Amino acid sequence of human thrombin A chain

BIOCHEMICAL AND BIOPHYSICALRESEARCHCOMMUNICATIONS

VoL60,No. 2, 1974

AMINO ACID SEQUENCE OF HUMANTHROMBINA CHAIN Daniel A. Walz and Walter H. Seegers Depart~nt of Physiology, Thro~osis Specialized Center of Research, Wayne State University School of Medicine, Detroit, Michigan 48ZDl

Received

August

6,1974 SUMMARY

The A chain isolated from biologically active human thro~in is thirteen residues shorter from the amino terminus than the A chain isolated from bovine thrombin. Automated Edman degradation of the reduced, alkylated A chain permitted the direct identification of 34 out of the 36 residues and established all tryptic overlaps. The NH2-terminal residue is threonine. This residue is homologous to threonine-14 of the A chain of bovine thrombin. Human thrombin A chain has one half cystine linking it to the larger B chain. ~thionine, histidine, valine and tryptophan are not present. There are nine acid residues (Glu, Asp) but none of their respective amides.

INTRODUCTION The generation prothrombin defined

of biologically

proceeds through a series

for bovine prothrombin

human prothrombin been isolated

thrombin

(l-5).

The final

bovine thrombin

is the hydrolysis

autoprothrombin

C (Factor

as the bond joining

similar

of corunercially

An altered

(1,5)

or

of biologically.active

of an arginine-isoleucine peptide

available

human zymogen has

to bovine prethrombin

step in the development

Xa) {3*5) and this

from

steps which have been well

The activation

complex has been examined (6).

1 (3,4).

(E.G. 3.4.4.13)

of activation

17) which has properties

intermediate

lished

active

peptide

linkage

bond by

has been estab-

the COOH-terminus of the A chain to the NH2-terminus

of the B chain (8). Human thrombin are similar

to bovine thrombin,

the properties citrate.

Copyrighi Ail rights

Lanchantin

of human prothrombin

and its

et al.

Multiple

@ 1974 by Academic Press, Inc. of reproduction &z any form reserved.

717

to thrombin

on

in 25% sodium

two forms of human thrombin

forms of human thro~in

(13).

properties

(10,111 have reported

conversion

They have also given evidence (12) that

have been isolated. elsewhere

has been prepared on a large scale (9) and its

have also been reported

VoL60,No.

2, 1974

These various the

forms

first

This

of human thrombin

of

bovine

enzyme

was called

classical only

may be that

and that

A chain

pared

with

arises,

activity

is

of

this

49 for

the

in nature

(5,14-16).

of a single

It

be obtained

from

is to

on the

contains chain

36 amino

from

bovine

of

the

the

thrombin-E

thrombin.

report

bond,

and loss

is called

species

activity.

Arg-Ile

of classical

conmiunication

bovine

esterase

by autolysis

The latter

to the

In the

By cleavage

one form

corresponding

similar

had only

can also

of human thrombin.

RESEARCH COMMUNICATIONS

prothrombin

and then

enzymes only

are

known

remains.

three

really

The purpose of the

(5).

activity

these

there

already from

prethrombin-E

esterase

AND BIOPHYSICAL

apparently

thrombin

to be derived

thrombin

chain, It

forms

BIOCHEMICAL

Bl (5).

human prothrombin

amino

acid

acid

sequence

residues

thrombin

as com-

(17).

MATERIALS AND METHODS Human prothrombin whose

properties

and dialyzed

were in the

was converted activating (19)

complex studied cold

to thrombin mixture

and the

purified

(20)

removed

using

was removed

x 91 cm; flow

last

peak

equilibrated

eluted

substrate

solution

in distilled

product, factors

acid

G-10

excess

in 50% acetic

and the

sample acid

equilibrated and it

at

on IRC-50 pH B.Z.

and reduced

and the

acid.

was then

units/ml,

in a five-component

was chromatographed

in 8 M urea

water

15,000

and freeze-dried

iodoacetic

S-CM-A chain

California),

was dissolved

coagulation

in 50% acetic

7 ml/hr)

was the

in 0.1

The alkylated

preparative

evaporation

rate

purified

Sephadex

Berkeley,

The final

was dialyzed

labeled

was dissolved

(2.5

(6),

mg) was dissolved

over

by rotary

S-CM-thrombin

enzyme

(300

by filtration

extensively

The thrombin

thrombin

tritium

Laboratories,

C) overnight.

using

(1B).

