(Received for publication, September 8, 1995; and in revised form, March 13, 1996)
From the
Fibrinogen is a dimer with each half-molecule composed of three
different chains (A, B
,
). Previous studies showed that
amino-terminal disulfide bonds, as well as the disulfide rings that
flank the ``coiled-coil'' region, are necessary for chain
assembly and secretion (Zhang, J. Z., and Redman, C. M.(1994) J.
Biol. Chem. 269, 652-658). We now determine whether other
amino-terminal domains are involved in linking the half-molecules.
Fibrinogen chains, with deletions at the amino terminus, were
co-expressed in COS cells together with normal fibrinogen chains.
Elimination of the first 8 amino acids of the B
chain did not
affect dimer assembly, but deletion of amino acid residues 9-72
had a small inhibitory effect on dimer formation. Deletion of the first
72 amino acids of the B
chain further inhibited dimer formation
and resulted in nearly equal amounts of half-molecule and dimeric
fibrinogen being formed and secreted. Deletion of the first 80
residues, which includes the cysteine residues that form the
amino-terminal disulfide ring, completely eliminated dimer formation,
and only half-molecules were secreted. By contrast deletion of the
first 41 amino acid residues of the A
chain or the first 15
residues of the
chain, which correspond to B
1-72,
did not affect chain assembly and secretion. However, co-expression of
both A
1-41 and
1-15 with normal B
,
inhibited dimer formation. Taken together, these results indicate that
in addition to disulfide bonds, noncovalent interactions of other
amino-terminal amino acid residues in the three fibrinogen chains also
participate in dimer formation.
Human fibrinogen is a dimer with each half-molecule containing
three different polypeptide chains A, B
, and
(for
reviews, see (1, 2, 3) ). Each of the
fibrinogen chains has an amino-terminal segment followed by an
-helical domain of about 111 amino acids that forms a 3-chain
``coiled-coil'' region flanked by interchain disulfide
rings(4) . On the carboxyl-terminal end, the B
and
chains have homologous globular domains, while the A
chain has a
long segment that is not equivalent to that of B
and
.
However, in normal plasma there are small amounts of a fibrinogen with
an extended A
chain, which is homologous to the carboxyl termini
of the B
and
chains(5) . Structural studies indicate
that fibrinogen is elongated and trinodal. The central node (E domain)
contains the amino termini of the 6 polypeptide chains, and the two
terminal nodes (D domains) are formed by globular carboxyl-terminal
domains of B
and
chains. The carboxyl-terminal domain of the
large A
chain is thought to fold back and contribute to the
structure of the central
node(12, 13, 14, 15, 16) .
The fibrinogen chains are held together by 29 inter- and intra-chain
disulfide
bonds(6, 7, 8, 9, 10, 11) .
Previous studies showed that intracellular assembly of the 2 half-molecules into a dimer requires not only the amino-terminal disulfide bonds but also that the disulfide rings that flank the coiled-coil region remain intact(17, 18, 19) . Disruption of the disulfide rings at the amino-terminal end of the coiled-coil region prevents dimer formation, and the elimination of the disulfide rings, at the carboxyl-terminal end of the coiled-coil region, allows dimer formation, but the 6-chain molecule that is assembled is not secreted (19) . To determine whether other amino-terminal domains are involved in fibrinogen assembly, we have constructed a series of deletion mutants and transiently co-expressed the mutant chains in COS cells together with two other normal or mutant chains. The half-molecules and dimeric fibrinogen, formed intracellularly and secreted into the medium, were determined.
A diagram, depicting the deletion mutants
constructed, is shown in Fig. 1. All normal and mutant chains
were expressed to approximately the same extent (Fig. 2).
Deletion mutant B1-192 was less radioactive, which
could be due to fewer methionine residues (Fig. 2A, lane 9). Lower molecular weight proteins, due to either
degradation or to incomplete translation, were noted in normal and
mutant A
and B
chains (Fig. 2A).
