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Formation of the Human Fibrinogen Subclass Fib 420 : Disulfide Bonds and
Glycosylation in Its Unique (a E Chain) Domains
By Yiping Fu, Jian-Zhong Zhang, Colvin M. Redman, and Gerd Grieninger
COS cell transfection has been used to monitor the assembly
and secretion of fibrinogen molecules, both those of the
subclass containing the novel aE chain and those of the more
abundant subclass whose a chains lack aE’s globular Cterminus. That region, referred to as the aEC domain, is
closely related to the ends of b and g chains of fibrinogen (bC
and gC). Transfection of COS cells with aE, b, and g cDNAs
alone results in secretion of the symmetrical molecule (aEbg)2,
also known as Fib420. Cotransfection with cDNA for the
shorter a chain yielded secretion of both (abg)2 and (aEbg)2
but no mixed molecules of the structure aaE(bg)2. Exploiting
the COS cells’ fidelity with regard to Fib420 production,
identification was made of the highly conserved Asn667 as
the sole site of N-linked glycosylation in the aE chain. No
evidence from Cys 8 Ser replacements was found for
interchain disulfide bridges involving the four cysteines of
the aEC domain. However, for fibrinogen secretion, the aE, b,
and g subunits do exhibit different requirements for integrity
of the two intradomain disulfide bridges located at homologous positions in their respective C-termini, indicating dissimilar structural roles in the process of fibrinogen assembly.
r 1998 by The American Society of Hematology.
F
identity); the truncated C-terminus of the common a chain (aC)
has a very different character.
Early investigations with transfected COS cells seemed to
suggest the obligatory presence of common a chains for the
assembly and secretion of recombinant aE-containing fibrinogen.6 The subsequent unexpected finding that two aE chains are
incorporated per molecule of Fib4201 prompted the current
investigation using batches of COS cells that more closely
resemble hepatic cells in their overall efficiency of fibrinogen
assembly. In this study, we show that such cells incorporate two
aE subunits per molecule of fibrinogen, whether the common a
isoform is present or not. Further use of the system to explore
the role of specific residues in Fib420 assembly and secretion
was undertaken based on an extensive series of studies showing
an absolute requirement for particular sets of interchain and
intrachain disulfide bonds in assembly and secretion of the more
abundant Fib340,7-10 which has 29 disulfide bonds in all and no
free cysteine residues.11 In this study, we examine the roles of
the four additional cysteines and two potential glycosylation
sites contributed by each of the aEC domains unique to Fib420.
IBRINOGEN, THE protein that forms the matrix of a blood
clot, is a complex molecule composed of paired sets of
three subunits (a, b, and g), each encoded by a separate gene. A
few years ago we identified a subclass of native fibrinogen
molecules based on a subunit differences.1 Molecules of this
subclass are characterized by their greater mass (,420 kD) and
lower plasma levels relative to the more abundant form (,340
kD), and we have coined the terms Fib420 and Fib340 to
distinguish between them. Whereas the novel Fib420 molecules’
three subunits all end in globular domains that resemble each
other, those of the more commonly known subclass Fib340 have
truncated a subunits that lack such a domain.
Although Fib420 molecules represent only one of every 100
fibrinogen molecules in the blood of normal, healthy adults,2
counterparts have been found throughout the vertebrate kingdom.3,4 This implies an important function for the novel
subclass that is distinct from that of the more abundant Fib340,
but that is as yet undefined.
Discovery of Fib420 in our laboratory evolved from the
seminal finding that the complete sequence of the human gene
encoding the a subunit contains an additional exon (exon VI)
undetected by previous investigators.5,6 Alternative splicing to
include exon VI gives rise to the isoform of the a chain (aE)
with a globular carboxy-terminal extension. It is the 236
residues encoded by exon VI (technically the aEC domain, but
often referred to as the VI-domain) that are as similar to the
sequences of the C-terminal domains of the fibrinogen b and g
chains (bC and gC) as the latter two are to each other (40%
From the Lindsley F. Kimball Research Institute of the New York
Blood Center, New York, NY.
Submitted April 28, 1998; accepted July 6, 1998.
