(Received for publication, February 9, 1995; and in revised form, July 28, 1995)
From the
Fibrinogen (340 kDa) is a plasma protein that plays an important
role in the final stages of blood clotting. Human fibrinogen is a dimer
with each half-molecule composed of three different polypeptides
(A, 67 kDa; B
, 57 kDa;
, 47 kDa). To understand the
mechanism of fibrinogen chain assembly and secretion and to obtain a
system capable of producing substantial amounts of fibrinogen for
structure-function studies, we developed a recombinant system capable
of secreting fibrinogen. An expression vector (pYES2) was constructed
with individual fibrinogen chain cDNAs under the control of a Gal-1
promoter fused with mating factor F
1 prepro secretion signal (SS)
cascade. In addition, other constructs were prepared with combinations
of cDNAs encoding two chains or all three chains in tandem. Each chain
was under the control of the Gal-1 promoter. These constructs were used
to transform Saccharomyces cerevisiae (INVSC1; Mat
his3-
1 leu2 trp1-289 ura3-52) in selective media.
Single colonies from transformed yeast cells were grown in synthetic
media with 4% raffinose to a density of 1
10
cells/ml and induced with 2% galactose for 16 h. Yeast cells
expressing all three chains contained fibrinogen precursors and nascent
fibrinogen and secreted about 30 µg/ml of fibrinogen into the
culture medium. The B
and
chains, but not A
, were
glycosylated. Glycosylation of B
and
chains was inhibited by
treatment of transformed yeast cells with tunicamycin. Intracellular
B
and
chains, but not the A
chains in secreted
fibrinogen, were cleaved by endoglycosidase H. Carbohydrate analysis
indicated that secreted recombinant fibrinogen contained N-linked asialo-galactosylated biantennary oligosaccharide.
Recombinant fibrinogen yielded the characteristic plasmin digestion
products, fragments D and E, that were immunologically indistinct from
the same fragments obtained from plasma fibrinogen. The recombinant
fibrinogen was shown to be biologically active in that it could form a
thrombin-induced clot, which, in the presence of factor XIIIa, could
undergo
chain dimerization and A
chain polymer formation.
Human fibrinogen is a large plasma glycoprotein with diverse
physiological functions. Its primary roles are in the final stages of
blood coagulation, when it forms a fibrin clot and participates in
platelet aggregation. Fibrinogen is a dimeric molecule with each
half-molecule composed of three different polypeptides. The A
chain has 610, the B
461, and the
411 amino acid residues.
The B
and
chains are N-glycosylated. The six chains
are connected by 29 disulfide bonds. The primary structure of
fibrinogen is known, and structural studies indicate that fibrinogen is
elongated and trinodal. The central E domain contains the NH
termini of the six polypeptide chains, and the two terminal
``D'' nodes are formed by carboxyl-terminal globular domains
of the B
and
chains and a small (12-kDa) segment of the
A
chain. The COOH-terminal regions of the A
chain extend
beyond the globular domains of the B
and
chains and may fold
back and contribute to the structure of the central node. Between the
central and terminal domains the three chains are coiled together in an
-helical, rope-like manner. This
-helical area, which
occupies about 111 amino acids in each chain, is termed the
``coiled-coil'' region and is flanked at either end by a set
of interchain disulfide bonds called the ``disulfide rings.''
Unfortunately, to date, high resolution structural analysis has not
been achieved since fibrinogen crystals have diffracted poorly.
Nevertheless, a combination of biochemical and electron microscopy
studies have reached a consensus on the general structure as described
above. The structure and physiology of fibrinogen has been reviewed (1, 2, 3, 4, 5, 6, 7) .
Biologically active recombinant fibrinogen has been expressed in several mammalian cell systems(8, 9, 10, 11) , and the individual component chains of fibrinogen have been expressed in Escherichia coli(12, 13, 14) . Although recombinant fibrinogen mutants have been used for structure/function studies (15, 16, 17, 18) the procedures are hampered by the fact that prokaryotic systems do not assemble the fibrinogen chains and that transfected mammalian cells only secrete small amounts of biologically active fibrinogen. To obtain substantial quantities of biologically active fibrinogen for structure/function studies and to develop a system in which a genetic approach to understanding fibrinogen chain assembly could be undertaken, we have expressed fibrinogen in yeast.
