Fibrinogen assembly and secretion. Role of intrachain disulfide loops.

Human fibrinogen is a homodimer composed of three different (Aα, Bβ, γ) polypeptide chains. The chains are linked by 29 inter- and intrachain disulfide bonds. Each half-molecule has 6 intrachain disulfide bonds, which form loops in the carboxyl-terminal region of each of the chains. Aα chain has one disufide loop (Cys442-Cys472), Bβ has three (Cys201-Cys286, Cys211-Cys240, and Cys394-Cys407), and γ has two loops (Cys153-Cys182 and Cys326-Cys339). The intrachain loops are conserved in fibrinogens of different species. We changed, by site-directed mutagenesis, the cysteines, which form the intrachain loops, to serine or alanine. Fibrinogen chain assembly and secretion was determined in transiently transfected COS cells expressing two normal and a mutant fibrinogen chain. In the Bβ and γ chains, disruption of the disulfide loops closest to the “coiled-coil” region (CysBβ211-Cys240, CysBβ201-Cys286, and Cysγ153-Cys182) abolished chain assembly and secretion, indicating that the disulfide loops closest to the coiled-coil region are essential for chain assembly. By contrast, preventing formation of the disulfide loops, which are toward the carboxyl termini of each of the chains, had different effects. Disruption of the single Aα disulfide loop had no effect, as did disruption of BβCys394-Cys407. However, disruption of Cysγ326-Cys339, which is similar in size and location to CysBβ394-Cys407, allowed chain assembly to occur, but the assembled chains were not secreted.

locations of the intrachain disulfide loops in the globular domains of the B␤ and ␥ chains have been conserved during evolution and are similar in diverse species such as human and lamprey (14). A diagram depicting the locations of the interand intrachain disulfide bonds of fibrinogen is shown in (Fig. 1).
Previous studies demonstrated the importance of aminoterminal interchain disulfide bonds on assembly of the two half-molecules to form 6-chain fibrinogen (15)(16)(17) and indicated that, in addition to the symmetrical disulfide bonds that link adjacent Cys A␣28 and Cys ␥8 and Cys ␥9 of one half-molecule to the other half-molecule, another disulfide interaction, Cys B␤65 to Cys A␣36 , also connects the two half-molecules of fibrinogen (15,17). Disruption of all of the disulfide interactions that connect the two half-molecules leads to the assembly and secretion of half-molecules and not of fully formed fibrinogen. However, the interchain disulfide rings at the amino-terminal side of the coiled-coil region (Fig. 1, arrow marked N) are also essential for dimerization of the two half-molecules. Maintaining all of the cysteine residues involved in disulfide linkage of the half-molecules (␥8 and 9, A␣28 and 36, and B␤65), but disrupting the disulfide rings that flank the amino-terminal side of the coiled-coil region, inhibits dimerization, and, again half-molecules but not fibrinogen are formed and secreted. On the carboxyl-terminal side of the coiled-coil region the interchain disulfide rings (Fig. 1, arrow marked C) are not needed for chain assembly, but if they are not present, fibrinogen is assembled but is not secreted, probably due to improper folding (15).
The present studies focus on the role of intrachain loops in fibrinogen assembly and secretion ( Fig. 1, shaded areas). We changed, by site-directed mutagenesis, the cysteines that form intrachain loops to serine or alanine and investigated in transiently transfected COS cells the assembly and secretion of fibrinogen containing the mutant chain.

EXPERIMENTAL PROCEDURES
Materials-Full-length A␣, B␤, and ␥ cDNAs, cloned into the PstI site of pBR322, were kind gifts from Dr. Dominic Chung (University of Washington, Seattle). Expression vector pED4-Neo (18) was obtained from the Genetics Institute (Cambridge, MA). Other reagents used have been described (15, 19 -23).
