BIOCHEMICAL

Vol. 84, No. 2, 1978

AND BlOPHYSlCAL

RESEARCH COMMUNICATIONS

Pages 495-500

September29,1978

SEQUENCE OF AMINO ACIDS COMPRISING THE SINGLE INTRA-CHAIN DISULFIDE

LOOP IN THE a-CHAIN

R. F. Doolittle, Department

B. A. Cottrell,

of Chemistry,

August

D. Strong

University

La Jolla,

Received

OF HUMAN FIBRINOGEN

and K. W. K. Watt

of California,

California

San Diego

92093

2,1978

SUMMARY. Amino acid sequence studies have revealed that the largest cyanogen bromide fragment from the a-chain of human fibrinogen contains two cysteine residues which are situated thirty residues apart and near the carboxy-termiIn contrast to a recent report, no other cysteines nal end of that fragment. The sequence has been compared to disulexist in this region of the a-chain. fide loops in the 8- and y-chains and some very marginal homology suggested.

The a-chains of the

three

non-identical

fibrinogens

polypeptide

sequence

studies

(1).

It has been thought

of which

exist

in

two cysteines

are

bromide

which

which

to plasmin includes

this

fibrinogen also

Fretto

a-chain

substantiated,

have a significant

would

contains

that

brief

Similarly,

(as opposed

cyanogen they

broare

exposure section

of

cyanogen

to isolated

a-chains)

(5).

et al.

the human fibrinogen

from

of a large

(3,4).

six

The other

have indicated

the release

same fragment

cysteines,

(1,2),

For example,

amino

of 625 f 20 are

resulting

of evidence

in

chain

largest

dimeric

preliminary

consists

of the

loop.

these

of these

fragment

results

of intact

however,

largest

molecule,

eight

quarter

the

comprise

the a-chain only

disulfide

to the same fragment

Recently,

that

lines

intrachain

fragmentation rise

in the

and several

fibrinogen

the a-chain

gives

located

in a single

of native

that

the amino-terminal

fragmentation,

joined

chains

have indicated

residues

mide

are characteristically

In the case of the human fibrinogen

molecules. acid

of vertebrate

(.6) have reported two disulfide bearing

that

loops. on the

nature

this This

region finding,

of this

of if

part

of

0006-291x/78/0842-0495$01,00/0

495

Copyright 0 1978 by Academic Press, Inc. AN rights of reproduction in any form reserved.

Vol. 84, No. 2, 1978

the a-chain,

BIOCHEMICAL

which

has a variety

characteristics. quence our

of interesting

Accordingly,

of this

portion

previous

by alkylation to account

dues

located

P4C]iodoacetic

for

all

which

apart

Moreover,

we have been able

remainder

of the carboxy-terminal

labeled

that in

this

section

region,

all

cysteines

we have been

two cysteine

the carboxy-terminus

to overlap

resi-

of the fragment.

of the fragment

of which

se-

As in

on their

in the fragment

and near

acid

in question.

We now report

the radioactivity

30 residues

the amino

fragment were

acid.

and physiological

examined

bromide

we used a-chains

with

able

cyanogen

RESEARCH COMMUNICATIONS

structural

we have carefully

of the

studies,

AND BIOPHYSICAL

with

the

we have sequenced

(7).

EXPERIMENTAL Most

of the

original

report

methodology on the eleven

of the CNBr fragments

teolytic

enzymes.

All

cysteine)

Peptides

were

ation

(CN-I)

were

an operation

after

in detail

starting

by treatments (i.e.,

and/or

by several

methods,

attachment

to glass

The largest

for

with

those

in our

(2).

material

filtration

strategy

(PI) (2)

(Fig.

protease

monitored

the

shown

1).

This

to a series

the present

a variety

of pro-

containing paper

carboxy-

electrophoresis.

including

beads

residues.