Supported in part by the National Institutes of Health through grants
to G.G. (HL 51050) and C.M.R. (HL 37457) and by the American Heart
Association through a grant to G.G.
Address reprint requests to Gerd Grieninger, PhD, New York Blood
Center, 310 E 67th St, New York, NY 10021; e-mail: ggrien@server.
nybc.org.
The publication costs of this article were defrayed in part by page
charge payment. This article must therefore be hereby marked ‘‘advertisement’’ in accordance with 18 U.S.C. section 1734 solely to indicate
this fact.
r 1998 by The American Society of Hematology.
0006-4971/98/9209-0039$3.00/0
3302
MATERIALS AND METHODS
The original a, b, and g cDNAs of human fibrinogen were generous
gifts from Dominic Chung (University of Washington, Seattle). Construction of vectors containing full-length cDNAs for the aE, a, b, and g
fibrinogen subunits has been described.6,12 If not otherwise indicated,
pED4-Neo vectors (Genetics Institute, Cambridge, MA) were used.13
Cys = Ser mutations in the a, b, and g chains were described
previously.7,9,10 The N-terminal and ‘‘aC’’-domain mutations of aE
were derivatized from the a mutants as done previously.6 The mutations
Cys = Ser, Cys = Ala, and Asn = Gln in the aE chain’s VI-domain
were generated by polymerase chain reaction (PCR)-directed mutagenesis according to standard procedures. As before, all constructs were
verified by sequencing.
Transient transfections of COS-1 cells were performed by the
calcium phosphate method used earlier.12 To achieve aE synthesis
comparable to that of the other subunits, an excess of aE-cDNA was
generally added, as indicated. Qualitative evaluation of fibrinogen
production was made by labeling the cells for 2 hours with [35S]methionine in the presence of 15 µg/mL heparin and 30 KIU aprotinin, then
immunoprecipitating fibrinogen from cell lysates or culture medium
with rabbit antibodies either to whole human fibrinogen (DAKO,
Carpinteria, CA) or to the VI-domain of aE.1,6,12 The intact fibrinogen
species were separated by sodium dodecyl sulfate (SDS)/4%PAGE
under nonreducing conditions; separation of the component subunits
was achieved by SDS/7.5% PAGE under reducing conditions. Signals
Blood, Vol 92, No 9 (November 1), 1998: pp 3302-3308
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Fib420: DISULFIDE BONDS AND GLYCOSYLATION
3303
were detected by autoradiography. The results reported here were
confirmed with several different passages of COS cells.
RESULTS
COS cells transfected simultaneously with either the aE/b/g
or the aE/a/b/g sets of cDNAs synthesized all three or four
fibrinogen subunits, respectively, as shown by SDS-PAGE
analysis of cell lysates immunoprecipitated with antifibrinogen
(Fig 1A). Predominantly aE chains were immunoprecipitated by
anti-VI, indicating that most of the intracellular aE subunits are
not assembled, ie, they exist as free polypeptides. Similar
intracellular pools of free aE chains have been found in the
human hepatocarcinoma cell line HepG2 (Grieninger et al, in
preparation).
Transfected COS cells secrete mostly fully assembled fibrinogen hexamers.12 To identify aE-containing fibrinogen from
among the other fibrinogen species, immunoprecipitation of
culture medium with two antibodies was used. Anti-VI immunoprecipitated only those consisting of aE, b, and g, whereas
antifibrinogen collected every fibrinogen species, yielding a
profile on reduced SDS-PAGE that included, when present, all
four fibrinogen subunits aE, a, b, and g (Fig 1B). In immunoprecipitates of the medium of cells transfected with aE/b/g cDNAs
alone, the three fibrinogen subunits were detected at roughly
similar molar ratios, irrespective of the antibody used (anti-VI
or antifibrinogen). Because anti-VI is highly specific for the aE
chain (see Fig 1A, lanes 1 and 3; Fig 2, lane 3), immunoprecipitating b or g only when bound to aE, it follows that the bulk of
the aE secreted by these cells is packaged in fully assembled
fibrinogen molecules. Cells transfected additionally with the
cDNA for common a (the aE/a/b/g-transfectants) secrete both
Fig 1. Synthesis and secretion of aE- and a-fibrinogen by transfected COS cells. Cells were transfected with stoichiometric proportions of pBC-vectors containing fibrinogen subunit cDNAs6,12 in
combination as indicated below the lanes. When all four subunit
cDNAs were cotransfected, aE cDNA was included at a twofold
excess. Fibrinogen was immunoprecipitated from cell lysates (A) and
culture medium (B) with either antifibrinogen (f) or anti-VI (VI) as
indicated above the lanes, and subunits were separated under
reducing conditions. Migration of the g chain as a doublet in cell
extracts (see also Figs 5A and 6A) may reflect the presence of the
nonglycosylated precursor.
