©1996 by The American Society for Biochemistry and Molecular Biology, Inc.
Assembly and Secretion of Fibrinogen
INVOLVEMENT OF AMINO-TERMINAL DOMAINS IN DIMER FORMATION (*)

(Received for publication, September 8, 1995; and in revised form, March 13, 1996)

Jian-Zhong Zhang Colvin M. Redman (§)

From the Lindsley F. Kimball Research Institute, The New York Blood Center, New York, New York 10021

ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
FOOTNOTES
REFERENCES

ABSTRACT

Fibrinogen is a dimer with each half-molecule composed of three different chains (Aalpha, Bbeta, ). Previous studies showed that amino-terminal disulfide bonds, as well as the disulfide rings that flank the ``coiled-coil'' region, are necessary for chain assembly and secretion (Zhang, J. Z., and Redman, C. M.(1994) J. Biol. Chem. 269, 652-658). We now determine whether other amino-terminal domains are involved in linking the half-molecules. Fibrinogen chains, with deletions at the amino terminus, were co-expressed in COS cells together with normal fibrinogen chains. Elimination of the first 8 amino acids of the Bbeta chain did not affect dimer assembly, but deletion of amino acid residues 9-72 had a small inhibitory effect on dimer formation. Deletion of the first 72 amino acids of the Bbeta chain further inhibited dimer formation and resulted in nearly equal amounts of half-molecule and dimeric fibrinogen being formed and secreted. Deletion of the first 80 residues, which includes the cysteine residues that form the amino-terminal disulfide ring, completely eliminated dimer formation, and only half-molecules were secreted. By contrast deletion of the first 41 amino acid residues of the Aalpha chain or the first 15 residues of the chain, which correspond to BbetaDelta1-72, did not affect chain assembly and secretion. However, co-expression of both AalphaDelta1-41 and Delta1-15 with normal Bbeta, inhibited dimer formation. Taken together, these results indicate that in addition to disulfide bonds, noncovalent interactions of other amino-terminal amino acid residues in the three fibrinogen chains also participate in dimer formation.


INTRODUCTION

Human fibrinogen is a dimer with each half-molecule containing three different polypeptide chains Aalpha, Bbeta, and (for reviews, see (1, 2, 3) ). Each of the fibrinogen chains has an amino-terminal segment followed by an alpha-helical domain of about 111 amino acids that forms a 3-chain ``coiled-coil'' region flanked by interchain disulfide rings(4) . On the carboxyl-terminal end, the Bbeta and chains have homologous globular domains, while the Aalpha chain has a long segment that is not equivalent to that of Bbeta and . However, in normal plasma there are small amounts of a fibrinogen with an extended Aalpha chain, which is homologous to the carboxyl termini of the Bbeta and chains(5) . Structural studies indicate that fibrinogen is elongated and trinodal. The central node (E domain) contains the amino termini of the 6 polypeptide chains, and the two terminal nodes (D domains) are formed by globular carboxyl-terminal domains of Bbeta and chains. The carboxyl-terminal domain of the large Aalpha chain is thought to fold back and contribute to the structure of the central node(12, 13, 14, 15, 16) . The fibrinogen chains are held together by 29 inter- and intra-chain disulfide bonds(6, 7, 8, 9, 10, 11) .

Previous studies showed that intracellular assembly of the 2 half-molecules into a dimer requires not only the amino-terminal disulfide bonds but also that the disulfide rings that flank the coiled-coil region remain intact(17, 18, 19) . Disruption of the disulfide rings at the amino-terminal end of the coiled-coil region prevents dimer formation, and the elimination of the disulfide rings, at the carboxyl-terminal end of the coiled-coil region, allows dimer formation, but the 6-chain molecule that is assembled is not secreted (19) . To determine whether other amino-terminal domains are involved in fibrinogen assembly, we have constructed a series of deletion mutants and transiently co-expressed the mutant chains in COS cells together with two other normal or mutant chains. The half-molecules and dimeric fibrinogen, formed intracellularly and secreted into the medium, were determined.


