Vol.

177,

June

No.

2, 1991

BIOCHEMICAL

AND

BIOPHYSICAL

RESEARCH

COMMUNICATIONS

Pages

14, 1991

CLONING

AND NUCLEOTIDE SEQUENCE OF A

HUMAN

696-703

Zn-C12-GLYCOPROTEIN

cDNA AND CHROMOSOMALASSIGNMENT OF ITS GENE Hisao

Ueyama'.

Masaaki

Niwa',

Toyohiro

Tada3.

Makoto

Sasaki'

and Iwao Ohkubo' 1Department

of Medical Biochemistry, Science, Seta. Otsu

'Department

of

3Department

Biochemistry, School,

of Pathology,

Received April

Shiga University 520-21, Japan

Nagoya City Nagoya 467, Japan

of

Medical

University

Nagoya City University Nagoya 467, Japan

Medical

Medical

School,

30, 1991

: A cDNA clone of Zn-CX2-glycoprotein (Zn a Zgp) was isolated from a human prostate library. The amino acid sequence of prostate Zna2gp deduced from the nucleotide sequence was identical to the one previously reported on the Zna2gp protein purified from human blood plasma, except at three positions: the 65th and 222nd amino acid residues were Gln (+Glu) and Glu (+Gln). and there was a two amino acid insertion (Ile-Phe) Southern blot between the 75th (Glu) and 76th (Met) amino acids. analysis of human genomic DNA, however, suggested a single gene encoding Zna2gp. Using a panel of rodent-human somatic cell hybrids, the Zna2gp gene was assigned to human chromosome 7. 0 1991 SUMMARY

Zn-a2-glycoprotein molecular

weight

Shibata

(1).

the

major

to amino acid

it

postulated

response

reported

al.

that

found

that

this

(3)

in

with blood

protein

urinary

Zna2gp (MHC)

a

plasma

has common

role

(or

is

related

complex

and domain structure.

ZnCLZgp may play

a

glycoprotein

showed that

histocompatibility

sequence

glycoprotein

with

Therefore, in

the

immune

(3).

ZnCi2gp in

et

a

and is

nephritogenic

respect is

(2)

Araki of

is

38.000-40,000

with

glycopeptides). antigens

of

and Miura

determinants

to

(Zna2gp)

seminal

has been detected plasma

(4.51,

0006-291X/91 $1.50 Copyright 0 1991 by Academic Press, Inc. All rights of reproduction in any form reserved.

urine

not

only (61,

696

in blood saliva

(6)

plasma and

but

sweat

also (f-5).

Vol.

177,

No.

Using

2, 1991

BIOCHEMICAL

anti-ZnaZgp

reactive

with

cells

of

antibody

this

bronchial,

(71,

antibody

various

and mammary,

AND

human

BIOPHYSICAL

we also

are widely

glands

(8).

exocrine

and apocrine

esophageal

glands,

RESEARCH

found

as

sweat, in

that

distributed

such

COMMUNICATIONS

proteins

in

epithelial

prostate,

pancreas,

salivary,

addition

to

tracheal being

found

and in

hepatocytes. In

this

study,

characterization the

gene

chromosome

Screening

we

describe

of a cDNA encoding

for

ZnaPgp 6 where

is

located

the

Zna2gp.

on human

genes

for

the

MHC antigens AND MEIllODS

of

cDNA

Zna2gp

We also

chromosome

MATERIALS and sequencing

isolation found

7. and not

and that on

reside.

cDNA libraries constructed from human prostate and liver were obtained from Clontech Laboratories, Inc. (Palo Alto, USA). Anti-human plasma Zna2 gp antibody was prepared as described previously (7). Screening of the prostate library with the antibody was carried out as described in "Molecular Cloning" (9). The secondary antibody conjugated with alkaline phosphatase was obtained from Zymed Laboratories, Inc. (San Francisco, USA). Screening of the liver library was carried out using Zna2gp cDNA isoh3ted from a prostate library (10). The cDNA was labeled with [aP]dCTP (Amersham Laboratories, Amersham. UK) using a Random Primer DNA labeling kit (Takara Shuzo Co., Ltd., Kyoto. Japan). After subcloning into the pUC119 vector, the nucleotide sequence of the cloned cDNA was determined by the chain termination method using Sequenase from United States Biochemical Corp. (Cleveland, USA). A set of amplification primers for the polymerase chain reaction (PCR) of the Xgtll insert was obtained from Takara Shuzo Co., Ltd. Southern