Fig 2. Secretion of Fib420 by transfected COS cells in the presence
and absence of the common a chain. Cells were transfected with
pED4–Neo-vectors containing fibrinogen subunit cDNAs in combination as indicated below each lane; aE cDNA was included at a fivefold
excess. Fibrinogen was immunoprecipitated from culture medium
with either antifibrinogen (f) or anti-VI (VI) as indicated and run under
nonreducing conditions. Traces of incompletely assembled or single
subunits could be seen in the lower part of the gel only after
overexposure of the film, confirming that most of the chains detected
in the medium upon reduction of the immunoprecipitates in Fig 1B
were indeed components of hexameric fibrinogen molecules.
a-fibrinogen and aE-fibrinogen as indicated by the presence of
aE, b, and g chains in the anti-VI immunoprecipitates and all
four chains in the antifibrinogen immunoprecipitates. Although
aE/a/b/g-transfectants expressed significant amounts of the
common a chain, none was found in their anti-VI–immunoprecipitated culture medium, suggesting that no mixed molecules
of the structure aaE(bg)2 were exported.
The issue of mixed versus symmetrical molecules was
explored further by separating the secreted fibrinogen species
under nonreducing conditions. As seen in Fig 2, aE/b/gtransfectants secreted a high molecular weight species (Fib420)
that is significantly larger than that of the a/b/g-transfectants
(Fib340). Most striking, however, is that only one aE-containing
species is secreted when all four chains are cotransfected
(aE/a/b/g-transfectants); this product comigrates with Fib420.
No mixed molecules, containing aE as well as a and therefore
migrating at a position intermediate between Fib420 and Fib340,
were detected.
These findings with heterologous host cells support our
earlier hypothesis that formation of symmetrical fibrinogen
molecules, (aEbg)2, is energetically favored over that of the
mixed molecules aaE(bg)2,1 driven perhaps by alternative
disulfide bond configurations either between the two aE chains’
VI-domains to form a fourth nodule and/or connecting the
VI-domains to N-terminal cysteines that could belong, in
principle, to any subunit chain, even aE itself. The latter would
serve to tether the VI-domains to the central nodule, consistent
with the tetranodular images observed in many published
electron micrographs of fibrinogen.14-16
To test this alternative disulfide bond hypothesis, we looked
for qualitative changes in the fibrinogen species secreted by
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3304
FU ET AL
COS cells that had been transfected with subunit cDNAs coding
for either wild-type chains or chains in which specific cysteines
were converted to serines (or alanines) by site-directed mutagenesis. All four cysteines in the VI-domain of human aE were
substituted, individually and in combination; changes in the
N-terminus and in the ‘‘aC’’ region of the aE chain were also
introduced (illustrated in Fig 3). In addition, N-terminal substitution mutants at sites in the b and g chains (b65S and g8S/9S)
were evaluated for their potential contribution to Fib420 formation.