EXPERIMENTAL PROCEDURES

Materials

Full-length Aalpha, Bbeta, and cDNAs, cloned into the PstI site of pBR322, were kind gifts from Dr. Dominic Chung (University of Washington, Seattle). Expression vector pED4-Neo (20) was obtained from the Genetics Institute (Cambridge, MA). Other reagents used have been described(17, 21, 22, 23, 24) .

Plasmid Construction and Mutagenesis

Full-length Aalpha, Bbeta, and cDNAs were subcloned into M13mp18 or M13mp19 (17, 18, 19) . To construct pED4-NeoAalpha, pED4-NeoBbeta, and pED4-Neo, EcoRI sites were introduced into the 5` end of Aalpha cDNA of M13mp19Aalpha, the 5` side of PstI of M13mp19Bbeta, and the 5` and 3` ends of cDNA of M13mp18 by site-directed mutagenesis(25, 26, 28) . The following oligonucleotides were used: CTTTTCTAAGAATTCGGCTGGCTC, GACCTGCAGGAATTCAAGCTTGGC, GAGTGCCCGGAATTCGAAAGCTTA, and CTTTGCAAGGAATTCCATTGTCCA. In order to eliminate an internal EcoRI site in Aalpha 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 was excised by digestion with EcoRI and inserted into the EcoRI site of expression vector pED4-Neo. The correct orientation was selected by restriction mapping.

Mutants of Aalpha Chain

Cysteine codons (TGC) in single strand DNA of M13mp19Aalpha were changed to serine (AGC) at positions 28 and 36. Two synthetic oligonucleotides, TGAATCTTTGCTGGCAGATTGA for Aalpha28s and TCATCAGAGCTGAAGGGCCA for Aalpha36s were used according to the method of Kunkel(25) . The mutations were confirmed by DNA sequencing (27) . A deletion mutant AalphaDelta1-41, was constructed using the oligonucleotide GAAGGGCATTTGTAGTTAGTCCATGCTGTGTGCCCA(17) . The mutant was screened by digestion with EcoRI and confirmed by DNA sequencing. All mutant cDNAs were released by digestion with EcoRI and ligated into the EcoRI site of pED4Neo. The proper orientation of mutant cDNAs and the mutations formed by simple base substitutions were confirmed by DNA sequencing.

Mutants of Bbeta Chain

Four deletion mutants M13mp19BbetaDelta1-8, M13mp19BbetaDelta9-72, M13mp19BbetaDelta1-80, and M13mp19BbetaDelta1-192 were prepared as described previously(17) . The oligonucleotides used for mutagenesis were CGGGCACTGAAGAAACCGGACTTAACTAGAAAAA for BbetaDelta1-8, GTAGGACACAACACCCCCTCCTCATTGTCGTTGA for BbetaDelta9-72, AAAGCCTCTTGCAACTGGGACTTAACTAGAAAAA for BbetaDelta1-80, and GTGCATGGGGTGCGACAGGACTTAACTAGAAAAA for BbetaDelta1-192. Mutant M13mp19BbetaDelta1-72 has been described(17) . All mutant cDNAs from bacteriophage M13mp19 were inserted into EcoRI site of pED4Neo. The right orientation of the expression vectors were determined by restriction mapping.

Mutants of Chain

To construct deletion mutant M13mp18Delta1-15, single strand DNA of bacteriophage M13mp18 with an EcoRI site at the 5` end was used as a template. Site-directed mutagenesis was performed using the oligonucleotide GTTGGACAATAACTACCTGCTACACATGTTGAAG. The deletion of codons encoding the first 15 amino acids was verified by DNA sequencing. The 1.4-kilobase DNA fragment, obtained from M13mp18Delta1-15, was cloned into the EcoRI site of expression vector pED4Neo.

Transfection and Immunoprecipitation

COS-1 cells were maintained in Iscove's media supplemented with 10% fetal calf serum, 100 units/ml penicillin, and 100 µg/ml streptomycin. Transfection was carried out as described previously(16, 17, 18) . Forty hours after transfection, the cells were washed 3 times in phosphate-buffered saline and incubated for 2 h in methionine-free Dulbecco's minimal essential medium (Life Technologies, Inc.) containing 200 µCi/ml of L-[S]methionine, 0.1 mg/ml heparin, 1% glutamine, and 100 units/ml Trasylol. Radioactive fibrinogen was immunoprecipitated using a rabbit polyclonal antibody against human fibrinogen as described previously(17, 21, 22, 23, 24) . Immunoprecipitates were isolated by SDS-PAGE (^1)under reduced and nonreduced conditions, and radioactive proteins were detected by autoradiography.