Figure 1:
Nomenclature and schematic
representation of deletion and substitution mutants of fibrinogen
A, B
, and
chains. Shown above are diagrams of
the fibrinogen A
, B
, and
chain mutants constructed. All
of the constructs had the appropriate signal sequences (S).
Deletion mutants are named based on the amino acids deleted.
Substitution mutants change cysteine residues to serine at the
positions listed. The coiled-coil region of each chain is marked by X, and the flanking cysteine residues involved in the
formation of disulfide rings are designated Cys*. Other
amino-terminal and carboxyl-terminal cysteine residues are also
shown.
Figure 2:
Expression of normal and mutant fibrinogen
chains. Single fibrinogen chain cDNAs were expressed in COS cells.
Radiolabeled fibrinogen chains from the cell lysate (panel A)
and from the culture media (panel B) were isolated by
immunoprecipitation and analyzed on 7-12% SDS-PAGE under reduced
conditions. Lane 1, normal A; lane 2,
A
28s,36s; lane 3, A
1-41; lane 4,
normal B
; lane 5, B
1-8; lane 6,
B
9-72; lane 7, B
1-72; lane
8, B
1-80; lane 9, B
1-192; lane 10, normal
; lane 11,
1-15.
Radioactive proteins were detected by autoradiography. Panel A was exposed overnight, and panel B was exposed for 5
days.
To
determine whether single mutant chains are secreted, the culture medium
was analyzed. A very small amount of normal A, A
28s,36s, and
A
1-41 were secreted (Fig. 2B, lanes
1, 2, and 3). Larger amounts of normal
and
1-15 were secreted (Fig. 2B, lanes
10 and 11). There was no secretion of normal B
,
B
1-8, B
9-72, B
1-72,
B
1-80, and B
1-192 chains. In all cases
the secreted chains were only detected on overexposure of the
autoradiogram, and we estimate that less than 10% of the
chain
synthesized was secreted into the medium. The large amount of protein
radioactivity at the top of the gel is a contaminant due to
cross-reaction of the polyclonal antibody with a protein secreted by
COS cells.
COS cells co-expressing B1-8 with normal
A
and
chains secreted the mutant dimeric fibrinogen and a
small amount of A
complex (Fig. 3A, lane
1). On reduction the component chains of fibrinogen were observed (Fig. 3B, lane 1). Free chains, intermediate
complexes, and dimeric fibrinogen occurred intracellularly (data not
shown). Expression of a mutant B
chain with an internal deletion
of amino-terminal residues 9-72, which is the same as the
naturally occurring mutant termed Fibrinogen New York 1(29) ,
allowed assembly and secretion of dimeric fibrinogen and small amounts
of half-molecules and of A
complex (Fig. 3A, lane 2). On reduction
B
9-72 co-migrates, on SDS-PAGE, together with the
chain (Fig. 3B, lane 2). However, deletion of
the first 72 amino acid residues (B
1-72) resulted in
inhibition of dimer formation and secretion of both half-molecules and
dimeric fibrinogen (Fig. 3, A and B, lane
3). Further deletion of another 8 amino acids
(B
1-80), which includes deletion of a pair of cysteines
flanking the amino-terminal side of the coiled-coil region, completely
eliminated the formation of dimeric fibrinogen, and only half-molecules
were assembled and secreted (Fig. 3, A and B, lane 4). In all cases small amounts of A
complexes were secreted. The radioactive protein at the top of the
gels, marked by arrows, is a non-fibrinogen contaminant
expressed by COS cells(19) .
Figure 3:
Deletion of amino-terminal amino acid
residues from the B chains: effect on chain assembly and
secretion. B
deletion mutant cDNAs were co-expressed in COS cells
with normal A
and
chain cDNAs. Transfected cells were
incubated for 2 h with L-[
S]methionine,
and radioactive fibrinogen complexes were isolated from the incubation
medium. Panel A, 5% SDS-PAGE, nonreduced conditions. Panel
B, 7.5% SDS-PAGE under reduced conditions. Panels A and B, lane 1, A
, B
1-8, and
; lane 2, A
, B
9-72, and
; lane
3, A
, B
1-72, and
; lane 4,
A
, B
1-80, and
; lane 5, A
,
B
1-192, and
. Panel B also shows normal
A
, B
,
as a control, in the first lane (lane 0). m
Fb, mutant half-molecule of
fibrinogen. The contaminant protein at the top of the gel is marked by an arrow.