(8)

thioacetyland the Dns-

of CN-I

the

fragment

(PII)

latter

fragment,

fragment

accounted

with into

containing

of independent

which digestions

plasmin, a large

all

the

consists with

of tryp-

In each case the radioactivity for

in purified

As a case in point,

CNBr = cyanogen bromide. chloride-phenylisothiocyanate.

496

digestion

splits

and chymotrypsin.

and subsequently

one of two cysteine

'Abbreviations used: naphthalene-l-sulfonyl

with

and a smaller

was subjected

staphylococcal

began

we have previously

piece

residues,

containing

by gel

reported

of the d-chain

peptides

and sequenced

carboxymethylcysteine

was carefully

as the

fragmented

purified

our

which

non-radioactive

sin,

is

(9).

In particular,

70-90

served

degradation

PhNCS procedure

study fragments

radioactive

analyzed

stepwise

this

CNBr'

It was further

investigation.

methyl

for

Dns-PhNCS

the

peptides chymotryptic

= 5-dimethylamino-

Vol. 84, No. 2, 1978

BIOCHEMICAL

AND BIOPHYSICAL

RESEARCH COMMUNICATIONS

1000

9

40

80 Fraction

Fig.

digest tained,

Gel filtration on Sephadex G-75 (2.5 x 110 cm.) of plasmin-treated cyanogen bromide fragment CN-I (left). The first peak (PI), which contains all the tryptophan and the bulk of the absorbance at A = 280 nM, has no radioactivity and no evidence of cysteine being The second peak (PII) contains all of the radioactivity. present. The material from pool II was subsequently treated with chymotrypsin and subjected to gel filtration on Sephadex G-50 (2.5 x 90 cm.) (right). Two radioactive peaks were observed. All the radioactivity in peak I was found associated with the carboxyterminal fragment shown in Fig. 2.

1.

was gel

filtered

each of which

peak was a virtually Only

one cysteine

of a repetitive

cent

acid

sequence

As a result

the 38-residue

segment

obtained

of this

as well

in

The first to Cl in Fig.

region

also

allowed

(Fig.

2).

disulfide

2.

overlaps,

Fig,

we have

2 to residues

us to deter-

The possibility

loops

as by establishing

of these

shown

ob-

in the peptide.

two identical

yields

peaks

radioactivity.

corresponding

of the peptides sequence

and two radioactive

of the total fragment

yielding

of peptide

peptides.

half

was identified

nature

amino

1, right)

(Fig.

29-residue

residue

the entire

on the basis

contained pure

The overlapping mine

on G-50

was ruled

overlaps

with

tentatively

462-499

out adja-

assigned

of the entire

CI-

chain.

It must be remarked portion Thus,

of the molecule chymotrypsin

second

occurring

larly,

the

tryptic

splits

that which after

at a relatively peptide

a number tended

of anomalous to complicate

two different slow

containing

rate

the

threonine and not

the cysteine

497

cleavages

occur

initial

interpretation.

residues

going adjacent

in this

(Fig,

2),

to completion. to a serine(Fig.

the Simi2)

BIOCHEMICAL

Vol. 84, No. 2, 1978

--Jr

Fig.

2.

arginine

residue extra

guises, on its

negative There

this

of the chain

part

Because identical

is

there

chains

(lo),

it

loop

region

is

are

with

disulfide

.6- and y-chains

(Fig.

spacing

in

in this

region

are

both

the cc-chain. (11,12),

of the a-chain.

of the chain a Cys-Ser-Lys residue

(I3),

at the

acid exist

The cysteines

13 residues

apart,

Whereas

B- and y-chains

just

as in

the slightest

common with

same spot

in all

the

s-chain

three

chains

498

in

this

disulfide

the corresponding

with are

has yet

hint

non-

a comnon ancestor

in the disulfide

the extreme

in unpublished).

the three

from

in contrast

homology

some circum-

&Doolittle,

sequence in

of an undeter-

and threonines

that

have evolved

which

the

under

(Jue

3).