RESULTS

Expression of Individual Mutant Chains

All normal and mutant fibrinogen chain cDNAs were subcloned into the expression vector pED4Neo. The vector was chosen because we anticipate, in future studies, selecting stable cell lines and also because it gives high levels of expression in COS cells. This vector contains TnF neomycin phosphotransferase (Neo), a selectable marker, and dehydrofolate reductase, an amplifiable gene. pED4Neo also has the SV40 origin of replication and enhancer and the adenovirus major late promoter(20) . Forty hours after transfection, COS cells were radiolabeled with L-[S]methionine for 2 h, and the radioactive fibrinogen chains were immunoprecipitated from both the detergent-treated cell lysate and from the culture medium, using a polyclonal antibody that recognizes the three fibrinogen chains. The radioactive chains were separated by SDS-PAGE and detected by autoradiography.

A diagram, depicting the deletion mutants constructed, is shown in Fig. 1. All normal and mutant chains were expressed to approximately the same extent (Fig. 2). Deletion mutant BbetaDelta1-192 was less radioactive, which could be due to fewer methionine residues (Fig. 2A, lane 9). Lower molecular weight proteins, due to either degradation or to incomplete translation, were noted in normal and mutant Aalpha and Bbeta chains (Fig. 2A).


Figure 1: Nomenclature and schematic representation of deletion and substitution mutants of fibrinogen Aalpha, Bbeta, and chains. Shown above are diagrams of the fibrinogen Aalpha, Bbeta, and chain mutants constructed. All of the constructs had the appropriate signal sequences (S). Deletion mutants are named based on the amino acids deleted. Substitution mutants change cysteine residues to serine at the positions listed. The coiled-coil region of each chain is marked by X, and the flanking cysteine residues involved in the formation of disulfide rings are designated Cys*. Other amino-terminal and carboxyl-terminal cysteine residues are also shown.




Figure 2: Expression of normal and mutant fibrinogen chains. Single fibrinogen chain cDNAs were expressed in COS cells. Radiolabeled fibrinogen chains from the cell lysate (panel A) and from the culture media (panel B) were isolated by immunoprecipitation and analyzed on 7-12% SDS-PAGE under reduced conditions. Lane 1, normal Aalpha; lane 2, Aalpha28s,36s; lane 3, AalphaDelta1-41; lane 4, normal Bbeta; lane 5, BbetaDelta1-8; lane 6, BbetaDelta9-72; lane 7, BbetaDelta1-72; lane 8, BbetaDelta1-80; lane 9, BbetaDelta1-192; lane 10, normal ; lane 11, Delta1-15. Radioactive proteins were detected by autoradiography. Panel A was exposed overnight, and panel B was exposed for 5 days.



To determine whether single mutant chains are secreted, the culture medium was analyzed. A very small amount of normal Aalpha, Aalpha28s,36s, and AalphaDelta1-41 were secreted (Fig. 2B, lanes 1, 2, and 3). Larger amounts of normal and Delta1-15 were secreted (Fig. 2B, lanes 10 and 11). There was no secretion of normal Bbeta, BbetaDelta1-8, BDeltabeta9-72, BbetaDelta1-72, BbetaDelta1-80, and BbetaDelta1-192 chains. In all cases the secreted chains were only detected on overexposure of the autoradiogram, and we estimate that less than 10% of the chain synthesized was secreted into the medium. The large amount of protein radioactivity at the top of the gel is a contaminant due to cross-reaction of the polyclonal antibody with a protein secreted by COS cells.

