Formation of the Human Fibrinogen Subclass Fib₄₂₀: Disulfide Bonds and Glycosylation in Its Unique (_EChain) Domains

FIBRINOGEN, THE protein that forms the matrix of a blood clot, is a complex molecule composed of paired sets of three subunits (α, β, and γ), each encoded by a separate gene. A few years ago we identified a subclass of native fibrinogen molecules based on α 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 Fib₄₂₀molecules’ three subunits all end in globular domains that resemble each other, those of the more commonly known subclass Fib₃₄₀ have truncated α 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 α 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 α chain (α_E) with a globular carboxy-terminal extension. It is the 236 residues encoded by exon VI (technically the α_EC domain, but often referred to as the VI-domain) that are as similar to the sequences of the C-terminal domains of the fibrinogen β and γ chains (βC and γC) as the latter two are to each other (40% identity); the truncated C-terminus of the common α chain (αC) has a very different character.

Early investigations with transfected COS cells seemed to suggest the obligatory presence of common α chains for the assembly and secretion of recombinant α_E-containing fibrinogen.6 The subsequent unexpected finding that two α_E 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 α_E subunits per molecule of fibrinogen, whether the common α 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 α_EC domains unique to Fib₄₂₀.

The original α, β, and γ cDNAs of human fibrinogen were generous gifts from Dominic Chung (University of Washington, Seattle). Construction of vectors containing full-length cDNAs for the α_E, α, β, and γ 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 α, β, and γ chains were described previously.7,9,10 The N-terminal and “αC”-domain mutations of α_E were derivatized from the α mutants as done previously.6 The mutations Cys → Ser, Cys → Ala, and Asn → Gln in the α_E 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 α_E synthesis comparable to that of the other subunits, an excess of α_E-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 α_E.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 were detected by autoradiography. The results reported here were confirmed with several different passages of COS cells.

COS cells transfected simultaneously with either the α_E/β/γ or the α_E/α/β/γ 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 α_E chains were immunoprecipitated by anti-VI, indicating that most of the intracellular α_E subunits are not assembled, ie, they exist as free polypeptides. Similar intracellular pools of free α_E 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 α_E-containing fibrinogen from among the other fibrinogen species, immunoprecipitation of culture medium with two antibodies was used. Anti-VI immunoprecipitated only those consisting of α_E, β, and γ, whereas antifibrinogen collected every fibrinogen species, yielding a profile on reduced SDS-PAGE that included, when present, all four fibrinogen subunits α_E, α, β, and γ (Fig 1B). In immunoprecipitates of the medium of cells transfected with α_E/β/γ 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 α_E chain (see Fig 1A, lanes 1 and 3; Fig 2, lane 3), immunoprecipitating β or γ only when bound to α_E, it follows that the bulk of the α_E secreted by these cells is packaged in fully assembled fibrinogen molecules. Cells transfected additionally with the cDNA for common α (the α_E/α/β/γ-transfectants) secrete both α-fibrinogen and α_E-fibrinogen as indicated by the presence of α_E, β, and γ chains in the anti-VI immunoprecipitates and all four chains in the antifibrinogen immunoprecipitates. Although α_E/α/β/γ-transfectants expressed significant amounts of the common α chain, none was found in their anti-VI–immunoprecipitated culture medium, suggesting that no mixed molecules of the structure αα_E(βγ)₂were 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, α_E/β/γ-transfectants secreted a high molecular weight species (Fib₄₂₀) that is significantly larger than that of the α/β/γ-transfectants (Fib₃₄₀). Most striking, however, is that only one α_E-containing species is secreted when all four chains are cotransfected (α_E/α/β/γ-transfectants); this product comigrates with Fib₄₂₀. No mixed molecules, containing α_E as well as α 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, (α_Eβγ)₂, is energetically favored over that of the mixed molecules αα_E(βγ)₂,1 driven perhaps by alternative disulfide bond configurations either between the two α_E 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 α_Eitself. 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 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 α_E were substituted, individually and in combination; changes in the N-terminus and in the “αC” region of the α_E chain were also introduced (illustrated in Fig 3). In addition, N-terminal substitution mutants at sites in the β and γ chains (β65S and γ8S/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 α_E/β/γ-transfectants, ie, in the absence of the major α 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 α_E613S and α_E780S/793S substitutions abolished secretion of Fib₄₂₀ (Fig 4B). Although the double mutation α_E613S/644S, which knocks out the putative first loop in the VI-domain, has no discernable effect on Fib₄₂₀secretion, the single α_E613S mutant and its mutated disulfide bond partner (α_E644S; 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 α_E780S/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 α_E780S/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 α_E780S/793A. This particular mutation also blocked secretion (Table 1), indicating that the missing (second) intrachain loop and not the carbohydrate attachment is responsible for lack of Fib₄₂₀ export in α_E780S/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 VI-domain, reasoning that those cysteines were not found to be critical for export of Fib₃₄₀. The first pair of these was further upstream in the α_E chain, Cys442 and Cys472, which correspond to positions in the αC region of the common α chain known to form an intrachain loop¹⁰; mutant α_E442S/472S had no effect on Fib₄₂₀ secretion in α_E/β/γ-transfectants (Fig 4). Other candidate cysteines that we evaluated, located in the N-termini of the constituent fibrinogen chains (α_E Cys28 and Cys36, β Cys65, and γ Cys8 and Cys9), are involved in forming the symmetrical disulfide bridges that hold the αβγ trimers together; only one out of the four symmetrical bonds is absolutely required for Fib₃₄₀ hexamer assembly and secretion.7,8,10None of these mutations, α_E28S/36S, β65S, or γ8S/9S, when cotransfected with complementary wild-type chains, interfered with proper secretion of Fib₄₂₀molecules in the α_E/β/γ-transfectants (Fig 4).