Plasmid Construction and Mutagenesis-Full-length A␣, B␤, and ␥ cDNAs were subcloned into M13mp18 or M13mp19 (15,16,24). To construct pED4-NeoA␣, pED4-NeoB␤, and pED4-Neo␥, EcoRI sites were introduced into the 5Ј-end of A␣ cDNA of M13mp19A␣, the 5Ј-side of PstI of M13mp19B␤, and the 5Ј-and 3Ј-ends of ␥ cDNA of M13mp18␥ by site-directed mutagenesis (25). The following oligonucleotides were used CTTTTCTAAGAATTCGGCTGGCTC, GACCTGCAGGAATTCAA-GCTTGGC, GAGTGCCCGGAATTCGAAAGCTTA, and CTTTGCAAG-GAATTCCATTGTCCA. To eliminate an internal EcoRI site in A␣ cDNA, an oligonucleotide (GGAAGGGAACTCAGCTATC) was used to change the nucleotide sequences GAA to GAG without changing glutamic acid at position 550. Each of the full-length cDNAs were excised by digestion with EcoRI and inserted into the EcoRI site of expression vector pED4-Neo. The correct orientation was selected by restriction * This work was partially supported by National Institutes of Health Grant HL37457. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18

mapping.
Mutants of A␣ Chain-Mutant A␣C472S (which encodes a serine instead of cysteine at position A␣472) was made by changing cysteine codon TGT in single strand DNA of M13mp19A␣ to serine codon AGT. A double mutant A␣C442S,C472S, which encodes cysteine to serine substitutions at amino acid positions 442 and 472 of A␣ chain, was also similarly prepared. Two synthetic oligonucleotides, GTTTTAGAGCTT-GAACGACGC for A␣C442S and GCCTCGGGACTGTCAGAACCA for A␣C472S, were used. Site-directed mutagenesis was performed according to the method of Kunkel (25). The mutations were confirmed by DNA sequencing (26). All mutant cDNAs were digested with EcoRI and inserted into the EcoRI site of pED4-Neo. The correct orientation of mutant cDNAs was confirmed by DNA sequencing.
Immunoprecipitation of nascent fibrinogen was carried out as described previously (15,16,19,24). The labeled cell lysates and the incubation medium were immunoprecipited using a rabbit polyclonal antibody to human fibrinogen (Dako Corp.) and protein A-Sepharose.
Immunoprecipitates were analyzed under reduced and non-reduced conditions and detected by autoradiography.
Characterization of Secreted Fibrinogens-Wild-type and mutant fibrinogens, which were secreted into the incubation media, were characterized, as described previously (23), by two-dimensional SDS-PAGE. 1 The first dimension was non-reduced, and the second dimension was with reduced gels (data not shown).

Expression of Mutant Chains-
The expression of mutant fibrinogen chains was compared with that of normal chains by transfection in COS cells. Nascent fibrinogen chains were isolated from radiolabeled cell lysates using polyclonal antibody to human fibrinogen, and the radioactive proteins were separated by SDS-PAGE (Fig. 2). All of the mutant chains were expressed at about the same level as the normal chains ( Fig. 2A). Four mutant chains, B␤C201S,C286S, B␤C394S,C407S, ␥C339S, and ␥C326S,C339S (marked by asterisks), migrated slower on SDS-PAGE than normal B␤ and ␥ chains, and two of them, ␥C339S and ␥C326S,C339S, migrated as doublets ( Fig Previous studies showed that small amounts of single A␣ and ␥ chains could be secreted by transfected COS cells (23). Therefore, we compared the secretion of single mutant A␣ and ␥ chains to the normal A␣ and ␥ chains. A small amount of normal A␣, A␣C472S, and A␣C442S,C472S were detected in the culture media on prolonged exposure of the autoradiogram (Fig. 2B, lanes 1-3). Larger amounts of normal ␥ chain was secreted (Fig. 2B, lane 9), but the mutant ␥ chains were not secreted, indicating that intact intrachain loops may be necessary for secretion of the ␥ chain (Fig. 2B, lanes 10 -14). Neither the normal nor the mutant B␤ chains were secreted (Fig. 2B, lanes 4 -8). Because the photographic film was exposed for a long time, a contaminant protein at the top of the gel is prominent and is marked by an arrow (Fig. 2B).