Role of Amino-terminal Domain of Bbeta Chain in Dimer Formation

Our previous study showed that deletion of the first 72 amino acids from Bbeta chain inhibits assembly of the two half-molecules and results in secretion of both half-molecules and dimeric fibrinogen (17) . To more narrowly define the domains involved in assembly of dimeric fibrinogen, Bbeta mutants with different deletions at the amino terminus were constructed and co-expressed with normal Aalpha and chains. The intracellular complexes were analyzed by immuno-precipitation and SDS-PAGE on nonreduced gels. Secretion was measured by immunoprecipitation of fibrinogen from the culture medium and analysis of the secreted fibrinogen chain complexes on nonreduced 5% SDS-PAGE or on reduced 7.5% SDS-PAGE.

COS cells co-expressing BbetaDelta1-8 with normal Aalpha and chains secreted the mutant dimeric fibrinogen and a small amount of Aalphabullet complex (Fig. 3A, lane 1). On reduction the component chains of fibrinogen were observed (Fig. 3B, lane 1). Free chains, intermediate complexes, and dimeric fibrinogen occurred intracellularly (data not shown). Expression of a mutant Bbeta chain with an internal deletion of amino-terminal residues 9-72, which is the same as the naturally occurring mutant termed Fibrinogen New York 1(29) , allowed assembly and secretion of dimeric fibrinogen and small amounts of half-molecules and of Aalphabullet complex (Fig. 3A, lane 2). On reduction BbetaDelta9-72 co-migrates, on SDS-PAGE, together with the chain (Fig. 3B, lane 2). However, deletion of the first 72 amino acid residues (BbetaDelta1-72) resulted in inhibition of dimer formation and secretion of both half-molecules and dimeric fibrinogen (Fig. 3, A and B, lane 3). Further deletion of another 8 amino acids (BbetaDelta1-80), which includes deletion of a pair of cysteines flanking the amino-terminal side of the coiled-coil region, completely eliminated the formation of dimeric fibrinogen, and only half-molecules were assembled and secreted (Fig. 3, A and B, lane 4). In all cases small amounts of Aalphabullet complexes were secreted. The radioactive protein at the top of the gels, marked by arrows, is a non-fibrinogen contaminant expressed by COS cells(19) .


Figure 3: Deletion of amino-terminal amino acid residues from the Bbeta chains: effect on chain assembly and secretion. Bbeta deletion mutant cDNAs were co-expressed in COS cells with normal Aalpha and chain cDNAs. Transfected cells were incubated for 2 h with L-[S]methionine, and radioactive fibrinogen complexes were isolated from the incubation medium. Panel A, 5% SDS-PAGE, nonreduced conditions. Panel B, 7.5% SDS-PAGE under reduced conditions. Panels A and B, lane 1, Aalpha, BbetaDelta1-8, and ; lane 2, Aalpha, BbetaDelta9-72, and ; lane 3, Aalpha, BbetaDelta1-72, and ; lane 4, Aalpha, BbetaDelta1-80, and ; lane 5, Aalpha, BbetaDelta1-192, and . Panel B also shows normal Aalpha, Bbeta, as a control, in the first lane (lane 0). mFb, mutant half-molecule of fibrinogen. The contaminant protein at the top of the gel is marked by an arrow.



The half-molecule, which contains BbetaDelta1-80, migrated more slowly than expected in comparison with the half-molecule that contains BbetaDelta1-72. However, two-dimensional gel electrophoresis confirms that they are both half-molecules and suggests that the differences in mobilities may be due to differences in conformation (Fig. 4).


Figure 4: Two-dimensional gel electrophoresis of secreted fibrinogen complexes. Fibrinogen complexes secreted into the medium were separated by SDS-PAGE in nonreduced conditions in the first dimension, followed by reducing conditions in the second dimension. The location of 69- and 46-kDa molecular mass markers is shown in the second dimension. The top left panel shows fibrinogen secreted on expression of normal Aalpha, Bbeta, and chains. The top right panel presents the expression of Aalpha, betaDelta1-72 and ; bottom left panel, Aalpha, betaDelta1-80 and ; bottom right panel, AalphaDelta1-41, beta, and Delta1-15. Fb, fibrinogen; 1/2Fb, half-molecule; mFb, mutant fibrinogen; m1/2Fb, mutant half-molecule; mbeta, mutant beta; mAalpha, mutant Aalpha; m, mutant .