Having established that Fib₄₂₀ production was possible despite substitution of particular residues, the same mutations were examined in α_E/α/β/γ-transfectants. This test of the alternative disulfide bond configuration hypothesis could be expected to yield secretion of mixed molecules (containing both α_E and α) 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 α_E, β, and γ chains, the levels of expression were comparable to those of the α_E/β/γ-transfectants (Fig4A). The medium of the transfected cells was then immunoprecipitated with anti-VI for detection of secreted α_E-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 α_E/β/γ-transfectant. It follows that selective incorporation of two α_E subunits in the presence of the abundant α chain is not dependent on disulfide bridges involving cysteines in the VI-domain of α_E or on available cysteine partners further upstream in the α_E 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 α′-fibrinogen,4 the lamprey equivalent of Fib₄₂₀, which appears to be a similarly symmetrical molecule but which has α_E-like chains derived from a separate α′ gene17 instead of being alternatively spliced α gene products as in higher vertebrates.3,6 

In contrast to the predominant human α chain, which has no carbohydrate, human α_E is N-glycosylated.1 We knocked out each of the two potential glycosylation sites, α_E667 and α_E812, by changing asparagines to glutamines. These mutated α_E chains showed a mobility shift comparable in magnitude to that fortuitously generated in mutant α_E780S/793S with its second active glycosylation site. Examination of lysates of α_E/β/γ-transfectants in Fig 6 revealed that the mutant α_E812Q comigrates with wild-type α_E, whereas α_E667Q migrates faster, suggesting that normally carbohydrate is attached to Asn667 and not to Asn812. α_E780S/793S, as noted above, is larger than wild-type α_E because of an additional carbohydrate moiety at Asn791. Thus, the stepwise increase in molecular weight of α_E667Q, α_E812Q, and α_E780S/793S reflects the attachment of zero, one, and two carbohydrate chains, respectively.

In mutant α_E667Q, 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 α_E667Q mutant Fib₄₂₀ results not from the absence of sugar but rather from a conformational change introduced by replacing Asn with Gln.