To establish whether any of the above cysteine mutations per
se affected Fib420 secretion, they were initially examined in
aE/b/g-transfectants, ie, in the absence of the major a chain
(Fig 4). All mutant fibrinogen subunits were expressed, as
shown by analysis of cell lysates (Fig 4A). Despite some
variation in the amount of fibrinogen secreted by the different
mutants, it is clear that aE613S and aE780S/793S substitutions
abolished secretion of Fib420 (Fig 4B). Although the double
mutation aE613S/644S, which knocks out the putative first loop
in the VI-domain, has no discernable effect on Fib420 secretion,
the single aE613S mutant and its mutated disulfide bond partner
(aE644S; Table 1) are inhibitory. In either of these single
mutations, a reactive thiol group introduced by an unpaired
cysteine appears to interfere nonspecifically with the assembly
and/or secretion process. Of note, the aE780S/793S mutation
creates a neo–N-glycosylation site (NNS) at Asn791. That
carbohydrate is attached to this site can be seen from the upward
mobility shift of aE780S/793S in Fig 4A, lane 5. To test whether
the extra carbohydrate chain is responsible for blocking secretion, we changed Cys793 to alanine instead of serine in mutant
aE780S/793A. This particular mutation also blocked secretion
(Table 1), indicating that the missing (second) intrachain loop
Fig 3. Schematic representation of targeted amino acid positions
in the aE subunit for site-directed mutagenesis. Residues are numbered as done previously.6 Arrows or simple vertical lines indicate
positions of those cysteines converted, either singly or in combination, to serine or alanine by site-directed mutagenesis. (A) The three
regions of the aE chain: the N-terminus (NT) containing a large
a-helical segment, the so-called ‘‘aC’’ region, and the C-terminal
globular VI-domain (aEC domain); (B) the VI-domain, potential glycosylation sites in the wild-type sequence at Asn667 and Asn812 are
marked, respectively, with closed and open diamonds. The glycosylation site at Asn791 introduced by the Cys793 8 Ser change is marked
with a striped diamond. Putative loops connecting cysteines aE613/
644 as well as aE780/793 are drawn by analogy with the intrachain
loops formed by homologous cysteines in the bC and gC domains.
Fig 4. Cys 8 Ser changes in the aE, b, and g subunits: Negative
and neutral effects on the secretion of Fib420. Cys 8 Ser changes were
made in the fibrinogen subunits at the positions indicated above the
lanes. COS cells were transfected with pED4-vectors containing aE, b,
and g (either wild-type or mutant) cDNAs as shown; aE constructs
were included at a fivefold excess. Fibrinogen was immunoprecipitated, from cell lysates (A) and culture medium (B), with antifibrinogen and run under reducing conditions. The size of protein markers
(lane 1) is displayed in kD in the left margin.
and not the carbohydrate attachment is responsible for lack of
Fib420 export in aE780S/793S mutants.
In addition to the VI-domain cysteines, we evaluated substitutions of cysteines in other parts of the Fib420 molecule that might
be available for alternative disulfide bonding with the VIdomain, reasoning that those cysteines were not found to be
critical for export of Fib340. The first pair of these was further
upstream in the aE chain, Cys442 and Cys472, which corre-
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Fib420: DISULFIDE BONDS AND GLYCOSYLATION
3305
Table 1. Effect of aE Subunit Mutations on Secretion of Fib420 in COS
Cells Transfected With aE, b, and g cDNAs
Mutation
Fib420 Secretion
Wild-type
28-S and 36-S
442-S and 472-S
613-S
644-S
780-S
793-S
613-S and 644-S
780-S and 793-S
780-S and 793-A
613-S and 780-S
644-S and 793-S
613-S, 644-S, 780-S, and 793-S
667-Q
812-Q
1
1
1
2
2
2
2
1
2
2
2
2
2
1
1
Cys = Ser (S); Cys = Ala (A); Asn = Gln (Q).
spond to positions in the aC region of the common a chain
known to form an intrachain loop10; mutant aE442S/472S had
no effect on Fib420 secretion in aE/b/g-transfectants (Fig 4).
Other candidate cysteines that we evaluated, located in the
N-termini of the constituent fibrinogen chains (aE Cys28 and
Cys36, b Cys65, and g Cys8 and Cys9), are involved in forming
the symmetrical disulfide bridges that hold the abg trimers
together; only one out of the four symmetrical bonds is
absolutely required for Fib340 hexamer assembly and secretion.7,8,10 None of these mutations, aE28S/36S, b65S, or g8S/9S,
when cotransfected with complementary wild-type chains,
interfered with proper secretion of Fib420 molecules in the
aE/b/g-transfectants (Fig 4).