A previous study showed that disruption of the disulfide rings that flank the carboxyl-terminal side of the coiled-coil region, by substituting cysteine with serine residues, allowed dimeric fibrinogen to be formed but prevented secretion. To further examine the role of the carboxyl-terminal disulfide rings in assembly and secretion, a Bbeta chain mutant (BbetaDelta1-192), which contains the carboxyl-terminal pair of cysteines at positions 193 and 196 but does not contain the amino-terminal domain or the coiled-coil region, was expressed. This deletion mutant did not assemble with normal Aalpha and chains, and no half-molecules or dimeric fibrinogen were detected on SDS-PAGE under nonreduced conditions either intracellularly (data not shown) or in the culture medium (Fig. 3A, lane 5). However, a small amount of Aalphabullet complex and free chains were secreted, and Aalpha and chains were noted on reduction (Fig. 3B, lane 5). The free chain is not noted in Fig. 3A, lane 5, because in order to separate the nonreduced fibrinogen complexes, a 5% gel was used, and the free chain migrated out of the system.

Analysis of the Secreted Bbeta Mutants by Two-dimensional Gel Electrophoresis

To further characterize the fibrinogen complexes secreted, the radioactive proteins were separated in the first dimension by nonreduced SDS-PAGE and in a second dimension in reducing conditions. Expression of normal Aalpha, Bbeta, and resulted in the secretion of dimeric fibrinogen. Detectable, only after overexposure of the film, were small amounts of half-molecule, Aalphabullet, and bullet complexes. The principal secreted product, dimeric fibrinogen, yielded, as expected, Aalpha, Bbeta, and chains on reduction (Fig. 4, top left panel). The area identified as Aalphabullet complex contained more radioactivity in the Aalpha than in the chain, and therefore it is possible that this area may also contain a mixture of AalphabulletAalpha dimer and small amounts of a homopolymer.

Expression of Aalpha, BbetaDelta1-72, and led to secretion of mutant dimeric fibrinogen, mutant half-molecule, and Aalphabullet complex. In both the mutant dimeric fibrinogen and mutant half-molecule, the BbetaDelta1-72 migrated faster in the second dimension than chain (Fig. 4, top right panel).

As shown in Fig. 3A, lane 4, expression of Aalpha, BbetaDelta1-80, and led to inhibition of dimer formation and secretion of half-molecules. The mutant half-molecule was characterized by its appropriate mobility in the first dimension and that it is composed of Aalpha, mutant Bbeta, and chains (Fig. 4, bottom left panel). Another fibrinogen complex migrated slightly faster than the half-molecule in the first dimension, and because of its size, chain composition, and variability in different experiments, it was tentatively identified as degraded half-molecule. Small amounts of mutant fibrinogen chains were also noted in the second dimension in a higher molecular weight complex, suggesting that larger complexes composed of Aalpha and mutant BbetaDelta1-80 chains may also occur.

Deletion of Amino-terminal Domains of Aalpha and Chains

To determine the effect of amino-terminal amino acid residues of Aalpha and chains in dimer formation, mutants were created, equivalent to BbetaDelta1-72, which deleted the amino-terminal amino acid residues upstream from the disulfide rings, which flank the amino-terminal side of the coiled-coil region. COS cells were co-transfected with a mutant chain and the two corresponding normal chains, and fibrinogen assembly and secretion were determined. In contrast to BbetaDelta1-72, (Fig. 5, lane 3) in which both half-molecules and dimeric fibrinogen were secreted, removal of the first 41 amino acids of Aalpha chain (AalphaDelta1-41) did not affect the assembly and secretion of dimeric fibrinogen (Fig. 5, lane 2). Dimeric fibrinogen was secreted into the culture medium. Similarly, deletion of the first 15 amino acids of chain (Delta1-15), did not interrupt dimer formation, and dimeric fibrinogen was secreted (Fig. 5, lane 4). However, COS cells co-expressing mutants AalphaDelta1-41, Delta1-15, and normal Bbeta chains, only assembled and secreted half-molecules and did not form dimeric fibrinogen (Fig. 5, lane 5). The half-molecule assembled in these conditions was further characterized by two-dimensional SDS-PAGE analysis (Fig. 4, bottom right panel). It had the appropriate mobility in the first dimension and yielded mutant Aalpha and chains and normal Bbeta on the second dimension. This is in agreement with previous studies in which amino-terminal cysteines were changed to serine(19, 30) . AalphaDelta1-41 and Delta1-15 do not contain the cysteine residues (Aalpha Cys, Aalpha Cys, and Cys^8 and Cys^9), which link, by disulfide bonds, the two half-molecules of fibrinogen.