Figure 1:
Expression vectors containing
fibrinogen chain cDNAs. The full-length cDNAs for individual fibrinogen
chains were inserted into multiple cloning sites at the 3`-end of
Gal-1-SS promoter (pYES2A, pYES2B
, and pYES2
). In the
other constructs, combinations of two chains (pYES2A
B
,
pYES2A
, and pYES2B
) and all three chains
(pYES2A
B
) were inserted in tandem. Each arrow indicates the cleavage site of the secretion
signal
The above digest was mixed with an equal volume
of buffer A (40 mM Tris-HCl, 110 mM NaCl, 0.1%
NaN, pH 7.5) and applied to a 2-ml column containing
approximately 40 mg of agarose-conjugated (Pierce AminoLink 228)
anti-fragment D monoclonal antibody (Fd4-7B3). Flow was stopped
for 2 h to allow maximum binding. Nonadsorbed protein was removed by
extensive washing with buffer A. Adsorbed protein was eluted with 4 ml
of 3 M NaSCN in buffer A.
The above nonadsorbed fraction was applied to a 2-ml column containing approximately 40 mg of agarose-conjugated anti-fibrinogen fragment E monoclonal antibody (2N3H10). The sample was recycled several times over this column to allow maximum binding. Nonadsorbed protein was removed by extensive washing with buffer A. Adsorbed protein was eluted with 4 ml of 3 M NaSCN in buffer A.
In human plasma fibrinogen the B and
chains,
but not A
, are N-glycosylated. To determine if correct N-glycosylation occurred the transformed cells expressing
single chains were incubated in the absence or presence of tunicamycin,
which prohibits N-glycosylation. Tunicamycin had no effect on
A
chain, but B
and
had faster electrophoretic
mobilities, indicating that they lacked N-linked carbohydrates (Fig. 2A).
Figure 2:
N-Glycosylation of B and
32 chains. Transformed yeast cells, expressing individual chains
and all three fibrinogen chains, were metabolically labeled with L-[
S]methionine in the presence or
absence of tunicamycin. Fibrinogen chains were isolated from the cell
lysate by immunoprecipitation and separated by 7.5% SDS-PAGE. An
autoradiogram is shown. Panel A, expression of individual
fibrinogen chains (reduced samples). Panel B, expression of
all three fibrinogen chains (nonreduced samples). The relative
positions of molecular size markers are shown on the left of
each autoradiogram. The locations of fibrinogen (Fbg), its
intermediates, and free chains are shown on the right.
INVSCIAB
expressed all
three fibrinogen chains, and analysis of intracellular fibrinogen
complexes on nonreduced SDS-PAGE demonstrated the presence of several
fibrinogen precursors. Free A
, B
, and
, two chain
complexes (B
-
and A
-
), a three-chain half-molecule,
and dimeric fibrinogen accumulated intracellularly (Fig. 2B). The intracellular complexes were
characterized by their estimated molecular weights, based on SDS-PAGE,
and the radioactive bands were excised and re-electrophoresed in
reduced conditions to determine the chain compositions (data not
shown).
On treatment with tunicamycin the A chain expressed by
INVSCIA
B
had a mobility similar to A
chains from
untreated cells, but both B
and
chains had faster
electrophoretic mobilities. Tunicamycin also affected the
electrophoretic mobilities of the higher molecular weight complexes. In
tunicamycin-treated cells the two-chain and three-chain fibrinogen
complexes were not as distinct as the corresponding complexes from
untreated cells (Fig. 2B).
Figure 3:
Endoglycosidase H treatment of
intracellular and secreted fibrinogen. INVSC1AB
cells
were metabolically labeled with L-[
S]methionine, and fibrinogen was
immunoprecipitated from the cell lysate and from the medium. The
isolated fibrinogen was treated with endoglycosidase H, and the
component chains were separated by 7.5% SDS-PAGE. An autoradiogram is
shown. The relative positions of molecular size markers are indicated
on the left, and those of component chains on the right.
Figure 4:
Carbohydrate analysis of purified
recombinant fibrinogen. Panel A, lane1,
glucose polymers obtained from partial wheat starch digest. The bands
are numbered on the leftside. Lane2, blank sample; lane3,
peptide-N-glycosidase F digest of recombinant fibrinogen. Two N-linked oligosaccharides are marked by asterisks. Lane4, 100 pmol of maltotetraose. Panel B, lane1, partial wheat starch digest; lane2, peptide-N-glycosidase F-released
oligosaccharide (starting material) treated with neuraminidase III; lane3, starting material digested with
endoglycosidase H; lane4, partial digestion with
-galactosidase; lane5, starting material
obtained by peptide-N-glycosidase F treatment; lane6, maltotetraose standard. Panel C, lane1, partial wheat starch digest; lane2,
complete digestion of starting material with
-galactosidase; lane3, digestion with combination of
-galactosidase and hexosaminidase III; lane4,
starting material obtained with peptide-N-glycosidase F
treatment; lane5, partial wheat starch
digest.