An Intact Intrachain Loop of A␣ Chain Is Not Necessary for Fibrinogen Assembly and Secretion-Fibrinogen A␣ chain has only one intrachain loop in the carboxyl-terminal domain spanning Cys 442 to Cys 472 . To determine if this A␣ intrachain loop is required for fibrinogen assembly and secretion, a mutant containing a single Cys to Ser substitution (A␣C472S) and another with two substitutions (A␣C442S,C472S) were made. COS cells were cotransfected with the mutant A␣ chain cDNA and with normal B␤ and ␥ chain cDNAs. Disruption of the A␣ intrachain loop by substituting cysteine with serine at amino acid position 472 or in both positions (442 and 472) did not affect fibrinogen assembly and secretion. In both cases, the A␣ mutant chains were assembled with the wild-type B␤ and ␥ chains forming, and accumulating, fibrinogen and A␣⅐␥ complex intracellularly (Fig. 3A, lanes 2 and 3). Small amounts of B␤⅐␥ complex and of free A␣, B␤, and ␥ chains were also detected intracellularly. There was less free A␣C472S (Fig. 3A, lane 2) than free A␣C442S,C472S or normal A␣ ( Fig. 2A, lanes  1 and 3). Mutants A␣C472S or A␣C442S,C472S (marked by circles) had slower migration on SDS-PAGE, under non-reduced conditions, than normal A␣ (Fig. 3A, lanes 2 and 3). This may be the result of a change in conformation resulting from loss of the intrachain disulfide loops since mutants A␣C472S or A␣C442S,C472S migrated similarly to normal A␣ chain under reduced conditions (Fig. 2, lanes 2 and 3).
Effect of Disrupting the Intrachain Loops of B␤ Chain on Fibrinogen Assembly and Secretion-Each fibrinogen B␤ chain has three intrachain loops, two of them near the coiled-coil region and the third located downstream toward the carboxyl terminus. To determine if fibrinogen assembly and secretion occurs when these loops are disrupted, mutants B␤C201S,C286S, B␤C240S, B␤C211S,C240S, and B␤C394S, C407S were constructed and co-expressed with normal A␣ and ␥ chains.
Disruption of the disulfide loop closest to the carboxyl terminus (Cys B␤394 -Cys 407 ), by substituting cysteine with serine, did not prevent fibrinogen assembly and secretion (Fig. 4, lane 5) but caused less fibrinogen and more A␣⅐␥ complex to be se-creted (Fig. 4B, lane 5). Also, there was a greater intracellular accumulation of the 2-chain intermediates A␣⅐␥ and B␤⅐␥ and of the free mutant B␤C394S,C407S chain (Fig. 4A, lane 5). It was also noted that, in the secreted material, B␤C394S,C407S migrated on SDS-PAGE as a doublet (marked by asterisks, Fig.  4C, lane 5), probably because of an extra N-linked oligosaccharide due to creation of an Asn-X-Ser motif at amino acid position 405-407.
Intrachain Disulfide Loops of the ␥ Chain-Fibrinogen ␥ chain has two intrachain disulfide loops that are similar in size and location to Cys B␤211 -Cys 240 and Cys B␤394 -Cys 407 of the B␤ chain. One intrachain disulfide loop (Cys ␥153 to Cys 182 ) is close to the coiled-coil region, and the other (Cys ␥326 -Cys 339 ) is located at the carboxyl terminus region of the ␥ chain. Preventing formation of disulfide loop Cys ␥153 -Cys 182 , which is closest to the coiled-coil region, by substituting cysteine with serine abolished fibrinogen chain assembly and secretion (Fig. 5, A-C, lane 2). Also, less A␣⅐␥ and B␤⅐␥ complexes were formed (Fig.  5A, lane 2). The free mutant ␥C153S,C182S chain (marked by circle) migrated slower than normal ␥ chain on SDS-PAGE under non-reduced conditions. Since there is no possibility for extra N-glycosylation in this mutant, a change in conformation may be responsible for its aberrant migration on SDS-PAGE.
