The human protein S locus: Identification of the PSα gene as a site of liver protein S messenger RNA synthesis

Vo1.157, No. 3,1988

BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS Pages 1033-1038

December 30,1988

THE HUMAN PROTEIN S LOCUS: IDENTIFICATION OF THE PS~ GENE AS A SITE OF LIVER PROTEIN S MESSENGER RNA SYNTHESIS

Hans K. Ploos van Amstel*, Pieter H. Reitsma and Rogier M. Bertina

Haemostasis and Thrombosis Research Unit, Leiden University Hospital, 2333 AA Leiden, The Netherlands

Received October 28, 1988

Summary: The protein S locus, situated on chromosome 3, consists of two protein S genes. Here, we report the cloning and complete nucleotide sequence of the 3'-untranslated region of the two genes designated PS~ and PSi. Both regions span approximately 1,200 nucleotides. They show a high degree (-97%) of homology, with deviations caused by small deletions, insertions and point mutations. Comparison of PS~ and PS~ with the reported protein S liver eDNAs, shows that the latter all originate from the PS~ gene. The PSe gene therefore is marked as the major site of synthesis of liver protein S mRNA. Sequence comparison with the bovine protein S eDNA reveals that the PS~ gene has accumulated a few more mutations than the PS~ gene since duplication of the ancestral protein S gene that seems to have occurred recently during primate evolution. © 1988 A c a d e m i c

Press,

Inc.

Human protein S is a vitamin K-dependent plasma glycoprotein that acts as a cofactor of activated protein C, the key component of the anti-coagulant pathway of the blood

coagulation system

(i). In plasma protein S cir-

culates both free and bound with C4b-binding protein, a component of the complement activated

system protein

( 2 ) . Only C,

thereby

free

protein

accelerating

S

serves

the

as

cofactor

inactivation

of

of the

coagulation factors Va (3) and Villa (4). Heterozygotes reported

to be

for a deficiency of either protein C or protein S are at risk to develop venous

thrombo-embolic disease

at a

relatively young age, probably through a shift in the hemostatic balance that favors the formation of insoluble fibrin (5-8). The nucleotide sequence of liver cDNA coding for human protein S has been

reported by

several

laboratories

(9-12).

Southern analysis

of the

protein S locus, with eDNA probes encompassing the 3'-untranslated region

*To whom correspondence should be addressed.

1033

0006-291X/88 $1,50 Copyright © 1988 by Academic Press, Inc. All rights of reproduction in any form reserved.

Vol. 157, No. 3, 1988

BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS

of protein S mRNA, has shown that it consists of two protein S genes both situated

on

chromosome

3

(12).

The

conservation

of

restriction

sites

suggested that the two genes are highly homologous. Recently, we demonstrated that in a family with hereditary protein S deficiency

the

reduced

plasma

protein

S

level

was

associated with

a

partial deletion in the protein S locus (13). For a proper understanding of the pathogenesis of the thrombotic disorder information is needed on the

relative

contribution

of

the

two

protein

S

genes

to

protein

S

synsthesis. Therefore, we started to isolate the protein S locus and to characterize it in greater detail. Here we report the nucleotide sequence of the complete 3'-untranslated regions of the two protein S genes, designated PS~ and PSfl. The

sequences

of PSi, PSfl and the liver cDNAs coding for protein S will be discussed.

Materials and Methods Materials Phage %EMBL4 DNA, packaging extracts, E.coli strain NM539 and restriction enzymes were purchased from Promega Biotech (Madison, USA). Labelling of the protein S cDNA fragments was performed with a random priming kit from Boehringer Mannheim (Mannheim, FRG) using (~-32p)dCTP (>3000 Ci/mmol) obtained from New England Nuclear (Boston, USA). The nucleotide sequence determinations were performed with a sequencing kit from Boehringer Mannheim using (~-35S)dATP (>600 Ci/mmol) from Amersham International (Amersham, UK). Genomic library: construction and screening High molecular weight DNA, isolated from peripheral blood leukocytes, was partially digested with the restriction enzyme EcoRl and ligated in phage AEMBL4 essentially as described (14). After in vitro packaging, E. coli NM539 bacterial cells were infected. The unamplified library, containing approximately 3x105 independent recombinant phages was screened by in situ hybridization using a probe, DS 400.2, representing the 3'-end of the 3'-untranslated region of human protein S cDNA (12). The positive reeombinants were plaque purified and DNA was isolated by the plate lysate method (14). Characterization and sequence determination of the recombinant clones DNA of the recombinant clones together with human total genomic DNA were characterized for their 3'-untranslated region by restriction mapping and Southern analysis. EcoRl restriction fragments were subcloned in phage MI3 mpl9 and the nucleotide sequence was determined using the dideoxy chain termination method (15). Based on the obtained nucleotide sequence oligonucleotides were synthesized and used as specific primers in the sequencing reactions. The nucleotide sequences were analyzed using the Microgenie program from Beckman Instruments (Fullerton, USA).