blot

of

genomic

DNA

High molecular weight DNA prepared from human peripheral blood leukocytes was digested with EcoRI, EcoRI + BamHI. BamHI. BamHI + PstI. PstI, or PstI + EcoRI and the digests were electrophoresed on 0.7% agarose gel. The DNA was then transferred to a nitrocellulose filter by the method of Southern Hybridization was carried out with the 731 bp EcoRI (11). derived from cloned Zna2gp dDNA. Finally. the filter fragment was washed at 65OC in a solution containing 15 mM NaCl. 1.5 mM sodium citrate, and 0.1% SDS. and exposed to X ray film (RX) (Fuji Photo Film Co. Ltd., Minamiashigara, Japan) for 3 days. Chromosomal

assignment

of

the

Zna2gp

gene

A set of amplification primers (5'-GAAGCAAGGGTTGGAGGCAA-3' 5'-GCTAGGCAAGGAGGGATGAT-3') corresponding to the 3' and untranslated region was prepared with a DNA synthesizer (System-l Beckman). PCR was carried out with an apparatus from Plus, 697.

Vol.

177,

No.

BIOCHEMICAL

2, 1991

Riko-Kagaku (Uji, Japan). were kindly provided by Boston) through Dr. Kanda Yoshida (Hokkaido Univ.),

AND

Xgtll

cDNA library

screened

with

positive

clones

and

one

(xZP15)

subjected strategy

is

internal

EcoRI

the

the

rabbit

a total

of

had

Fig.

and

the

1.

For

The

the

was

sequencing around

the

isolated

was

into

plaques,

insert,

sequencing

subcloned

Fifteen

800,000

longest

fragment

was

prostate IgG.

analysis.

96 bp TaqI

insert,

in

presence

the

primary

sequence

Fig.

ZnCt2gp

the

that

in

nucleotide

shown

length

ZnaZgp

sequence

outlined

human

AccI

from

site

of

the

vector. The

are

from

which

nucleotide

PCR-amplified

pUC119

obtained

site,

from

plasma

clone,

to

COMMUNICATIONS

AND DISCUSSION constructed

anti-human were

RESEARCH

DNAs Rodent-human somatic cell hybrid Dr. Bruns (The Children's Hospital, (Tokyo Women's Medical College), by Dr. (Tottori Univ.). and by Dr. Oshimura

RESULTS A

BIOPHYSICAL

of

2.

cDNA.

human

Although the

seminal

clone

plasma

Zna2gp

is

VT 4 t

sequence

contain

sequence of

Zna2gp.

acid

not

acid

consisting of

amino

does

amino

peptide products

deduced

the

predicted

a signal

translated

and the

full suggests

16 amino

acids

We reported

previously

missing

N-terminal

in

TTT ip IAIl \

. I

‘ /

1

.

\ A

, 100

bp

.

I

.

/

.

,

Fig. 1. Structure and sequencing strategy of cDNA for human prostate Zn-Ci -glycoprotein. The restriction sites are Mb011 (0). HaeIII (o 3 , HinfI (0). TaqI (B). MspI(v). and Sau3A (V). respectively. ZnCl2gp cDNA has one each of internal EcoRI and BamHI sites. Solid and open boxes show the coding region and 5’and 3’-untranslated regions, respectively. Sequenced regions are indicated by horizontal arrows. For confirmation of continuity at the internal EcoRI site, the 96 bp TaqI fragment was isolated from the PCR-amplified insert, subcloned into the AccI site of puc119, and sequenced.

698

Vol.

177,

No.

2, 1991

BIOCHEMICAL

AND

BIOPHYSICAL

RESEARCH

COMMUNICATIONS

GCA AGA

6

ATG GTG CCT GTC CTG CTG TCT CTG CTG CTG C'TT CTG GGT CCT GCT GTC CCC Met Val Pro Val Leu Leu Ser Leu Leu Leu Leu Leu Gly Pro Ala Val Pro

GAG AAC Glu Asn

66 3

CAA Gln

GAA GAC Glu Asp

126 23

GAT GGT CGT TAC TCT CTG ACC Asp Gly Arg Tyr Ser Leu Thr

GTC CCC Val Pro MA Lys

GCG TTT CAG GCC Cl-I' Ala Phe Gln Ala Leu

TAT ATC TAC ACT Tyr Ile Tyr Thr

GGC TCA CTC AAT GAC CTC CAG TX Gly Ser Leu Asn Asp Leu Gln Phe

GAC AGG AAG TCT CAG CCC ATG GGA CTC TGG AGA Asp Arg Lys Ser Gin Pro Met Gly Leu Trp Arg