Figure 5: Amino termini of Aalpha and chains participate in dimer assembly. COS cells, co-transfected with normal or mutant fibrinogen chain cDNAs were incubated with L-[S]methionine for 2 h. Fibrinogen complexes were immunoprecipitated, separated on SDS-PAGE under nonreduced conditions, and detected by autoradiography. Panel A shows intracellular fibrinogen chains on 7.5% SDS-PAGE and panel B shows secreted fibrinogen chains on 5% SDS-PAGE. Lane 1, normal Aalpha, Bbeta, and ; lane 2, AalphaDelta1-41, Bbeta, and ; lane 3, Aalpha, BbetaDelta1-72, and ; lane 4, Aalpha, Bbeta, and Delta1-15; lane 5, AalphaDelta1-41, Bbeta, and Delta1-15. Lanes 1, 2, and 3 are from the same experiment, and lanes 4 and 5 were analyzed at different times. Fb*, normal fibrinogen; mFb, mutant fibrinogen; mFb, mutant half-molecule of fibrinogen



Noncovalent Interactions in Dimer Assembly: Comparison of Amino-terminal Deletion Mutants with Corresponding Cysteine to Serine Substitutions

Previous studies indicated that the half-molecules of fibrinogen are linked by three symmetrical disulfide bonds between adjacent Aalpha and chains (Aalpha Cys-Aalpha Cys, Cys-Cys) and probably between Aalpha Cys of one half-molecule and Bbeta Cys of the other half-molecule. This conclusion was reached because substituting cysteine residues with serine at Aalpha 28 and 8 and 9 did not abolish dimer formation, but if the cysteines at Aalpha 36 or Bbeta 65 were also changed to serine, then half-molecules and not dimers were mostly formed(19, 30) . There was, however, a difference depending on whether the cysteines at Aalpha 36 or Bbeta 65 were substituted. When Aalpha Cys was changed to serine some dimer formation occurred, and when Bbeta Cys was changed to serine very few dimers were formed(30) . This could be interpreted as either due to a rearrangement of disulfide interactions or the result of other noncovalent interactions between the half-molecules. To determine if, in addition to disulfide linkages, noncovalent interactions also occur between the amino-terminal domains of the two half-molecules, we compared deletion mutants with substitution mutants, in which the participating cysteine residues were substituted with serine. As described earlier, truncation of the first 41 amino acids of the Aalpha chain, which eliminates Aalpha cysteines 28 and 36 (Fig. 6A, lane 2), or truncation of the first 15 amino acids of chain, which eliminates cysteines 8 and 9 (Fig. 6A, lane 6), did not prevent dimer formation and secretion if these mutants were co-expressed with the two other normal chains. Similar results were obtained by substituting serine for cysteine residues at Aalpha Cys and Cys (Fig. 6, lane 3) and at Cys^8 and Cys^9 (8s,9s, Fig. 6A, lane 7). However, co-expressing AalphaDelta1-41, Delta1-15, and BbetaDelta1-72, which removes all of the amino-terminal cysteine residues (Aalpha Cys and Cys, Cys^8 and Cys^9, and Bbeta Cys) that are thought to hold the half-molecules together, resulted in the formation and secretion of only half-molecules (Fig. 6A, lane 14). It should be noted (Fig. 6B, lanes 7, 11, 13, and 15) that expression of 8s,9s leads to the addition of an extra N-linked sugar and results in the mutant chain migrating close to normal Bbeta chains(18) .