Results and Discussion Approximately 3x105 independent recombinant phages of the ~EMBL4 library were screened with a cDNA fragment coding for part of the 3'-untranslated region of the protein S mRNA

(probe DS 400.2,

1034

Figure

I). Two positive

V o l . 1 5 7 , N o . 3, 1 9 8 8

BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS

cz'} ~/) ~E o_ o_

'ROBE DS I+00.2 IONE ~PSo(

1 KB i i P I ..................... P I...

i

....

P

E E

I

I .,I

PE

E

LONE ),PS/3

I

E

II

I

PE II

E I

L._

E/

P

I

I I

f,~ O_

~

E

1.1

0.7'~ o~

,3

o

o

i~

i

-

®

-

o

P '

0.2 KB, '

EcoR

I

Pst

Figure 1:

Partial restriction map and sequencing strategy of the 3'untranslated region of the phage IEMBL4 clones PS~ and PSi. The black box indicates the exon for the 3' -untranslated region. Arrows indicate the direction and extension of the sequencing reactions. Open circles denote reactions primed by specific oligonucleotides. The position of probe DS 400.2 is indicated at the top. E,EcoRI; P,Pstl.

Figure 2:

Southern blot analysis of human total DNA (HMW) and the phage clones IPS~ and IPS~, digested with the enzymes EcoRl (left panel) and Pstl (right panel). Numbers indicate the calculated molecular weights (in kilobases) of the fragments hybridizing with probe DS 400.2 (see Figure i).

recombinants were obtained, plaque purified and subjected to Southern blot analysis

(Figure

restriction

2).

enzymes

in the visualization APS~

and

IPS#

Digestion EcoRl

of two EcoRl

(Figure

performed with

of

the phage

and Pstl.

2,

left

the enzyme Pstl,

DNA was

performed

Hybridization with

fragments panel).

of 0.77

DS

with

the

results

and I.i kb for both

However,

when

digestion

was

IPS~ shows a 2.4 kb fragment hybridizing

with DS 400.2, whereas IPS# shows a 5.6 kb Pstl fragment panel).

400.2

(Figure 2, right

All fragments are in accordance with the h y b r i d i z a t i o n p a t t e r n of

h u m a n total DNA fragments

(HMW in Figure 2).

present

in human

Earlier we reported that the two Pstl

total DNA and hybridizing with DS 400.2,

find

their origin in the presence of two protein S genes per h a p l o i d genome on chromosome Pstl

as

(12).

restriction

confirmed IPS#

3

by

the

It was postulated pattern

in

isolation

their

and

far

as

characterization

with

DS

400.2

differ

regions. of

the

in their

This

clones

is

now

IPS~

and

identical EcoRl restriction patterns

but

deviate

when

digested

with

Pstl.

the EcoRl fragments encompassing the 3'-untranslated region were

subcloned sequencing (Figure

detected

two genes

3°-flanking

(Figure i). The two clones have

Next,

that the

in

phage

strategy

MI3 is

and

the

outlined

3) of the fragments

nueleotide in

containing

1035

Figure

sequence I.

The

determined. sequence

the 3'-untranslated

The

analysis

region of PS~

I

Vol. 157, No. 3, 1988

BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS

-AsnSerEnd 60 GAATT CTTAAGG CATC TTTTCTCTG CTTATAATACCTTTTCCTTG TGTG TAATTATACTT .............. C ............ G ..................C ............. -HisThrEnd

PSc~ PSB

Bovine

-C-

PS~ PSB Bovine

ATGTTTCAATAACAGCTGAAG G GTTTTATTTACAATGTGCAGTCTTTGATTATTTTG . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

PS~ PSB Bovine

TACA A~A

PSc, PSB Bovine

GATATAAATCACAGTAAAGAAATTCTTACTTCTCTTG

PS~ PSB Bovine

ATAACAATTTTAAATTTGAATTTTTTTG

-A .........