Human thrombin ated

(4'

(Cutter

and alkyl-

salts

were

The acetic

acid

freeze-dried.

and passed

over

in 50% acetic

a G-100

acid

was refiltered

The column

(14).

The

on a G-50 column

M NH4HC03 pH 7.8. A chain

ratio

electrophoresis

of

was digested 1:50

with

trypsin

The tryptic

(w/w).

or preparative

718

thin-layer

for

6 hours

fragments

were

chromatography.

at 37' purified

C at an by The amino

BIOCHEMICALAND BIOPHYSICALRESEARCHCOMMUNICATIONS

VoL60,No. 2, 1974

TABLE I AMINO ACID SEQUENCES OF HUMANAND BOVINETHROMBINA CHAINS HUMAN:

Sequence Absent

BOVINE:*

THR-SER-GLU-ASN-H~S-PHE-GLU-PRO-PHE-PHE-ASN-GLU-LYS-

HUMAN:

1 THR-PHE-GLY-SER-GLY-GLU-ALA-ASP-CYS-GLY-LEU-ARG:

BOVlNE:

THR-PHE-GLY-ALA-GLY-GLU-ALA-ASP-CYS-GLY-LEU-ARG-

HUMAN:

2 +3+ 4 PRO-LEU-PHE-GLU-LYS-LYS-SER-LEU-GLU-ASP-LYS~THR-

BOVINE:

PRO-LEU-PHE-GLU-LYS-LYS-GLN-VAL-GLN-ASP-GLU-THR---

HU~N:

GNU-LYS~GL"-LEU-LEU-GLU-SER~TYR-*Lo-ASP-GLY-ARG-

BOVINE:

GLJ-LYS-GLU-LEU-PHE-GLU-SER-TYR-ILE-GLU-GLY-ARG-

*Bovine sequence from ~gnusson (17). Arrows indicate tryptic cleavage points for human chain only. Numbers refer to order of tryptic fragments used for amino acid analyses. Underlined residues are points of mutation. Only one of these involves more than one base change; namely, residue number 32, GLN to SER.

acid compositions fragments,

of reduced,

were determined

alkylated

A chain,

as well

using a Beckman 12lM analyzer

as the purified

tryptic

(21).

The S-CM-A chain was degraded with the use of a Beckman 89OC sequencer using a modified

DMAApeptide

program.

matography (22) and a color-coded

PTH derivatives thin-layer

were identified

chromatography

using gas chro-

technique

(23).

RESULTSAND DISCUSSION The amino acid sequence of the A chain of human thrombin The sequence of the bovine chain (17) is also presented

It is most striking NH2-te~inus differ

to note that

of the human chain.

from the bovine.

there are thirteen

In addition,

719

for comparative

residues

there are eight

All but one of these differences

is shown in Table I. purposes.

missing from the residues

represents

which

a single

Vol.&C&No. 2, 1974

BIO~EMI~L

AND BIOPHYSI~L

RESEARCH CO~UNI~TIONS

TABLE II AMINO ACID COMPOSITIONOF HUMANTHROMBINA CHAIN AND ITS TRYPTIC FRAGMENTS S-CMA Chain

TRY-1

Aspartic

2.91 (3)

?*?3(i)

Threonine* Serine*

1.90 (2)

0.89(l)

2.64 (3)

0.76(l)

Glutamic**

1.00(l)

Proline

5.42 (6) 1.09+(l)

Glycine

3.55 (4)

3.16(3)

Alanine

1.07+(l)

0.85(l)

EM-Cysteine

0.89+(l)

0.92(J)

TRY-2

TRY-3

TRY-4

TRY-5

TRY-6

0*92(l)

0*97(T) 0.90(l)

0.72(l) 1.00(l)

0.88(J)

1.00(l)

?.OO(J)

2.00(Z)

0.87(l) 1*35(l)

Valine Methionine Isoleucine

1.08+(l)

Leucine

4-23 (5)

Tyrosine Phenyla?anine

0.93 (1)

0,76(l) l.J6{?)