This study shows, in transfected COS cells, that formation of secreted α_E-containing fibrinogen hexamers is possible with the α_E, β, and γ subunit building blocks alone. Importantly, the predominant secreted species of α_E-containing fibrinogen is the homodimeric (α_Eβγ)₂, even in the presence of the common α chain (ie, in α_E/α/β/γ-transfectants); no mixed molecules of the composition αα_E(βγ)₂ are observed (Figs 1 and 2). In other words, the heterologous COS cell system mimics the hepatic Fib₄₂₀ assembly process as evaluated in the cell line HepG2, where α_E-homodimeric fibrinogen molecules are formed preferentially in the presence of an abundant supply of α chains, the α_E:α chain ratio being roughly 1:20.1 

This investigation of human α_E-containing fibrinogen contradicts prior notions regarding the obligatory presence of common α chains for assembly and secretion.6 Inability of cells used in previous COS experiments to match the performance of hepatoma cells (HepG2), which assemble α_E and α 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 α_EC 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 α_E 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 α and β, lysine in γ. Whereas the γ chain bears carbohydrate in a more upstream region of the chain, both α_E and β chains use sites in the C-terminal region. Although the α_EC and βC 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 α_EC domain, Fib₄₂₀ has the potential to form 33 disulfide bonds. The positions of the four cysteines in α_EC are invariant among α_E homologs throughout vertebrate evolution and align precisely with cysteines in the βC and γC 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 α_E613/α_E644 and at α_E780/α_E793. The finding that the individual α_E613S and α_E644S mutations block secretion but not the double mutation α_E613S/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 (α_E780/793), 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 α_EC, βC, and γC. 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 β chain actually has three intrachain disulfide bonds. In the large loop formed by cyteines β201/286, which is essential for secretion,10only β Cys286 is part of the βC domain proper.6Although the loop is conserved among all vertebrate fibrinogen β chains, there is no cysteine corresponding to β Cys286 in any other member of the FRED family, implying a highly specific function.

Structurally, the α_E chain differs significantly from the β and γ chains by virtue of the large “αC” region (identical to the truncated C-terminus of the common α chain) that tethers the subunit’s globular C-terminus (α_EC) to its α-helical N-terminus. Overall, the “αC” tether, encoded by exon V of the α 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 (Figs4 and 5). An apparent conformational change in Fib₄₂₀occurs in the α_E442S/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 α442S/472S mutant relative to wild-type α chains under nonreducing SDS-PAGE conditions10 and gives credence to the hypothesis that the loops in the “αC”-region of α_E make the Fib₄₂₀ molecule more compact. It remains to be seen whether the α442S/472S Fib₄₂₀ mutant displays neoepitopes and/or is more susceptible to proteolytic attack.

Based on sequence comparison,6 the globular fold of the α_EC domain is expected to be very similar to the folds of the βC and γC 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 βC region, only the first loop is required; in the α_EC region, it is the second loop; and in the γC 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 α_E/α-containing molecule in the presence of excess α 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 α_E/α/β/γ-transfectants (Fig 5) do not support that hypothesis; specific mutations in α_E’s VI-domain and the N-termini of all the α_E, β, and γ chains, designed to remove cysteines potentially available for alternative disulfide bridges, failed to reduce production of (α_Eβγ)₂ in favor of mixed αα_E(βγ)₂ molecules. Thus, a different mechanism based on noncovalent interactions must be advanced to explain symmetrical incorporation of α_E chains into Fib₄₂₀ against all stoichiometric odds. Given the high negative charge borne by each α_EC domain,6 it is conceivable that chaperone proteins associated with nascent fibrinogen26,27 may play a critical role in balancing the spatial charge distribution.

As this report was being processed for publication, the assignments of bound cysteine pairs and carbohydrate attachment site in the α_EC domain were confirmed by x-ray crystallographic analysis of a recombinant version of the domain.28 

We thank our colleague K.M. Hertzberg for many valuable contributions to the manuscript.