AAG CAG GAC AGC CAA Lys Gin Asp Ser Gin

CAG Gin t GGG CTG TCC AAG CAT'GTT Gly Leu Ser Lys His Val

TTT AGA TAC AAC Phe Arg Tyr Asn

AGT Ser

186 43

CAG GTG GAA GGA ATG GAG GAT TGG Gin Val Glu Gly Met Glu Asp Trp

246 63

CTT CAG AAG GCC AGG GAG GAC ATC TTT ATG GAG ACC Leu Gln Lys Ala Arg Glu Asp Ile Phe Met Glu Thr

GAC Asp

306 81

GGG TCT CAC GTA TTG CAG GGA AGG TTT GGT TGT Gly Ser His Val Leu Gln Gly Arg Phe Gly Cys

366 101

GAG ATC GAG AAT AAC Glu Ile Glu Asn Asn

AGA AGC AGC GGA GCA TTC TGC AAA TAT TAC TAT GAT CGA AAG GAC Arg Ser Ser Gly Ala Phe Trp LYS Tyr Tyr Tyr Asp Gly Lys Asp

426 121

TAC ATT GAA TTC AAC Tyr Ile Glu Phe Asn

AAA GAA ATC CCA LYS Glu Ile Pro

GCA GCC CAG ATA Ala Ala Gln Ile

486 141

GTC TAC GTG CAG CGG GCC AAG GCT TAC CTG GAG Val Tyr Val Gln Arg Ala Lys Ala Tyr Leu Glu

546 161

GAG GAG TGC CCT GCG ACT CTG CGG AAA Glu Glu Cys Pro Ala Thr Leu Arg Lys

TAC CTG AAA Tyr Leu Lys

606 181

CAA Gln

GAT CCT CCC TCT GTG GTG GTC ACC Asp Pro Pro Ser Val Val Val Thr

AGC CAC Set- His

AAG Lys

TGC CTG GCC TAC GAC TTC TAC CCA GGG AAA Cys Leu Ala Tyr Asp Phe Tyr Pro Gly Lys

ATT GTG GAG TAT TAC AAC Ile Val Glu Tyr Tyr Asn

ACC Thr

GAC AGT AAC Asp Ser Asn

AAG CAG AAG TGG GAG GCA GAA CCA Lys Gin Lys Trp Glu Ala Glu Pro

GCC TGG GTC CCC TX Ala Trp Val Pro Phe

GAC CCA ASP Pro

TAC AGC AAA Tyr Ser Lys

CAG GCC CCA Gln Ala Pro

CTG AAA Leu Lys

AAT ATC CTG GAC CGG Asn Ile Leu Asp Arg

GGA GAA AAG AAG AAA Gly Glu Lys Lys Lys

ATT GAT GTG CAC Ile ASP Val His

CTG Leu

666 201

TGG ACT CGG GCC GGC Trp Thr Arg Ala Gly

726 221

GAG GTG CAG GAG CCT GAG TTA CGG GGA GAT GTT CTT CAC AAT GGA AAT GGC ACT TAC Glu Val Gin Glu Pro Glu Leu Arg Gly Asp Val Leu His Asn Gly Asn Gly Thr Tyr TCC TGG GTG GTG GTG GCA GTG CCC Ser Trp Val Val Val Ala Val Pro CAC His

AGC AGC CTG GCC CAG CCC Ser Set- Leu Ala Gln Pro

CCG CAG GAC ACA Pro Gln Asp Thr

GCC CCC Ala Pro

TAC TCC TGC CAC Tyr Ser Cys His

CAG Gln

766 241

GTG CAG Val Gln

646 261

CTC GTG GTG CCC TGG GAG GCC AGC TAG GAAGCAAGGGTTGGA Leu Val Val Pro Trp Glu Ala Set- END

909 276

GGCMTGTGGGATCTCAGACCCAGTAGCTGCCCTTCCTGCCTGATGTGGGAGCTG~CCACAG~ATCACAGTCAATGG

988

ATCCACN\GGCCTGAGGAGCAGTGTGGGGGGACAGACAGGAGGTGGATTTGGAGACCG~GACTGGGATGCCTGTC~rG

1067

AGTAGACTTGGACCCAAAAAA

TCATCTCACCTTGAGCCCACCCCCACCCCATTGTCTAATCTAAATA

1146

ATCATCCCTCCTTGCCTAGC

1166

Nucleotide sequence of human prostate Zn-(x2-glycoprotein . Nucleotides are numbered in the 5’ to 3’ direction. The deduced amino acid sequence is shown below the nucleotide sequence. Amino acid residues are numbered as reported by Araki et al. (3). The cleavage slte by signal peptidase is indicated by a solid arrow. The two amino acids (Ile-Phe, underlined) were inserted into the previously reported sequence (3). The two amino acid substitutions are also underlined. The possible polyadenylation signal (AATAAA) is doubly underlined.

