Isolation and characterization of a partial cDNA clone for heparin cofactor II

Vol. 137, No. 1, 1986 May 29, 1986

BIOCHEMICAL

ISOLATION

RESEARCH COMMUNICATIONS Pages 431-436

AND BIOPHYSICAL

AND CHARACTERIZATION OF A PARTIAL FOR HEPARIN COFACTOR 111

Roger

C. Inhorn

and Douglas

cDNA CLONE

M. Tollefsen2 of Internal University 63110

Division of Hematology-Oncology, Departments Medicine and Biological Chemistry, Washington School of Medicine, St. Louis, Missouri

Received April

12, 1986

Summary: A human fetal liver cDNA library constructed in Xgtll was screened with affinity-purified rabbit antibodies raised against heparin cofactor II. One positive clone was plaque purified and the cDNA insert The clone encodes the C-terminal 167 amino acid was completely sequenced. residues of heparin cofactor II as well as the entire 3'-untranslated Proline and leucine were identified in the P2 and region of the message. P positions of the protease cleavage site, providing a possible exlanat t on for the ability of heparin cofactor II to inhibit both thrombin and chqmotrypsin-like proteases. The coding sequence is identical to that of the recently published human leuserpin 2 (Ragg (1986) Nucl. Acids Res. I& 0 1986 Academic Press, Inc. 1073).

Heparin

cofactor

one of several like

gel tion

protease

complex

that

electrophoresis. by both

specifically

and trapping

inhibitors

is

a 65,600 found

(ATIII),

inhibits

stable

Heparin

increases effect

The plasma substrates

is

the

accelerates

inhibitors the

the

polyacrylamide

In contrast,

are

cleavage

the appropriate

surrounding

sulfate

of thrombin

inactiva-

dermatan

complex

is

HCII, of an

rate

of thrombin-HCII

(l-5).

by formation

dodecyl the

that

sulfate

formation

but

has

on AT111 (6).

protease

by mimicking

glycoprotein

in human plasma

to sodium

rate

dalton

thrombin

HCII and AT111 slOOO-fold.

no significant

acids

III

antithrcnnbin

equimolar

II (HCII)3

cleavage

protease site,

sites

thought

to function

of physiological

in a stable particularly

complex. the

residue

as suicide substrates The amino in the

P‘1

lA preliminary report of this work was presented at the annual meeting of the American Society of Hematology and was published in abstract form in Blood 66 (Suppl l), 336a (1985). ?To whomcorrespondence should be addressed. JAbbreviations: HCII, heparin cofactor II; ATIII, antithranbin III; hLS2, hunan leuserpin 2; dATPCa3%], deoxyadenosine 5'-[[a3SS]thio]triphosphate.

431

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Vol. 137, No. 1, 1986

position, the

BIOCHEMICAL

are thought

inhibitor

(7).

position

(8).

clotting

cascade,

to be critical

ATIII,

inhibits

residues.

Thrombin

inhibited

by HCII (11);

cleave

(11) and chymotrypsin

ability part

by the

inhibitor

These

amino

acid

proteases

hydrophobic

known to be

leukocyte

in the

cathepsin proteases

amino proteases

of a leucine

10).

trypsin-like

chymotrypsin-like

upon limited

amino

acid

sequence

as a member of the

gene

family

a-1-antitrypsin, isolated

acid

G in

residues.

The

may be explained

PI position

in

of the

and sequenced

a 2.1

an oligonucleotide

family.

In the

encoding

the

region prepared

probe

current

from

human fetal

antibodies

against

identical,

firmly

determine

by the

Ragg isolated

the

region

inhibitor

as the

was isolated using

We report

entire from

that

we propose the

unusual

that

the

kb cDNA clone

a Xgtll

cDNA library

polytzlonal

HCII and hLS2 are

PI leucine

bifunctional

clone

3'-untranslated

affinity-purified here

2 (hLS2)

of the

a 1.2

HCII as a member of this

establishing

Furthermore,

HCII together

liver

HCII.