TT ...........

G ............

T-C-

- -G . . . . . . . . . . . . . 120

............

T ........

AA .....

CC ...........

TATC

TG G

. . . . . . . . . . . . . . . 180

-A-

CTTTCCTGGGATTTTTA.AAAGGTCCTTTGTGAAGG~TTCTGTTGT

AAA,AJk

- A- TTTAA

. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

........

AA .....

A ............

•

T .....

. . . . . . . . . . . . .

A- - -C-

-G .....

C .... 240

. . . .

G . . . . . . . . . . . . . . . . . . .

....

TG .....

T-T

....

~

A-TG

CTATCTAAAGAATAGTG~

. . . . . . . . . . . . . . . .

....... .o..

C- - -G .....

•

G ....

. . . . .

AA-

G . . . . . .

G-A-T-AA

.....

CCT 3OO

PS~ PSB Bovine

CTACAAATGACAGT

. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

A&TTCAATTTTT

A A G TTT

aA

AA

. . . . . . . . . .

. . . .

....A ...... T ........ AA ..... GTG .......... T-TC- --TT ..... AT .... 360 GTAAAACTAAATTTTAATTTTATCATCATGAACTAGTGTCTAAATAC CTATG TTTTTTTC --G ......................................................... - -A-

- -G ....

G . . . . . . . . . . . . . . . . . .

420

PS~ PSB PS~ PSB

AGAAAG CAAGGAAGTAAACTCAAACAAAAGTGCG TGTAATTAAATACTATTAAT CATAGG ......... C .......................A .......................... 480 CAGATACTATAAAATTTGTTTATGTTTTTGTTTTTTTCCTGATGAAGG CAGAAGAGATGG .......... TTTG . . . . . . . T. . . . . . . . . . . . . . . . . . . . . . . . T ............ A 540

PSa PSB

TGGTCTATTAAATATGAATTGAATGGAGGGTCCTAATGCCTTATTTCAAAACAATTCCTC ............................................................ 60O

PSc~ PSB

AGGGGGACCAGCTTTGGCTTCATCTTTCTCTTGTGTGGCTTCACATTTAAACCAGTATCT ............................................................ 660

PS~ PSB

TTATTGAATTAGAAAACAAGTGGGACATATTTTCCTGAGAGGAGCACAGGAATCTTACTT

PS~

CTTGGCAGCTGCAGTCTGTCAGGATGAGATATCACATTAGGTTGGATAGGTGCGGAAATC

-

-

-C

. . . . . . . . . . . . . . . . . . . . . . .

A-

-

-G

. . . . . . . . . . . . . . . . . . . . . . . .

A-

-

-

720

PSB

. . . . . . . . . . . . . . . . . . . . .

PS~

TGAAGTCGGTACATTTTTTAAATTTTGCTGTGTGGGTCACACAAGGTGTACATTACAAAA

PSB

. . . . . . . . . . . . . . .

PS~ PSB

GACAGAATTCAGGGATGGAAAGGAGAATGAACAAATGTGGGAGTTCATAGTTTTCCTTGA ............................................................

PSc~

A .......

C . . . . . . . . . . . . . . . . . . . . .

AT . . . . . . 780

AC ........

C . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 840

900 PSB

ATCCAACTTTTAATTACCAGAGTAAGTTGCCAAAATGTGATTGTTGAAGTACAAAAGGAA ............................................................

PSce

C TATGAAAACCAGAACAAATTTTAACAAAAG

PSB

...........

PS~

1020 G TATCATTG TAATCAAAGAAGTAAGGAGGTAAGATTG CCACGTG C CTG CTG GTAC TGTGA

PSB

.............

960 TGAATATC

A .......

A . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

A- - -

PSB

PS~

ATAAA

PSB

.....