Based on the
relative migration of standard oligosaccharides, as compared with the
bands obtained from partial digestion of wheat starch (data not shown),
the main N-linked oligosaccharide (marked by an asterisk) obtained by peptide-N-glycosidase F
digestion is consistent with it being an asialo-galactosylated
biantennary oligosaccharide. Subsequent experiments confirmed the
structure. Digestion with neuraminidase III, (panelB, lane2) had no effect on the major
band, indicating lack of sialic acid, and treatment with
endoglycosidase H (panelB, lane3)
also did not affect the mobility of the N-linked
oligosaccharide, indicating, as was shown in a previous experiment (Fig. 3), that secreted recombinant fibrinogen glycoprotein is
not of the high mannose type. Partial digestion with
-galactosidase (panelB, lane4) showed the starting material and the appearance of two
lower bands. This suggests that at least two galactose monomers were
cleaved.
Further confirmation of the oligosaccharide structure was
obtained by complete digestion with -galactosidase (panelC, lane2), which demonstrates removal
of approximately two galactose units. Treatment of the starting
material with a combination of
-galactosidase and a hexosaminidase
(hexosaminidase III) (panelC, lane3) indicated complete removal of galactose and GlcNAc
from the nonreducing end of the starting oligosaccharide. Taken
together these results are consistent with the recombinant fibrinogen
being a glycoprotein that is not of the high mannose type but contains
an asialo-galactosylated biantennary oligosaccharide.
The absence of sialic acid was confirmed by assaying the recombinant yeast fibrinogen by the thiobarbituric acid method(27) . Plasma fibrinogen, used as a control, yielded about 1.25 µmol of sialic acid/µmol of fibrinogen, but sialic acid was not detected in yeast fibrinogen. Also a monosaccharide composition assay, performed by Glyko Inc., failed to detect sialic acid (data not shown). It was not possible, however (because of a high background of glucose, which was also present in the blank sample and is possibly due to contamination from the dialysis membranes), to accurately determine the molar concentration of the sugars (data not shown).
Figure 5:
Standard curves used to quantitate
fibrinogen in yeast culture medium. Fibrinogen in the culture medium (CCM) was determined by an enzyme-linked immunosorbent
assay-based competitive assay using two different antibodies. A, anti-B 1-42; B, anti-fragment
D.
In some cases the secreted fibrinogen was isolated
from the incubation medium by affinity chromatography using protamine
sulfate conjugated to Sepharose. Fibrinogen was the principal protein
product present in the incubation medium although there was a large
amount of low molecular weight material, which did not bind to the
protamine-sulfate column and which absorbed at 280 nm (Fig. 6A). Analysis of the component fibrinogen chains
by SDS-PAGE indicated that they had similar electrophoretic mobilities
as human plasma fibrinogen (Fig. 6B) and that only
B and
, and not A
, reacted with periodic acid-Schiff
stain.
Figure 6:
Analysis
of purified fibrinogen secreted by INVSC1AB
cells.
Secreted fibrinogen produced by yeast cells was purified by adsorption
on a protamine sulfate-Sepharose column. The chains were separated by
7.5% SDS-PAGE and stained with Coomassie Blue or with periodic
acid-Schiff stain. Panel A, typical elution profile of
fibrinogen from protamine sulfate coupled to Sepharose 6B. The
fibrinogen peak (Fbg) was the only protein eluted at pH 4.5. Panel B, plasma fibrinogen and yeast recombinant fibrinogen
were reduced and separated by SDS-PAGE and stained with periodic
acid-Schiff base (lanes1 and 2) and with
Coomassie Blue (lanes3 and 4). Lanes1 and 3, human plasma fibrinogen (Imco); lanes2 and 4, recombinant yeast
fibrinogen.
Figure 7:
Clotting properties of recombinant yeast
fibrinogen. Purified recombinant fibrinogen secreted by yeast cells was
incubated with thrombin or thrombin + factor XIIIa at 37 °C
for 4 h. After clotting, each sample was solubilized in a
dithiothreitol- and SDS-containing buffer separated by SDS-PAGE
(5-15% gradient gel), and Western blot analyses were performed
with two monoclonal antibodies. One monoclonal antibody reacts with
fibrin chain, and the other reacts with fibrinogen
chain
and fibrin
-dimer. Panel A, stained with Coomassie blue; panel B, immunoblot reacted with fibrin
-chain antibody
(T2G1); panel C, immunoblot reacted with fibrinogen
chain/fibrin
-dimer antibody). Lane1, molecular
size markers; lane2, plasma fibrinogen; lane3, yeast fibrinogen; lane4,
non-cross-linked fibrin prepared from yeast fibrinogen; lane5, factor XIIIa-cross-linked yeast fibrinogen. The
position of cross-linked
chain dimer is
shown.