(17).

we have characterized

The clone

Ragg

human leuserpin

a conserved

of HCII as well

C-terminus

of the message.

inhibitors.

study

Recently,

termed

family

against

ATIII,

includes

(14-16).

kb cCNA clone

a new member of this

HCII has been

information, that

and a-1-antichymotrypsin

represents

exhibited

(9,

inhibits

P1

intrinsic

basic

above

to

(13).

classified

using

in the VIIa

from

specificity

in the

following

differ

following

identification

Based

that

and factor

HCII also

COMMUNICATIONS

protease

proteases

one of the

which

HCII to inhibit

of

serine

however,

cleave

RESEARCH

an arginine

proteins

only

(12),

generally

contains

as plasmin

is the

BIOPHYSICAL

in conferring

of the

as well

preferentially

they

which

all

proteases

that

AND

protease

family

in fact of

protease

and P2 proline specificity

inhibitor. Materials

--and Methods

Materials Restriction of Escherichia coli Laboratories. m

endonucleases, T4 DNA ligase, and the Klenow fragment DNA polqmerase were from Bethesda Research intestine alkaline phosphatase was frcm Boehringer

432

of

Vol. 137,No.

BIOCHEMICAL

1, 1986

AND BIOPHYSICAL

Deoxyadenosine 5'-[[a35S]thio]triphosphate Mannheim. Deoxynucleotides NaI251 were from Pmersham. Nitrocellulose from Pharmacia P-L Biochemicals. and Schuell.

RESEARCH COMMUNICATIONS

(dATP[a-35]S) and and dideoxynucleotides were BA-85 was from Schleicher

Isolation of clone The humanfetal liver xgtll library was kindly provided by Dr. Savio Rabbit antiserum against HCI I was prepared as Woo, Baylor University. described by Jaffe --et al. (18) and was affinity purified on an The library was plated at a density of 50,000 HCII-Sepharose column. recombinants'per 150-mm plate, and nitrocellulose lifts were hybridized sequentially with affinity-purified antibody and I25I-labeled (+S x lo6 cpm/ng) Staphylococcus aureus protein A as described by Young and Davis One positive cTo~CII.1, was plaque purified and DNA was ::$&I by a plate lysis'method (20,21). DNA s;;&t& insert from iHCII.1 was subcloned directly into Ml3 mp18, and sequencing was performed by the dideoxynucleotide chain termination method (22) using dATPCa35S) and buffer gradient gels (23). 20-base oligonucleotides were synthesized with a DNA synthesizer (Applied Biosystems, Foster City, CA) and used as sequencing primers. The identity of each nucleotide was confirmed by sequencing both strands in their entirety. DNA sequences were analyzed with the computer programs of CompuGene (St. Louis, IQ) and the Protein Identification Resources (24).

--Results A human liver

cDNA library

affinity-purifed

rabbit

positive

were

plaques

approximately xHCII.1,

insert

was subcloned

dideoxy its

of the

sequence

using

of HCII

AATAAA was identified

HCII,

I251-labeled

screened,

purified.

(Fig.

a 501-nucleotide

codon 1).

(TAG)

frame

matches

the

prior

Among

one positive

The 1.2

kb cDNA

sequenced open

amino

in 34 of 37 residues. 20 nucleotides

A.

acid

published

by the

reading

and 659 nucleotides

The predicted

with

and antibody-

protein

recombinants

contains

reading

(14)

against

and plaque

by a stop

open

was screened

MI3 mp18 and was completely

~HC11.1

sequence

in Xgtll

raised

independent

into

followed

3'-noncoding 3'-end

detected

was identified

method.

5'-end

prepared

antibodies

100,000

phage,

and Discussion

frame of

sequence carboxy

34-nucleotide

to nucleotides

942-2081

at the

terminal

A polyadenylation to the

at

signal

of

poly

(A)

tail. ~HC11.1 with

the

(nucleotide

is

exception

identical

in sequence

of nucleotide

584 in Fig.

