amino acid sequences.

However detailed analyses of the genomic

sequences are needed to elucidate the origin of these exons. To characterize the corresponding proteins of W211, W233 and W239, we tansfected COS-1 cells with the expression vectors, pSG5-W211, pSG5-W233 and pSG5-W239.

A rabbit antibody raised against CEA precipitated proteins from the

culture medium of the metabolically labeled transfectants, as seen by diffuse bands on 583

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' BIOCHEMICALAND BIOPHYSICAL RESEARCH COMMUNICATIONS

W211 -

Mr

1

Medium W233

TM

2

-

3

TM

4

W239 -

TM

5

6

W211 -

7

TM

8

Cell W233 -

TM

9

10

W239 -

TM

11 12

92.5 K69 K46 K30 K-

Fig. 4. SDS-PAGE of the recombinant proteins of W211, W233 and W239 expressed on COS-1 cells. COS-1 cells transfected with expression vectors pSG5-W211 (lanes 1,2,7 and 8), pSG5-W233 (lanes 3, 4, 9 and 10) and pSG5-W239 (lanes 5, 6, 11 and 12) were labeled with [3sS]methionine in the presence (lanes of even numbers) or absence (lanes of odd numbers) of tunicamycin (TM). Immunoprecipitates with rabbit anti-CEA antibody from spent culture medium (lanes 1-6) and cell lysates (lanes 7-12) were subjected to SDS-PAGE (10% gel)/fluorography. Molecular weight standards (Mr) are phosphorylase b (92.5 K), bovine serum albumin (69 K), ovalbumin (46 K) and carbonic anhydrase (30 K). SDS-PAGE under reducing conditions (Fig. 4). Each band apparently consists of two or three components with different molecular masses: about 6 5 - 9 0 kDa for W211, 4 5 - 7 0 kDa for W233, and 5 0 - 7 5 kDa for W239.

However, the components

synthesized in the presence of tunicamycin gave single bands of about 45, 35 and 40 kDa for W211, W233 and W239, respectively (Fig. 4).

Furthermore, practically the

same patterns were obtained under nonreducing conditions (not shown). Therefore, the broadness of the bands observed for the glycosylated components probably reflect heterogeneous glycosytation rather than dimeric structure possibly formed by a disulfide linkage between the extra Cys residue in B1 domain (6,9). In the cells, the bands of the glycosylated forms of the proteins were barely visible (Fig. 4), indicating that they are secretory proteins; this is in accordance with the hydrophilic nature of their C-terminal residues. On the other hand, their unglycosylated peptides could be seen in the cells (Fig. 4), suggesting that glycosylation is requisite for the rapid secretion of these glycoproteins. The serum level of BGPa in normal healthy individuals was reported to be more than 500 ng/ml, which was at least 100 times higher than that of CEA (4). Although the corresponding proteins of our three clones have not yet been identified at protein level in granulocytes or other blood cells, it is possible that, besides biliary tracts and gall bladder, leukocytes are one of the major sources of the BGP proteins in serum. The three new species of BGP identified in this study may correspond to the low molecular 584

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weight species of BGP found in bile (2) or BGPa-like antigens immunologically similar but nonidentical to BGPa in serum (4).

ACKNOWLEDGMENTS This work supported in part by a Grant-in-Aid for Cancer Research from Ministry of Education, Science and Culture, Japan and Fukuoka Cancer Association, Japan. We thank Dr. Yoshio Misumi for helpful discussion and Miwa Soda for technical assistance.

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7. 8. 9. 10. 11. 12. 13. 14. 15. 16. 17. 18. 19. 20. 21.

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