Having established that Fib420 production was possible
despite substitution of particular residues, the same mutations
were examined in aE/a/b/g-transfectants. This test of the
alternative disulfide bond configuration hypothesis could be
expected to yield secretion of mixed molecules (containing both
aE and a) whenever cysteines involved in bonds favoring
homodimer formation were replaced. For each set of mutations,
it was first shown that simultaneous transfection with all four
cDNAs leads to expression of all four subunits (Fig 5A) and that
for the subunits comprising Fib420, ie, the aE, b, and g chains,
the levels of expression were comparable to those of the
aE/b/g-transfectants (Fig 4A). The medium of the transfected
cells was then immunoprecipitated with anti-VI for detection of
secreted aE-containing fibrinogen (Fig 5B). Although secretion
with mutated subunits was less efficient, in no case were mixed
molecules detectable as species migrating faster than that of the
aE/b/g-transfectant. It follows that selective incorporation of
two aE subunits in the presence of the abundant a chain is not
dependent on disulfide bridges involving cysteines in the
VI-domain of aE or on available cysteine partners further
upstream in the aE chain itself or in the other two subunits
present in the central core of the fibrinogen molecule. These
results with recombinant molecules are consistent with trypsin
digests of a8-fibrinogen,4 the lamprey equivalent of Fib420,
which appears to be a similarly symmetrical molecule but
which has aE-like chains derived from a separate a8 gene17
B
Fig 5. Effect of aE, b, and g subunit mutations on Fib420 secretion in
presence of the wild-type a subunit: No secretion of mixed molecules. Cys 8 Ser changes were made in the fibrinogen subunits at
the positions indicated above the lanes. COS cells were transfected
with either wild-type or mutant aE, b, and g cDNAs together with
wild-type a cDNA in the combinations shown; aE constructs were
included at a fivefold excess. Fibrinogen was immunoprecipitated
from cell lysates (A) with antifibrinogen and run under reducing
conditions; from the culture medium (B), fibrinogen was immunoprecipitated with anti-VI and run under nonreducing conditions. As
described previously,1 due to the differential proteolytic susceptibility
of a and aE subunits in cell lysates, a proteolytic fragment derived
only from the a chain, not from aE, appears as a band migrating
below the g chain doublet in panel A (compare also Fig 1A).
instead of being alternatively spliced a gene products as in
higher vertebrates.3,6
In contrast to the predominant human a chain, which has no
carbohydrate, human aE is N-glycosylated.1 We knocked out
each of the two potential glycosylation sites, aE667 and aE812,
by changing asparagines to glutamines. These mutated aE
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3306
FU ET AL
chains showed a mobility shift comparable in magnitude to that
fortuitously generated in mutant aE780S/793S with its second
active glycosylation site. Examination of lysates of aE/b/gtransfectants in Fig 6 revealed that the mutant aE812Q comigrates with wild-type aE, whereas aE667Q migrates faster,
suggesting that normally carbohydrate is attached to Asn667
and not to Asn812. aE780S/793S, as noted above, is larger than
wild-type aE because of an additional carbohydrate moiety at
Asn791. Thus, the stepwise increase in molecular weight of
aE667Q, aE812Q, and aE780S/793S reflects the attachment of
zero, one, and two carbohydrate chains, respectively.
In mutant aE667Q, secretion of Fib420 is reduced to less than
20% of wild-type levels (Fig 6; upon overexposure of the film
the subunits are clearly detectable), suggesting possible involvement of carbohydrate attachment at this site in secretion.
However, from an earlier finding that Fib420 secretion was not
appreciably inhibited when tunicamycin blocked N-glycosylation in HepG2 cells,1 it is apparent that reduced secretion of
aE667Q mutant Fib420 results not from the absence of sugar but
rather from a conformational change introduced by replacing
Asn with Gln.