E&p

acid

pyroglutamic

and the

in human blood

plasma

suggests

presence

prostate

the

carbohydrate

ZnCE!gp (5). of

chains

The deduced

glutamine

at

the

which

are

present

amino acid

N-terminus

sequence of

human

ZnCUgp.

The amino acid was

identical

the

ZnWgp

sequence

to the protein

deduced

previously purified

from

reported from 699

human

the nucleotide amino acid blood

sequence

sequence

plasma(3).

on The

Vol.

177,

No.

amino

2, 1991

acid

composition

endoplasmic ThrS.

BIOCHEMICAL

p

Leulg,

This

corresponds

Tyr18.

carbohydrate

Glu

observed.

These than

liver

(Glu)

in Asnll

Va121,

Trp8. of

of Zna2gp

.

Met3,

and l/2-Cys4.

32.142

without

order

had both

sequence

of

(8).

of

DNA

revealed

fragments

were

and

the

3'

that

there

is

support

and we presume

hZP15.

a simple

detected

in

untranslated a single

three

at

Southern

blot

the

existence

that

there

or

i.e.,

3 exons (Fig.

Zna2gp

of plural

3).

700

randomly

and showed

the

HaeIII

the

types

as

the

nucleotide

clones

(hZL1)

analysis

of

only (for

of

was human

three

EcoRI

B and C domains,

strongly

in humans.

was a misinterpretation

we screened synthesizes

sites,

liver

region)

secreted

cDNA clones

these

least

gene for

Znaagp

Moreover,

pattern,

controlled

liver

Sau3A.

is

focusing,

plasma,

why

there

tears

be

the

in blood

exhibited.

of

and

could

whether

was

Ferrell

by isoelectric

MspI,

of

acids

and

saliva,

In

(Ile-Phe)

that

Kamboh

and BamHI

with

(3).

possibility

proteins

the

EcoRI

one representative that

the

humans.

that

All

XZP15.

with

amino

to determine

pattern

clone,

(Met)

obtained

from

internal

restriction

and 76th

the

sequencing

respectively acid

these

from

acid

amino

to clarify

different

amino

in plasma,

that

deduced

a two

in

in patterns

Zna2gp

but

suggest

cDNA library

plasma

genomic

75th

Zna2gp

In

is

prostate

do not

the

loci.

consistent

two positions,

these

suggested

prostate

same

ASP189

acids

at

that

also

by separate

chosen

Arg12,

amino

substitutions,

as seen

blood

expressed

Alal8,

weight

222nd

discrepancies

observed

human

His7.

and Glu.

one form

dissimilar,

a

ZnCL2gp

G1~16.

were Gln

these

between

from

COMMUNICATIONS

as follows:

molecular

the

and Gln

insertion

they

prostate

Pro17,

LYS~~.

a

and

to

and

Pheg.

sequence

addition

(12)

Gln20.

to

65th

nucleotide

more

human

RESEARCH

(5).

The

revealed

BIOPHYSICAL

was calculated

Gl~2~.

Ileg.

is

for

reticulum

Serl8

AND

suggesting The above

Zna2gp in

in the

data

humans, previous

Vol.

177,

No.

2, 1991

BIOCHEMICAL

AND

BIOPHYSICAL

RESEARCH

COMMUNICATIONS

123456

2.32.0-

0.6hb

Fig. 3. Southern blot of human genomlc DNA. High molecular weight DNA from peripheral blood leukocytes (5 pg each) was digested with EcoRI (lane 11, EcoRI + BamHI (lane 2). BamHI (lane 31, BamHI + PstI (lane 4). PstI (lane 5). or PstI + EcoRI (lane on 0.7% 6). separated agarose and transferred to a gel. nitrocellulose filter. The 731 bp EcoRI fragment from ZnCi2gp cDNA corresponding to the 124th amino acid residue through the end of the 3’ untranslated region was used as the hybridization probe.

Table

I.