The half-molecule, which
contains B1-80, migrated more slowly than expected in
comparison with the half-molecule that contains B
1-72.
However, two-dimensional gel electrophoresis confirms that they are
both half-molecules and suggests that the differences in mobilities may
be due to differences in conformation (Fig. 4).
Figure 4:
Two-dimensional gel electrophoresis of
secreted fibrinogen complexes. Fibrinogen complexes secreted into the
medium were separated by SDS-PAGE in nonreduced conditions in the first
dimension, followed by reducing conditions in the second dimension. The
location of 69- and 46-kDa molecular mass markers is shown in the
second dimension. The top left panel shows fibrinogen secreted
on expression of normal A, B
, and
chains. The top
right panel presents the expression of A
,
1-72 and
; bottom left panel, A
,
1-80 and
; bottom right panel,
A
1-41,
, and
1-15. Fb,
fibrinogen; 1/2Fb, half-molecule; mFb, mutant
fibrinogen; m1/2Fb, mutant half-molecule; m
,
mutant
; mA
, mutant A
; m
, mutant
.
A previous
study showed that disruption of the disulfide rings that flank the
carboxyl-terminal side of the coiled-coil region, by substituting
cysteine with serine residues, allowed dimeric fibrinogen to be formed
but prevented secretion. To further examine the role of the
carboxyl-terminal disulfide rings in assembly and secretion, a B
chain mutant (B
1-192), which contains the
carboxyl-terminal pair of cysteines at positions 193 and 196 but does
not contain the amino-terminal domain or the coiled-coil region, was
expressed. This deletion mutant did not assemble with normal A
and
chains, and no half-molecules or dimeric fibrinogen were detected
on SDS-PAGE under nonreduced conditions either intracellularly (data
not shown) or in the culture medium (Fig. 3A, lane
5). However, a small amount of A
complex and free
chains were secreted, and A
and
chains were noted on
reduction (Fig. 3B, lane 5). The free
chain is not noted in Fig. 3A, lane 5, because
in order to separate the nonreduced fibrinogen complexes, a 5% gel was
used, and the free
chain migrated out of the system.
Expression of A, B
1-72, and
led to
secretion of mutant dimeric fibrinogen, mutant half-molecule, and
A
complex. In both the mutant dimeric fibrinogen and
mutant half-molecule, the B
1-72 migrated faster in the
second dimension than
chain (Fig. 4, top right
panel).
As shown in Fig. 3A, lane 4,
expression of A, B
1-80, and
led to
inhibition of dimer formation and secretion of half-molecules. The
mutant half-molecule was characterized by its appropriate mobility in
the first dimension and that it is composed of A
, mutant B
,
and
chains (Fig. 4, bottom left panel). Another
fibrinogen complex migrated slightly faster than the half-molecule in
the first dimension, and because of its size, chain composition, and
variability in different experiments, it was tentatively identified as
degraded half-molecule. Small amounts of mutant fibrinogen chains were
also noted in the second dimension in a higher molecular weight
complex, suggesting that larger complexes composed of A
and mutant
B
1-80 chains may also occur.
Figure 5:
Amino
termini of A and
chains participate in dimer assembly. COS
cells, co-transfected with normal or mutant fibrinogen chain cDNAs were
incubated with L-[
S]methionine for 2 h.