DISCUSSION
This study shows, in transfected COS cells, that formation of
secreted aE-containing fibrinogen hexamers is possible with the
aE, b, and g subunit building blocks alone. Importantly, the
predominant secreted species of aE-containing fibrinogen is the
homodimeric (aEbg)2, even in the presence of the common a
chain (ie, in aE/a/b/g-transfectants); no mixed molecules of the
composition aaE(bg)2 are observed (Figs 1 and 2). In other
words, the heterologous COS cell system mimics the hepatic
Fig 6. Mutation of potential N-glycosylation sites in the aE VIdomain: Determination of carbohydrate attachment site. The VIdomain’s two potential glycosylation sites, at Asn667 and Asn812,
were changed to Gln; a new site at Asn791 had been introduced by
the Cys793 8 Ser change. COS cells were transfected with either
wild-type or these mutant aE cDNAs together with the b and g
subunit cDNAs. Fibrinogen was immunoprecipitated from cell lysates
and culture medium with antifibrinogen and run under reducing
conditions. Upon overexposure of the film, Fib420 subunits are clearly
detectable in the culture medium of the Q667 mutant.
Fib420 assembly process as evaluated in the cell line HepG2,
where aE-homodimeric fibrinogen molecules are formed preferentially in the presence of an abundant supply of a chains, the
aE:a chain ratio being roughly 1:20.1
This investigation of human aE-containing fibrinogen contradicts prior notions regarding the obligatory presence of common a chains for assembly and secretion.6 Inability of cells
used in previous COS experiments to match the performance of
hepatoma cells (HepG2), which assemble aE and a chains into
fibrinogen with equal efficiency, may account for the anomalous
earlier findings. Exploiting the current COS cells’ fidelity with
regard to Fib420 synthesis and secretion, studies were extended
to analyze the molecule’s glycosylation sites as well as the
requirement for specific cysteines in connection with its secretion.
Of the two potential glycosylation sites in the Fib420’s unique
aEC domain, the tripeptide at Asn667 is conserved in all
vertebrates, including lamprey, whereas the one at Asn812 is
conserved only in mammals.3,17 By substituting Gln for Asn we
have now shown that it is the highly conserved Asn667 site that
has carbohydrate attached in the aE chain of human Fib420
(Fig 6). Thus, Fib420 has a total of six carbohydrate moieties
among its six component chains. All of the sites have the
consensus tripeptide sequence NXT, with X being a positively
charged residue: arginine in a and b, lysine in g. Whereas the g
chain bears carbohydrate in a more upstream region of the
chain, both aE and b chains use sites in the C-terminal region.
Although the aEC and bC attachment sites do not align with
each other, they are both located between the homologous first
and second disulfide loops.
With its four extra cysteines per aEC domain, Fib420 has the
potential to form 33 disulfide bonds. The positions of the four
cysteines in aEC are invariant among aE homologs throughout
vertebrate evolution and align precisely with cysteines in the
bC and gC domains,3,6,17 each of which has two intrachain
disulfide bridges. The results from substituting serine or alanine
residues for the VI-domain cysteines (Figs 4 and 5, Table 1) are
consistent with the existence of similar bonds between the
cysteine pairs at aE613/aE644 and at aE780/aE793. The finding
that the individual aE613S and aE644S mutations block secretion but not the double mutation aE613S/644S strongly suggests
that there is an intrachain loop formed by Cys613 and Cys644.
Fib420 secretion is prevented by double mutation of the cysteines presumably forming the domain’s second disulfide loop
(aE780/793), indicating a vital role for them, and not those of
the potential first loop, in Fib420 export from the cell.
A number of protein sequences have been identified that bear
C-terminal domains homologous to aEC, bC, and gC. In this
family of fibrinogen-related domains (FREDs),18 which contains at least 14 unique members at latest count, the cysteines of
the first and second disulfide loops are preserved. With only one
exception,19 the 12 residues of the second loop are highly
conserved; those of the first loop, varying in number from 24 to
30, constitute the sequence with the lowest conservation in the
entire family of FREDs. In the context of our findings regarding
Fib420 secretion, the second loop’s higher degree of conservation may reflect a more critical role in secretion of the parent
molecule.
It should be mentioned that the C-terminal region of the b
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Fib420: DISULFIDE BONDS AND GLYCOSYLATION
chain actually has three intrachain disulfide bonds. In the large
loop formed by cyteines b201/286, which is essential for
secretion,10 only b Cys286 is part of the bC domain proper.6
Although the loop is conserved among all vertebrate fibrinogen
b chains, there is no cysteine corresponding to b Cys286 in any
other member of the FRED family, implying a highly specific
function.