I),

where

1525 in our

the,3'-untranslated

analysis 433

reveals

of hLS2 (17) region

a T rather

than

a

Vol. 137, No. 1, 1986

8lOCHEMlCALAND8lOPHYSlCAL

RESEARCH COMMUNICATIONS

CTC GCA GCA AAT GAC CAG GAG CTG GAC TGC GAC ATC CTC CAG CTG GAA TAC GTG Leu Ala Ala Asn Asp Gln Glu Leu Asp Cys Asp Ile Leu Gln Leu Glu Tyr Val

: 55

GGG GGC ATC AGC ATG CTA ATT GTG GTC CCA CAC AAG ATG TCT GGG ATG AAG ACC Gly Gly Ile Set- Met Leu Ile Val Val Pro His Lys Met Ser Gly Met Lys Thr

19

CTC GAA GCG CAA CTG ACA CCC CGG GTG GTG GAG AGA TGG CAA AAA AGC ATG ACA Leu Glu Ala Gln Leu Thr Pro Arg Val Val Glu Arg Trp Gln Lys Ser Met Thr

55

163

AAC AGA ACT CGA GAA GTG CTT CTG CCG AAA TTC AAG CTG GAG AAG AAC TAC AAT Asn Arg Thr Arg Glu Val Leu Leu Pro Lys Phe Lys Leu Glu Lys Asn Tyr Asn

217 73

CTA GTG GAG TCC CTG AAG TTG ATG GGG ATC AGG ATG CTG TTT GAC AAA AAT GGC Leu Val Glu Ser Leu Lys Leu Met Gly Ile Arg Met Leu Phe Asp Lys Asn Gly

271

91

AAC ATG GCA GGC ATC TCA GAC CAA AGG ATC GCC ATC GAC CTG TTC AAG 'XC Asn Met Ala Gly Ile Ser Asp Asn Arg Ile Ala Ile Asp Leu Phe Lys His

325 109

GGC ACG ATC ACA GTG AAC GAG GAA GGC ACC CAA GCC ACC ACT GTG ACC ACG GTG Gly Thr Ile Thr Val Asn Glu Glu Gly Thr Gln Ala Thr Thr Val Thr Thr Val

379 127

GGG TTC ATG CCG CTG TCC ACC CAA GTC CGC TTC ACT GTC GAC CGC CCC TTT CTT Gly Phe Met Pro Leu Ser Ile Gln Val Arg Phe Thr Val Asp Arg Pro Phe Leu

CAA Gln

9 TTC CTC ATC TAC GAGCAC CGCACC AGCTGC CTG CTC TTC ATG GGA AGA GTG GCC

433 145

Phe

Leu

Ile

Tyr

Glu

His

A~J

Thr

Ser

t&

Leu

Leu

Phe

Net

Gly

Arg

487 163

AAC CCC AGC AGG TCC TAG Asn Pro Ser Arg E Stop

551

TGTTTCCATTCCAACAACGAGAACAGAGATGTTTTGGTCATCATTTTAC~AGTTTACGCTACCAATCTGA ATTCGAGGCCCATATGAGAGGAGCTTAGAAACGACCAAGAAGAGAGGCTTGTTGGAATCAATTCTGCACAA TAGCCCATGCTGTAAGCTCATAGAAGTCACTGTAACTGTA~GTGTCTGCTGTTACCTA~GGGTCTCACC TCCCCACTCTTCACAGCAAACCTGAGCAGCGCGTCCTAAGCACCTCCCGCTCCGGTGACCCCATCCTTGCA CACCTGACTCTGTCACTCAAGCCTTTCTCCACCAGGCCCCTCATCTGAATACCAAGCACAGAAATGAGTGG TGTGACTAATTCCTTACCTCTCCCAAGGAGGGTACACAACTAGCACCATTCTTGATGTCCAGGGAAGAAGC CACCTCAAGACATATGAGGGGTGCCCTGGGCTAATGTTAGGGCTTAATTTTCTCAAAGCCTGACCTTTCAA ATCCATGATGAATGCCATCAGTCCCTCCTGCTGTTGCCTCCCTGTGACCTGGAGGACAGTGTGTGCCATGT CTCCCATACTAGAGATP(AAtAAPGTAGCCACATTTACTGTG (A)34