0*91(J)

1.06(l)

1,12(l)

Lysine

?.68 (2) 3.47 (4)

Histidine Arginine

2.11 (2)

1.09(J)

TOTALS

0.73(J)

0.86(l)

36

0.76(l)

0.82(J) 0.94(l)

12

' Average amino acid residue

1,00(l)

J.77(2) 0*88(J)

5

J

5

3

10

value used as integer.

* Threonine and serine values extrapolated from 72 hours hydrolysis. ** Glutamic acid used a& integer for the tryptic fragments. Numbers in parentheses

base change for the mutation to human Lys-23)

results

represent

nearest

integer.

One of the changes (bovine GJx-36

to have occurred.

in the fo~tion

of an additional

tryptic

frag~nt

in the

human chain. The amino acid compositions tryptic

fragments

of the reduced,

human A chain and the

The amino acid differences

are given in Table II.

human and bovine chains (residues

alkylated

14-49 only)

720

are minor.

between the

There is an increase

VoL60,No.

2, 1974

(by 1) in basic residues.

residues

of

the

bovine

As compared activation

known

0 fragment

indicate

COOH-terminus. missing

in the

autolysis the

of

based latter

but

not

glutamine

contain

of

it

does not for of

on the

there

predicted

or asparagine

is evidently

acidic

so the

acid

results

for

contain

an extra

these

by the

a difference

The final chain

the

residues

human prothrombin

human prothrombin

(by 2) in the

net

similar.

a 109 amino

Our preliminary that

are

RESEARCH COMMUNICATIONS

a decrease

human prothrombin.

removal

biosynthesis of

human chain

prothrombin,

The possibilities

activation

favor

bovine

in the (24).

AND BIOPHYSICAL

and human chains

with

results

already

does

characteristics

thrombin

of

in the

The human chain

charge

the

BIOCHEMICAL

sequence

is

acid

sequence

of

thirteen are

or (b)

specificity

of bovine

whose

amino

five-component

proteolytic

generation

in the

(a) they

amino they are

system of

acids

human at

its

are genetically removed

during

or as a result thrombin

(25).

We

interpretation.

ACKNOWLEDGEMENTS This investigation was supported by research grant HL 14142-04 from the National Heart and Lung Institute, National Institutes of Health, U. S. Public Health Service. We wish to thank Drs. Lowell E. McCoy, David Hewett-Esunett, and Jan Reuterby for helpful discussion.

REFERENCES

;: i: 5. 6. 7. 8. 9. 10. 11. 12.

Marciniak, E., and Seegers, W.H. (1966) Nature x, 621-622. Aronson, D.L., and Menache, D. (1966) Biochemistry !j, 2635-2640. Heldebrant, C.M., and Mann, K.G. (1973) J. Biol. Chem. 248, 3642-3652. Dwen, W.G., Esmon, C.T., and Jackson, C.M. (1974) J. Bix Chem. E, 594-605. Seegers, W-H., Walz, D.A., Reuterby, J., and McCoy, L.E. (1974) Thrombosis Res. 5, 829-859. Seegers, W.H., Sakuragawa, N., and McCoy, L.E. (1972) Thrombosis Res. l-, 33-46. Tishkoff, G.H., Williams, L.C., and Brown, D.H. (1971) Thromb. Diath. Haemorrh. g, 325-333. McCoy, L.E., Walz, D.A., and Seegers, W.H. (1973) Thrombosis Res. 2, 357361. Fenton, J.W., Campbell, W.P., Harrington, J.C., and Miller, K.D. (1971) Biochim. Biophys. Acta m, 26-32. Lanchantin, G.F., Friedman, J.A., and Hart, D.W. (1965) J. Biol. Chem. m, 3276-3282. Lanchantin, G.F., Friedman, J.A., and Hart, D.W. (1967) J. Biol. Chem. z, 2491-2501. Lanchantin, G.F., Friedman, J.A., and Hart, D.W. (1973) J. Biol. Chem. m, 5956-5966. 721

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BIOCHWICAL AND ~OPHYSICALRESEARCH~O~UNICATIONS

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