Structurally, the aE chain differs significantly from the b and
g chains by virtue of the large ‘‘aC’’ region (identical to the
truncated C-terminus of the common a chain) that tethers the
subunit’s globular C-terminus (aEC) to its a-helical N-terminus.
Overall, the ‘‘aC’’ tether, encoded by exon V of the a gene, is
poorly conserved among the fibrinogens of higher vertebrates.20
However, the disulfide loop it contains, homologous to the
human cysteines at positions 442/472, is highly conserved,
although it does not play a role in either Fib340 or Fib420
secretion10 (Figs 4 and 5). An apparent conformational change
in Fib420 occurs in the aE442S/472S mutant as a result of
eliminating a disulfide-bridged loop, and it is reflected, even in
this high-molecular-weight range, in distinctly slower migration
on SDS-PAGE relative to all the other species that were
generated (Fig 5). This is consistent with the slower migration
noted previously of the a442S/472S mutant relative to wildtype a chains under nonreducing SDS-PAGE conditions10 and
gives credence to the hypothesis that the loops in the ‘‘aC’’region of aE make the Fib420 molecule more compact. It remains
to be seen whether the a442S/472S Fib420 mutant displays
neoepitopes and/or is more susceptible to proteolytic attack.
Based on sequence comparison,6 the globular fold of the aEC
domain is expected to be very similar to the folds of the bC and
gC domains, which were recently determined at atomic resolution and which were found to be virtually superimposable.21-23
However, the dependence of secretion on the integrity of the
two homologous intradomain disulfide bridges is distinctly
different for each subunit. In the bC region, only the first loop is
required; in the aEC region, it is the second loop; and in the gC
region both loops play a critical part. These observations
support different structural roles for the subunits in the process
of fibrinogen assembly, a notion put forward more than a decade
ago.24,25
The mechanism that favors formation of the symmetrical
Fib420 molecule over that of a mixed aE/a-containing molecule
in the presence of excess a chains is not known at present. We
previously speculated that alternative disulfide bridges, either
between the VI-domains and/or the VI-domain and the center of
the molecule, might provide the impetus for homodimer
formation. Without directly determining disulfide bridges between particular cysteines, the experiments using aE/a/b/gtransfectants (Fig 5) do not support that hypothesis; specific
mutations in aE’s VI-domain and the N-termini of all the aE, b,
and g chains, designed to remove cysteines potentially available
for alternative disulfide bridges, failed to reduce production of
(aEbg)2 in favor of mixed aaE(bg)2 molecules. Thus, a
different mechanism based on noncovalent interactions must be
advanced to explain symmetrical incorporation of aE chains
into Fib420 against all stoichiometric odds. Given the high
negative charge borne by each aEC domain,6 it is conceivable
that chaperone proteins associated with nascent fibrinogen26,27
3307
may play a critical role in balancing the spatial charge
distribution.
NOTE ADDED IN PROOF
As this report was being processed for publication, the
assignments of bound cysteine pairs and carbohydrate attachment site in the aEC domain were confirmed by x-ray crystallographic analysis of a recombinant version of the domain.28
ACKNOWLEDGMENT
We thank our colleague K.M. Hertzberg for many valuable contributions to the manuscript.
REFERENCES
1. Fu Y, Grieninger G: Fib420: A normal human variant of fibrinogen
with two extended a chains. Proc Natl Acad Sci USA 91:2625, 1994
2. Grieninger G, Lu X, Cao Y, Fu Y, Kudryk BJ, Galanakis DK,
Hertzberg KM: Fib420, the novel fibrinogen subclass: Newborn levels
are higher than adult. Blood 90:2609, 1997
3. Fu Y, Cao Y, Hertzberg KM, Grieninger G: Fibrinogen a genes:
Conservation of bipartite transcripts and carboxy-terminal-extended a
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1998 92: 3302-3308
Formation of the Human Fibrinogen Subclass Fib420: Disulfide Bonds and
Glycosylation in Its Unique (? EChain) Domains
Yiping Fu, Jian-Zhong Zhang, Colvin M. Redman and Gerd Grieninger
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