622 693 764

a35 906 977 1046 1119

Val

Ala

AGGTGGAGGTCTAGGTGTCTGAAGTGCCTTGGGGGCACCCTCATTT

Figure 1 Nucleotide sequence for the cDNA insert of xHCII.1. Nucleotide 1 corresponds to nucleotide 942 in the sequence reported by Ragg (17). The numbering of amino acids is arbitrary, with amino acid 1 representing residue 314 of the mature protein. The arrow represents the site of thranbin cleavage, and the polyadenylation signal of AATAAA is enclosed by a box. Amino acid residues downstream of the thrombin cleavage site have been determined for heparin cofactor II (14); the sequence predicted by xHCII.1 is identical except for three discrepancies whose positions are indicated by underlining.

C.

Since

recognize medium perfect the

our

clone

only (18), match

published

between clones

the

was isolated

one protein

from

we argue

that

between

the

24 amino published

probably

using either

hLS2 is predicted

terminal

from

plasma

in

fact

residues

errors

This

is

that

post-culture supported

by the

terminal

sequence

of hLS2 and

of HCII.

The three

discrepancies

sequence in the

antibodies

or hepatoma

HCII.

amino

HCI I protein

result

affinity-purified

and that amino

acid

predicted

by the

sequence

detennin-

cDNA

ation. Our sequence of HCII

to inhibit

analysis both

provides thrombin

a possible

explanation

and chymotrypsin-like

434

for proteases.

the

ability The

BIOCHEMICAL

Vol. 137, No. 1, 1986

Table

I. Cleavage

sites

Substrate

Pp'

Reference

-Pro

Leu

Ser

Thr

this

-Pro

Met

Ser

Ile

25

G1Y

Arg

Ser

Leu

8

-Pro

Aw

Ser

Glu

26

-Pro

Arg

Thr

Phe

26

C

-Pro

Au

Leu

Ile

27

VIII

cofactor

II

III

prothroinbin I, protein

-Pro

Arg

Ser

Phe

28

II

-Pro

Arg

Ser

Phe

28

I,

Gln

Am

Glu

Ile

28

-Pro

Arg

Val

Val

29

Val

Arg

G1Y

Pro

29

Ala

At-9

GUY

His

30

-Pro

Am

GUY

Val

31

fibrinogen,

Acl

II fibrinogen, factor

8s

XIII

The frequent occurrence by underlining,

is

cathepsin confers the

(13),

consistent

the

with

anti-thrombin

P2 position the

values

containing

residues

(32).

together

determine

provides

a model

in the P2 Position

is

ability

higher

(11,lZ).

Our prediction the that

that

bifunctional can be directly

to

for

in a-1-antichyntotrypsin leukocyte

that is

the

P2 proline

frequently

substrates

thrombin

cleavage

in the

found

with

peptides

containing

the

proline

tested

and leucine

using

I).

in In

of peptide

homologs

specificity

found

(Table

or proline

protease

paper

is emphasized

inhibit

Proline

thrombin

measured

those

found

We propose

upon HCII.

proline than

also

of HCII

known physiological

kcat&

consistently

which

activity

of

p-nitroanilides are

of proline

of a Pl leucine

G and chymotrypsin

addition,

substrates

PI'

antithrombin

(16),

thrombin

PI

a-1-antitrypsin

presence

of physiological

RESEARCH COMMUNICATIONS

Pp

heparin

factor

AND BIOPHYSICAL

P2 positions other

residues of

site-specific

HCII muta-

genesis. Acknowledgments We thank Dr. Evan Sadler for invaluable discussions, and John Bell for synthesis of oligonucleotides. This investigation was supported in part by NIH Research Service Award GM 07200, Medical Scientist, NIH grants HL 27589 and HL 01079, and a grant from the Monsanto Company. 435

P2

Vol. 137, No. 1, 1986

BIOCHEMICALAND

BIOPHYSICALRESEARCH

COMMUNICATIONS

References 1. 2. 43: 5. 6. ;: 9. ::: 12. 13. 14. 15. 16. :;: 19. 20. 21. 22. 23. 24. 25. 26. 27. 28. 29. 30. 31. 32.

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