Zn-

a2-glycoprotein in 24 rodent-human

segregation somatic

with cell

human hybrids

chromosomes

signal/chromosome Chromosome

Concordant +/+

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 X Y

2 4 6 4 7 5 11 1 3 6 6 6 8 7 7 6 5 6 7 4 7 2 7 1

Discordant -/10 12 9 7 12 8 13 10 8 10 10 10 10 4 11 11 10 8 3 7 4 11 4 13

% discordancy

+/-

-/+

8 6 4 7 4 6 0 9 6 4 4 5 3 4 4 5 4 4 3 6 4 7 3 9

1 1 4 6 1 5 0 3 4 3 3 3 2 8 2 2 2 4 9 5 9 2 8 0

One of the Zn- a 2-glycoprotein exons containing untranslated region was amplified by PCR in DNA independent rodent-human somatic cell hybrids. Occurrence expected product (272 bp) that is cleavable with BamHI and 95 bp fragments correlated well with human chromosome

701

43 30 35 54 21 46 0 52 48 30 30 33 22 52 25 29 29 36 55 50 54 41 50 39

the from of to 177 7.

3’ 24 the bp

Vol.

177,

No.

BIOCHEMICAL

2, 1991

analysis

Several

(3).

microheterogeniety the

of

variant

type

11/2926).

to

Araki closely

et related

structure.

It

gene

for

chromosome

6.

of

panel the

antigens, control

for

(3)

Yoshida providing

reported

on

were

quite

low

that

was,

of

interest

therefore, located

from

located

of

We thank

Dr.

Univ.),

(Tokyo

for

reason

or these

complex

on

human

using

we established on human its

thought

whether

assignment

hybrids,

is

and domain

determine

MHC

of

molecule

sequence to

the

spite

that

chromosome

similarity

to

a

be under

to

7 MHC

a different

MHC.

Bruns

(The

Women's

and Dr. somatic

acid

somewhere

ZnCXZgp is that

Kanda

rodent-human

of

16/2448,

ZnC12gp

chromosomal

cell

in of

near

PCR for somatic

ZnCXZgp is

(Hokkaido

the

the

amino

expression

(l/890,

ascribe

the

frequencies

However,

in

By applying

Dr.

have

to MHC antigens

Acknowledgments: Boston),

papers

reported

ZnC%Zgp is

mechanism

COMMUNICATIONS

heterogeneity.

Furthermore,

I).

RESEARCH

we do not

rodent-human

gene

(Table

ZnaZgp

such al.

BIOPHYSICAL

ZnCXZgp (12-14).

Accordingly,

discrepancies

the

of

AND

cell

Medical

Oshimura hybrid

Children's

(Tottori

Hospital, College), Univ.)

Dr. for

DNAs.

REFERENCES 1.

2. 3.

4. 5. 6. 7. 8.

1066BKrgi, W. and Schmid, K. (1961) J. Biol. Chem. 236, 1074. Shibata, S. and Miura, K. (1982) Nephron 31, 170-176. Araki. T., Gejyo, F., Takagaki, K., Haupt, H., Schwick, G., Burgi. W., Marti, T., Schaller, J., Rickli, E., Brossmer, R . I Atkinson, P.H., Putnam, F.W., and Schmid, K. (1988) Proc. Natl. Acad. Sci. USA 85, 679-683. Dube, J.Y., Lazure, C., Frenette, G., Paradis, G., Chretien, M., and Tremblay, R.R. (1987) The Prostate 11, 257-270. Ohkubo, l., Niwa, M., Takashima, Gasa A., Nishikimi, N., S and Sasaki, M. (1990) 152Biochim. Biophys. Acta 1034, 156. Poortmans. J.R. and Schmid, K. (1968) J. Lab. Clin. Med. 71, 807-811. Prep. Biochem. Ohkubo, I.. Niwa. M., and Sasaki, M. (1988) 413-430. 18, Tada, T., Ohkubo, I., Niwa, M., Sasaki, M., Tateyama, H., and Eimoto, T. (1991) J. Histochem. Cytochem., submitted. 702

Vol.

177,

9.

10. 11. 12. 13. 14.

No.

2, 1991

BIOCHEMICAL

AND

BIOPHYSICAL

RESEARCH

COMMUNICATIONS

Sambrook, J., (1989) Fritsch, E.F., and Maniatis, T. Molecular Cloning. A laboratory manual (2nd ed.) Chap. 12, Cold Spring Harbor Lab. Press. Benton, W.D. and Davis, 196. 180-182. R.W. (1977) Science Southern, E.M. Biol. 78, 503-517. (1975) J. Mol. Kamboh, M.I. and Ferrell. R.E. (1986) Biochem. Genet. 24, 849-857. 293Nakayashiki, N. and Katsura, S. (1989) Hum. Genet. 82, 295. Ding, M.. Umetsu, K., Nakayashiki, N., Choi, W.Y., Jia, J., and Suzuki, T. (1990) Hum. Hered. 40, 311-312.

703