is in

peptide

indicating

the amino

loops

because

some of the serines

fibrinogen

Nonetheless,

sequence

the

body of data

no convincing

there

and partly

phosphorylated

to compare

the

with

that

partially

a large

was of interest

side,

associated

a possibility

of the incomplete cleavage of the

because

amino-terminal

of vertebrate

B- and y-chains

dine

partly

charge

stances.

part

m

Sumnary of data used to determine the sequence of the a-chain region including the single disulfide loop which exists in this chain. T = trypsin; G = staphylococcal protease; C = chymotrypsin. Asterisks (*) indicate cysteine residues labeled with [I4diodoacetic acid.

in several

of the

RESEARCH COMMUNICATIONS

Tl

appeared

mined

AND BIOPHYSICAL

regions

loops the

homolgous

been observed

in this

carboxy-terminal

and the existence (Fig.

3).

the

30-residue

strikingly

of resemblance,

in

portions including of a histi-

In general,

BIOCHEMICAL

Vol. 84, No. 2, 1978

Fig.

3.

of this

a-chain chain

sequence

in the disulfide

in

its

carboxy-terminal

In summary,

the

a-chain

residues.

As reported

connections cysteines

apprOXimately

disulfide

loop

loops

a hint

previously,

which

six

in

with

contains

of these

quarter

exists

exemplifies

the uniqueness

portion.

located

of homology

found

loop

of human fibrinogen

in the amino-terminal are

has just fide

RESEARCH COMMUNICATIONS

Comparison of a-, B- and y-chains in the regions of intra-chain disulfide loops. Note the strong homology between B- and y-chains (11,12) and the marginal (at best) homology involving the u-chain. The y-chain section shown corresponds to residues y-324-361, and the B-chain to residues B-392-429. According to our preliminary numbering system, the a-chain section corresponds to residues 462499.

however,the

single

AND BIOPHYSICAL

eight

are involved

of the molecule.

at positions

in

interchain

The other

two

464 and 494 and form

in u-chains. the sequences

cysteine

The disulfide observed

loop

the

sequence

in the smaller

disul-

the B- and y-chains.

ACKNOWLEDGMENTS

tance

We thank

Marcia

in this

study.

Riley

and Dennis

Our work

is

Trovato

supported

for

excellent

by N.I.H.

technical

Grants

Nos.

assisHL-18,576

and GM-17,702. REFERENCES

1.

Iwanaga, S., Wall&, P., Grijndahl, Eur. J. Biochem., 8:189-199 (1969).

2.

Doolittl Hucko,

e, R . k.,

fissman,

J. T. and Takagi,

T.,

M. J.,

K. G., Cottrell, Biochemistry,

499

Henschen,

A. and Blombgck,

B. A., Friezner, S. J., &:1703-1709 (1977).

B.

Vol. 84, No. 2, 1978

;: 2: ;: 1:: ::: 13.

BIOCHEMICAL

AND BIOPHYSICAL RESEARCH COMMUNICATIONS

Harfenist, E. J. and Canfield, R. E., Biochemistr . , y, 14:4110-4117 (1975). Takagi, T. and Doolittle, R. F., Biochemistry, Q:5149-5156 (1975). Gollwitzer, R. and Timpl, R., FEBS Letters, 58:269-272 (1975). Fretto, L. J., Ferguson, E. W., Steinman, H.3. and McKee, P. A., -.A J Biol. Chem., %:2184-2195 (1978). Cottrell, B. A. and Doolittle, R. F., Thromb. Res., In press. Mross, G. A. and Doolittle, R. F., Mol. Biol., Biochem. and Biophys., 25:1-20 (1977). cay, W.; Meth. Enzymol., 25:333-344 (1972). Doolittle,-XX., Federation Proceedings, 35:2145-2149 (1976). Henschen, A. and Lottspeich, F., Thromb. Rz., 11:869-880 (1977). Watt, K. W. K., Takagi, T. and Doolittle, R. F.TProc. Natl. Acad. Sci., U.S.A., 75:1731-1735 (1978) Cottrell, B. A. and Doolittle, R. F., Biochem. Biophys. Res. Comm., -71: 754-761 (1976).

500