Preventing formation of the ␥ intrachain loops near the carboxyl terminus by substituting Cys ␥326 or Cys ␥339 , or both of them, with serine did not abolish fibrinogen assembly (Fig. 5A,  lanes 3, 4, and 6) but disallowed secretion (Fig. 5, B and C,  lanes 3, 4, and 6). In these conditions, less A␣⅐␥ and B␤⅐␥ complexes were formed (Fig. 5A, lanes 3-6). Unexpectedly, a small amount of free A␣ and B␤ chains were released into the medium by COS cells, which co-expressed normal A␣ and B␤ chains together with mutant ␥C326S,C339S (Fig. 5C, lane 6). On prolonged exposure (5 days) of the autoradiographic film, small amounts of free A␣ and B␤ could also be detected when ␥C326S or ␥C339A were co-expressed (data not shown). ␥C339S and ␥C326S,C339S, isolated from cell lysates (marked by asterisks), migrated on SDS-PAGE as doublets both under non-reduced conditions (Fig. 5A, lanes 4 and 6) and under reduced conditions (Fig. 2, lanes 12 and 14). The additional product may be due to an extra N-glycosylation caused by creation of a new Asn-Lys-Ser site on changing Cys ␥339 to Ser ␥339 .
To demonstrate that creation of the extra N-glycosylation at amino acid position 339 of ␥ chain did not, per se, affect fibrinogen secretion, a mutant (␥C339A), which encodes alanine instead of cysteine at ␥339 and which does not create an extra N-glycosylation site, was co-expressed together with normal A␣ and B␤ chains. Fibrinogen was assembled intracellularly, perhaps to a lesser degree than wild-type fibrinogen (Fig. 5, lane  5), but the mutant fibrinogen was not secreted (Fig. 5, B and C,  lane 5). This indicates that failure of this mutant fibrinogen to be secreted is not due to extra glycosylation but probably caused by misfolding due to disruption of the carboxyl-terminal intrachain disulfide loop. DISCUSSION The fibrinogen intrachain loop domains are conserved in different species, suggesting that they play important roles in maintaining structures which allow biological functions. The and ␣⅐␤394S,407S⅐␥) were expressed and isolated as described in Fig. 2. Panel A shows intracellular fibrinogen, and panels B and C show secreted fibrinogen. Panels A and B are non-reduced gels, and panel C contains reduced fibrinogen. The location of fibrinogen (Fb), A␣⅐␥ and B␤⅐␥ complexes, and free chains are indicated. In panel C, the B␤ mutant (␤394S,407S) separated, in reduced SDS-PAGE, as a doublet (␤* and ␤). strictest conservation of amino acid sequence and location of the intrachain loops are in the B␤ and ␥ chains. The intrachain loops close to the coiled-coil region show remarkable similarities in size and location in human, chicken, Xenopus, and lamprey fibrinogen (14). Interestingly, similar intrachain loops are found in fibrinogen-related proteins such as tenascin (27), the Sca protein in Drosophila (28), and in some other proteins (29). In the A␣ chain, a single intrachain loop (Cys 442 -Cys 472 in human fibrinogen) is similar in size and in amino acid sequence in nine other species (30). The A␣ chain intrachain loop is missing, however, in lamprey fibrinogen. Lamprey also expresses a minor form of the A␣ chain whose carboxyl-terminal half is homologous to the carboxyl-terminal globular domain of the B␤ and ␥ chains and contains two intrachain disulfide loops (14). Humans also express, in small quantity, a fibrinogen with an extended A␣ chain (␣ E ), and ␣ E also contains two intrachain disulfide loops that are similar to those in B␤ and ␥ chains (31,32). In this study, we demonstrate that the conserved intrachain loops, particularly those in the B␤ and ␥ chains in the predominant form of human fibrinogen, play important roles in fibrinogen assembly and secretion.