PSB

PSc~ PSB

PS~

3:

GGATATAG

1080 TGCATTTCAAGTGGCAGTTTTATCACGTTTGAATCTACCATTCATAGCCAGATGTGTATC ............................................................ 1140 AGATGTTTGAC TGACAGTTTTTAACAATAAATTCTTTTCACTGTATTTTATATCACTTAT .............. G ..... G ....................................... 1200 AATAAATCGGTGTATAATTTTAAAATGCATGTGAATATCTTTATTATATCAACTGTTTGA ....... T-A . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . G ..........

PS~

Figure

GACAAC CACAGAO

G . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Jc~xx

Nucleotide sequences of the 3'-untranslated human PS~ and PS~ genes, and of the bovine (19). The bovine sequence extends to

region of the protein S cDNA the site of

polyadenylation and is inherently shorter than sequence. A dash indicates nucleotide similarity, triangle indicates a nucleotide differences

single between

base deletion. Dots the human and bovine

the human a closed indicate sequences

that are unique for either the PS~ or PS~ gene. The Mspl (CCGG) restriction site present in the PS~ gene at position 9 1 0 is i n d i c a t e d by asterisks. Two putative polyadenylation signals are underlined. The arrow indicates the site of polyadenylation.

1036

Vol. 157, No. 3, 1988

BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS

and PSi, shows that the two genes are highly homologous

(-97%). Both PS~

and PS~ possess the two putative AATAAA polyadenylation signals and the homology between the two genes extends downstream from the putative site of adenylation. When the reported sequences of the 3'-untranslated regions of liver protein S cDNAs (9-12) are compared with the corresponding region of the PS~ and PS~ genes, they all show complete similarity with the PS~ gene.

This

observation

suggests

that

the

liver

mRNAs

that

served as

template for the protein S cDNA synthesis, have been transcribed from the

PS~ gene. The

survey

of

human

liver

protein

S

cDNAs

was

extended

to

eight

additional clones also containing the 3'-untranslated region but of which no nucleotide sequences were determined (ii). Analysis was performed by making use of a Mspl restriction site distinguishing the PS~ gene from the PSe

gene

at position

910

(Figure

3),

i.e.

upstream

from

the putative

polyadenylation signals. None of the eight protein S cDNA clones contained the additional Mspl site and therefore they can not originate from the PS~ gene.

If the PS~ gene is expressed in the liver it must be less than ten

percent when compared with the level of transcription of the PSe gene. No data are available on the identity of protein S mRNA of endothelial cells and megakaryocytes that both have been shown to be, like the liver (16), sites of protein S synthesis (17,18). When part of the 3'-untranslated regions of PS~ (Figure 3, nucleotide 1-316)

and

of

PS~

(Figure

corresponding complete

3,

nucleotide

1-312)

3'-untranslated region

are

compared with

in bovine

protein

the

S cDNA

(19) about 80% homology is found for both genes. Most of the mutations that underlie the -20% divergence of the 3'-untranslated region of PS~ and PS~ from their bovine counterpart, are the same for the two human genes (Figure

3).

However,

of

the

differences

two

point

mutations

are

only

found in the PS~ gene, whereas the PS~ gene contains five unique point mutations and one unique deletion of four nucleotides. Since the moment of duplication

of

the

ancestral

protein

S

gene,

apparently

a

few

more

mutations have accumulated in the PS~ gene than in the PS~ gene. The high degree of homology between the PSe and PS~ region,

where

most

mutations

are

thought

to

have

no

3'-untranslated effect

on

the

phenotype and can be considered neutral, suggests that the duplication of the

ancestral

protein

S

gene has

taken place

recently

during

primate

evolution (20). The elucidation of the nucleotide sequence of the exons encompassing

the

3'-untranslated region

of

the

PS~

and

PS~

genes

now

offers the tools to study tissue dependent protein S mR_NA synthesis using gene specific oligonucleotide probes. The analysis of the protein S mRNA population in the respective tissues (16-18) will give us further insight

1037

Vol. 157, No. 3, 1988

in the physiological

BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS

role of each of the two protein S genes PSa and PSfl

of the human protein S locus.

Acknowledgements We thank Mr. W. te Lintel Hekkert for excellent technical assistance and Mrs. M.J. Mentink for her help in preparing the manuscript.

This work was

supported by a grant (no. 86.015) from the Trombose Stichting Nederland.

References i. 2. 3. 4.

5. 6. 7. 8. 9.

I0. ii. 12. 13. 14.

15. 16. 17. 18. 19. 20.

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