Figure 8: Plasmin digestion of recombinant fibrinogen. Purified fibrinogen from yeast culture medium was digested with plasmin. The digested material was divided into two parts. The first part was adsorbed with antibody to fragment D, and the second part was adsorbed with antibody to fragment E coupled to Agarose columns. The bound materials from these two affinity columns were eluted and run on SDS-PAGE followed by Coomassie Blue staining and Western blot analyses. Panel A, protein stain; panel B, immunoblot reacted with anti-fragment D; panel C, immunoblot reacted with anti-fragment E. Lane1, plasmin digest of yeast fibrinogen; lane2, material absorbed by anti-fragment D; lane3, material absorbed by anti-fragment E.
Human fibrinogen has been expressed in a number of different
recombinant systems(8, 9, 10, 11) .
Although these procedures usually only produce small amounts of
secreted fibrinogen they can be scaled up, using cells in suspension
and roller bottles, to yield sufficient quantities to study
structure/function relationships. The yeast system offers an advantage
in that it is more easily adaptable to express and secrete milligram
quantities of fibrinogen. The fibrinogen expressed in yeast is
biologically active in that it forms a thrombin-induced clot and
undergoes factor XIIIa cross-linking. Carbohydrate processing,
composition, and sequence was determined by several different methods.
Periodic acid-Schiff staining of the separated chains, treatment of
transformed yeast cells with tunicamycin, and endoglycosidase H
digestion of intracellular and secreted fibrinogen showed that only
B and
chains are glycosylated. In addition, tunicamycin and
endoglycosidase H treatment suggest that initial N-linked
glycosylation of recombinant fibrinogen occurs in a manner similar to
that in hepatocytes. Tunicamycin treatment only affected the processing
of B
and
chains, and digestion with endoglycosidase H
indicated that mannose-rich fibrinogen precursors are present in the ER
and are processed before secretion occurs. Carbohydrate analysis
demonstrated that recombinant fibrinogen, unlike plasma fibrinogen,
does not contain terminal sialic acid but otherwise may be similar in
composition and sequence to plasma fibrinogen(30) . These
results are in keeping with the synthesis of N-linked glycans
by yeast. The early stages of N-glycosylation in yeast and
animal systems are similar, but further oligosaccharide processing,
which occurs in the Golgi, differs. In yeast, mannose-rich
oligosaccharides are usually formed, although galactose and N-acetylglucosamine residues may be added(31) . Our
studies indicate that recombinant yeast fibrinogen is not of the high
mannose variety and is similar but not identical to that of plasma
fibrinogen, since it lacks terminal sialic acid.
Biological activity
of recombinant fibrinogen was shown by its ability to form a
thrombin-induced clot and to undergo factor XIIIa-catalyzed
cross-linking of fibrin chains. In addition the response of recombinant
fibrinogen to thrombin and factor Xllla shows that recombinant
fibrinogen has a structure similar to that of plasma fibrinogen.
Cleavage of fibrinopeptides A and B by thrombin, polymerization, and
correct alignment of and
chains for participation in factor
XIIIa-catalyzed cross-linking, requires proper chain assembly and
folding. Further evidence that recombinant fibrinogen has a structure
similar to plasma fibrinogen was obtained by determining that fragments
D and E are produced when recombinant fibrinogen is digested with
plasmin. Fragments D and E are characteristic products when
fibrin(ogen) is treated with plasmin and can only be obtained if the
fibrinogen chains are organized in the correct configuration.
Fibrinogen chains are assembled in a series of stepwise reactions in
which single chains are linked into two-chain complexes, followed by
the addition of a third chain to form half-molecules, which are
subsequently joined to produce dimeric
fibrinogen(19, 20, 21, 32, 33, 34) .
In the yeast recombinant system, the same intermediates that accumulate
in HepG2 cells are noted. Free A, B
, and
chains,
two-chain complexes (A
and B
), and
half-molecules as well as dimeric fibrinogen accumulated in transformed
yeast cells. This suggests that the sequence of chain assembly in yeast
is similar to that in mammalian cells and that this recombinant system
will be useful for studying the mechanisms of fibrinogen chain assembly
and folding. Folding and assembly probably involve several chaperones
present in the endoplasmic reticulum, and the yeast system allows the
use of a genetic approach to studying this process. Yeast mutants,
defective in secretory factors or chaperones, can be prepared and used
to analyze chain assembly, folding, and
secretion(35, 36, 37, 38, 39, 40) .
Knowledge gained from congenital dysfibrinogens, from analyzing evolutionary conserved domains, and from biochemical and structural determinations has led to assignment of specific domains as important in the functional properties of fibrinogen. However, many of these assignments were reached by inference and have not been unambiguously elucidated. It is obvious that the recombinant systems provide the opportunity to mutate specific domains and study functional modifications. The yeast system should prove useful in this regard since it produces relatively large amounts of secreted fibrinogen that is biologically active.