A prominent feature of fibrinogen is an ␣-helical coiled-coil domain spanning about 111 hydrophobic amino acids in each of the chains and flanked on either end by a pair of cysteine residues (Cys-X-X-X-Cys), which form interchain disulfide linkages and are commonly termed "disulfide rings" (3,33). The coiled-coil region links the central E domain to the terminal D domain, providing fibrinogen with its well known elongated, trinodal structure. Our present studies show that not only the interchain disulfide bonds but also some of the intrachain disulfide interactions are necessary for assembly and secretion (see (Fig. 6) for summary). Preventing formation of the intrachain disulfide loops closest to the coiled-coil region (Cys B␤201 -Cys 286 , Cys B␤211 -Cys 240 , and Cys ␥153 -Cys 182 ) does not allow fibrinogen to be assembled, and, in these conditions, neither the 3-chain (A␣⅐B␤⅐␥) complex nor the 6-chain fibrinogen is formed. Preventing formation of these intrachain disulfide bonds may change the conformation of the chains so that complexes larger than two chains cannot be assembled. These B␤ and ␥ chain mutants can, however, form B␤⅐␥ and A␣⅐␥ complexes, although the formation of these 2-chain complexes appears to be also inhibited. Fibrinogen assembly is a stepwise process, proceeding from single chains to 2-chain complexes (predominantly A␣⅐␥ and B␤⅐␥) to 3-chain half-molecules (A␣⅐B␤⅐␥), which dimerize to form fibrinogen (A␣⅐B␤⅐␥) 2 (21,34,35). Disruption of the B␤ and ␥ intrachain loops closest to the coiled-coil region appears to mostly affect the addition of a third chain to A␣⅐␥ and to B␤⅐␥ complexes, thus inhibiting the formation of A␣⅐B␤⅐␥ half-molecules. The mechanisms by which the 3-chain coiled-coil region is assembled and stabilized by the disulfide rings is not well understood, but these studies suggest that, in addition to the amino acid sequence responsible for ␣-helix formation and the flanking interchain disulfide rings, the B␤ and ␥ intrachain loops closest to the coiled-coil region are also needed to allow proper alignment and formation of the 3-chain coiled-coil structure.
In addition to the intrachain loops close to the coiled-coil region, each of the chains has distal intrachain loops toward the carboxyl terminus (Cys A␣442 -Cys 472 , Cys B␤394 -Cys 407 , and Cys ␥326 -Cys 339 ). Cys B␤394 -Cys 407 and Cys ␥326 -Cys 339 are similar in size and in location and are different from Cys A␣442 -Cys 472 , which spans a larger amino sequence (see Figs. 1 and  6). Disruption of these carboxyl-terminal disulfide loops have different effects on fibrinogen assembly and secretion. Cys A␣442 -Cys 472 may be very important in fibrinogen function since this domain is conserved in different species, but disruption of this disulfide loop has little or no effect on chain assembly and secretion. On the other hand, disruption of Cys B␤394 -Cys 407 also does not block fibrinogen assembly and secretion but does influence the assembly process and leads to intracellular accumulation of A␣⅐␥ complex, some of which is secreted. By contrast, disruption of Cys ␥326 -Cys 339 allows fibrinogen assembly but completely blocks secretion. This shows that intact Cys ␥326 -Cys 339 is not necessary for fibrinogen chain assembly, but if this intrachain loop is not formed, the structure of fibrinogen is sufficiently altered so as to prevent it from being secreted. It should be noted that small amounts of free ␥ chains are secreted by transfected COS cells (Fig. 2B) and that preventing formation of the intrachain disulfide loops also abolishes secretion of the free ␥ chain.
A previous study showed that a truncated B␤ chain, lacking the carboxyl-terminal amino acids 208 -461, could assemble with wild-type A␣ and ␥ chains, and the mutant fibrinogen was secreted (24). Since the deleted segment contains all three B␤ intrachain loops, this suggested that this domain is not necessary for assembly and secretion. Yet, our present studies demonstrate that the intrachain loops Cys B␤201 -Cys 286 and Cys B␤211 -Cys 240 , which are present in this region, are needed for fibrinogen assembly. Taken together, this indicates that a truncated B␤ chain containing amino acids 1-207, which includes the coiled-coil region and the flanking disulfide rings but lacks the carboxyl-terminal domain, is sufficient for assembly; but if the entire B␤ chain is expressed, then the intrachain loops are necessary to maintain the structure of the carboxylterminal domain so that it does not interfere with formation of the coiled-coil region.