Fibrinogen complexes were immunoprecipitated, separated on SDS-PAGE
under nonreduced conditions, and detected by autoradiography. Panel
A shows intracellular fibrinogen chains on 7.5% SDS-PAGE and panel B shows secreted fibrinogen chains on 5% SDS-PAGE. Lane 1, normal A
, B
, and
; lane 2,
A
1-41, B
, and
; lane 3, A
,
B
1-72, and
; lane 4, A
, B
, and
1-15; lane 5, A
1-41, B
,
and
1-15. Lanes 1, 2, and 3 are from the same experiment, and lanes 4 and 5 were analyzed at different times. Fb*, normal fibrinogen; mFb, mutant fibrinogen; m
Fb, mutant
half-molecule of fibrinogen
Figure 6:
Comparison of deletion and substitution
mutants. Combinations of deletion and substitution mutant(s) and of
normal fibrinogen chain(s) were co-expressed in COS cells. L-[S]methionine-labeled fibrinogen
chains were analyzed on SDS-PAGE. Autoradiograms are shown. Panel A contains secreted fibrinogen chains in nonreduced conditions, and panel B shows them in reduced conditions. Lane 1,
normal A
, B
, and
; lane 2,
A
1-41, B
, and
; lane 3,
A
28s,36s, B
, and
; lane 4, A
,
B
1-72, and
; lane 5, A
, B
65s,
and
; lane 6, A
, B
, and
1-15; lane 7, A
, B
, and
8s,9s; lane 8,
A
1-41, B
1-72, and
; lane
9, A
28s,36s, B
65s, and
; lane 10,
A
1-41, B
, and
1-15; lane
11, A
28s,36s, B
, and
8s,9s; lane 12,
A
, B
1-72, and
1-15; lane
13, A
, B
65s, and
8s,9s; lane 14,
A
1-41, B
1-72, and
1-15; lane 15, A
28s,36s, B
65s, and
8s,9s; lane
16, A
1-41,B
and
8s,9s; lane 17,
A
28s,36s, B
, and
1-15. Fb*, normal
fibrinogen; mFb, mutant fibrinogen; m
Fb,
mutant half-molecule of fibrinogen.
To determine whether the first 41 amino acids
of the A chain and the first 15 amino acids of
chain
interact noncovalently and assist dimeric fibrinogen assembly, a series
of co-transfections were performed. COS cells co-expressing A
28s,36s, normal B
, and
8s,9s (Fig. 6A, lane 11) secreted a mixture of half-molecules and dimeric
fibrinogen, indicating that dimer formation was affected but not
completely abolished. Similar results were obtained with
A
1-41, normal B
, and
8s,9s (Fig. 6A, lane 16). By contrast COS cells
co-expressing A
1-41, normal B
, and
1-15 (Fig. 6A, lane 10)
predominantly secreted half-molecules and very little dimeric
fibrinogen. The chain composition of the mutant half-molecule is shown
in two-dimensional gels in Fig. 4. Also, co-expression of
A
28s,36s, normal B
, and
1-15 (Fig. 6A, lane 17) led to the formation and
secretion of half-molecules, with very little dimeric fibrinogen. These
results indicate that noncovalent interactions at the amino-terminal
domain of the
chain, besides covalent interactions of cysteines,
may also be involved in the formation of the dimeric complexes.
Truncation of the first 72 amino acids of the B chain also
inhibited dimer formation, and both half-molecules and dimers were
secreted (Fig. 6A, lane 4). This region of the
B
contains a cysteine at position 65 that is thought to be linked
to the cysteine at A
36 of the other half-molecule of fibrinogen.
However, only substituting B
cysteine 65 to serine did not affect
dimer formation and secretion (Fig. 6A, lane
5). This again suggests that amino acid residues within B
1-72 other than the cysteine residues are involved in dimer
formation by noncovalent interactions.
On reduction, the secreted fibrinogen complexes in all cases only contained the expected mutant or normal fibrinogen chains (Fig. 6B).
The two half-molecules of fibrinogen are linked by
amino-terminal disulfide bonds. Earlier studies indicated that the
three symmetrical disulfides, between adjacent cysteines at A 28
and
8 and 9 were the principal disulfide interactions holding the
two half-molecules together. However, substituting these cysteine
residues with serine did not abolish dimer formation in transfected COS
cells(18) . It was later shown that if, in addition, the
cysteine residues at A
36 or B
65 were also substituted with
serine, then dimer formation was inhibited. This led to the suggestion
that, in addition to the symmetrical disulfide bonds between adjacent
A
and
chains, cysteine A
36 of one half-molecule is
linked to cysteine B
65 of the other
half-molecule(19, 30) . However, these amino-terminal
disulfide bonds may not be the only interactions holding the two
half-molecules. For example, substitution of B
Cys 65 with serine
did not affect dimer formation, but deleting the first 72
amino-terminal amino acids inhibited dimer formation, and equal amounts
of half-molecule and dimer were secreted. This suggested the
involvement of some of the amino-terminal amino acid residues of the
B
chain, other than cysteine, in dimer formation.
In this
report we test other B deletion mutants and also amino-terminal
A
and
deletion mutants and demonstrate that, in addition to
the cysteine at position 65, sequences within the first 72 amino acid
residues of B
participate in dimer formation as do amino-terminal
residues in the A
and
chains. The results further suggest
that a linear amino acid sequence within the B
amino-terminal
region may not be responsible for linking the two half-molecules, since
B
1-8 had little or no effect on dimer formation;
B
9-72 slightly inhibited dimer formation, but
B
1-72 significantly interrupted dimer formation.
B9-72 is the same as a naturally occurring mutant
fibrinogen, termed Fibrinogen New York 1, which was identified as a
heterozygous congenital dysfibrinogenemia with thrombotic tendency. In
the patient, Fibrinogen New York 1 was present, in plasma, as a dimer.
In the recombinant system small amounts of half-molecules are also
secreted when B
9-72 was co-expressed with normal A
and
chains. If some half-molecules were present in the plasma of
Fibrinogen New York, they could remain undetected because plasma
fibrinogen was not analyzed in nonreduced SDS-PAGE gels. Alternatively, in vivo, the half-molecule may not be secreted by hepatocytes
or may be quickly cleared from the circulation.
Clearly the major
link between the two half-molecules is the amino-terminal disulfide
bonds(19, 30) . However, our current results indicate
that noncovalent interactions in all three chains also participate. For
example, COS cells co-expressing A28s,36s,
8s,9s, and normal
B
chains are capable of assembling and secreting equal amounts of
dimeric fibrinogen and half-molecules. By contrast, cells that
co-expressed A
1-41,
1-15, and normal
B
chains only assembled and secreted half-molecules. This
indicates that besides cysteines at A
28 and 36 and
8 and 9,
noncovalent interactions of other residues in the regions of
A
1-41 and
1-15 participate in dimer
formation. Further analysis showed that COS cells co-expressing
A
1-41,
8s,9s, and normal B
chains secreted
both dimer and half-molecules, while COS cells co-expressing
A
28s,36s,
1-15, and normal B
chains only
secreted half-molecules, indicating that noncovalent interactions of
1-15 residues may play a more important role than that
of A
chain in holding the two half-molecules together.
A
structural feature of the three fibrinogen chains is that each chain
contains about 111 hydrophobic amino acids, which intertwine to form an
-helical coiled-coil region. The coiled-coil region is flanked by
a pair of cysteine (Cys-X-X-X-Cys) residues,
which form interchain disulfide bridges, termed disulfide rings.
Theoretical considerations suggested that the coiled-coil domain and
the disulfide ring may play a key role in fibrinogen
assembly(4) . Our early studies demonstrated that the first 207
amino acids of the B
chain, which contains the amino-terminal
residues and an intact coiled-coil domain with disulfide rings, can
form a dimeric structure when co-expressed with normal A
and
chains(17) . The present data show that removal of the first 72
amino acids of the B
chain results in assembly of the three chains
but that dimer formation is inhibited. This indicates that amino acid
residues 73-207 of the B
chain are sufficient for assembly
of half-molecules, while a subset of amino acid residues 1-72 of
B
chain are necessary for dimer formation. Our data also
demonstrate that deletion of an amino-terminal segment that contains
the cysteine residues that are part of the disulfide rings completely
eliminates dimer formation. This is consistent with our earlier
studies, which showed that disruption of the disulfide rings by
substituting cysteine residues with serine abolished dimer formation.
Portions of this study were presented at the annual meeting of the American Society of Biochemistry and Molecular Biology (May 1994).