Figure 6: Comparison of deletion and substitution mutants. Combinations of deletion and substitution mutant(s) and of normal fibrinogen chain(s) were co-expressed in COS cells. L-[S]methionine-labeled fibrinogen chains were analyzed on SDS-PAGE. Autoradiograms are shown. Panel A contains secreted fibrinogen chains in nonreduced conditions, and panel B shows them in reduced conditions. Lane 1, normal Aalpha, Bbeta, and ; lane 2, AalphaDelta1-41, Bbeta, and ; lane 3, Aalpha28s,36s, Bbeta, and ; lane 4, Aalpha, BbetaDelta1-72, and ; lane 5, Aalpha, Bbeta65s, and ; lane 6, Aalpha, Bbeta, and Delta1-15; lane 7, Aalpha, Bbeta, and 8s,9s; lane 8, AalphaDelta1-41, BbetaDelta1-72, and ; lane 9, Aalpha28s,36s, Bbeta65s, and ; lane 10, AalphaDelta1-41, Bbeta, and Delta1-15; lane 11, Aalpha 28s,36s, Bbeta, and 8s,9s; lane 12, Aalpha, BbetaDelta1-72, and Delta1-15; lane 13, Aalpha, Bbeta65s, and 8s,9s; lane 14, AalphaDelta1-41, BbetaDelta1-72, and Delta1-15; lane 15, Aalpha28s,36s, Bbeta65s, and 8s,9s; lane 16, AalphaDelta1-41,Bbeta and 8s,9s; lane 17, Aalpha28s,36s, Bbeta, and Delta1-15. Fb*, normal fibrinogen; mFb, mutant fibrinogen; mFb, mutant half-molecule of fibrinogen.



To determine whether the first 41 amino acids of the Aalpha chain and the first 15 amino acids of chain interact noncovalently and assist dimeric fibrinogen assembly, a series of co-transfections were performed. COS cells co-expressing Aalpha 28s,36s, normal Bbeta, and 8s,9s (Fig. 6A, lane 11) secreted a mixture of half-molecules and dimeric fibrinogen, indicating that dimer formation was affected but not completely abolished. Similar results were obtained with AalphaDelta1-41, normal Bbeta, and 8s,9s (Fig. 6A, lane 16). By contrast COS cells co-expressing AalphaDelta1-41, normal Bbeta, and Delta1-15 (Fig. 6A, lane 10) predominantly secreted half-molecules and very little dimeric fibrinogen. The chain composition of the mutant half-molecule is shown in two-dimensional gels in Fig. 4. Also, co-expression of Aalpha28s,36s, normal Bbeta, and Delta1-15 (Fig. 6A, lane 17) led to the formation and secretion of half-molecules, with very little dimeric fibrinogen. These results indicate that noncovalent interactions at the amino-terminal domain of the chain, besides covalent interactions of cysteines, may also be involved in the formation of the dimeric complexes.

Truncation of the first 72 amino acids of the Bbeta chain also inhibited dimer formation, and both half-molecules and dimers were secreted (Fig. 6A, lane 4). This region of the Bbeta contains a cysteine at position 65 that is thought to be linked to the cysteine at Aalpha 36 of the other half-molecule of fibrinogen. However, only substituting Bbeta cysteine 65 to serine did not affect dimer formation and secretion (Fig. 6A, lane 5). This again suggests that amino acid residues within Bbeta 1-72 other than the cysteine residues are involved in dimer formation by noncovalent interactions.

On reduction, the secreted fibrinogen complexes in all cases only contained the expected mutant or normal fibrinogen chains (Fig. 6B).


DISCUSSION

The two half-molecules of fibrinogen are linked by amino-terminal disulfide bonds. Earlier studies indicated that the three symmetrical disulfides, between adjacent cysteines at Aalpha 28 and 8 and 9 were the principal disulfide interactions holding the two half-molecules together. However, substituting these cysteine residues with serine did not abolish dimer formation in transfected COS cells(18) . It was later shown that if, in addition, the cysteine residues at Aalpha 36 or Bbeta 65 were also substituted with serine, then dimer formation was inhibited. This led to the suggestion that, in addition to the symmetrical disulfide bonds between adjacent Aalpha and chains, cysteine Aalpha 36 of one half-molecule is linked to cysteine Bbeta 65 of the other half-molecule(19, 30) . However, these amino-terminal disulfide bonds may not be the only interactions holding the two half-molecules. For example, substitution of Bbeta Cys 65 with serine did not affect dimer formation, but deleting the first 72 amino-terminal amino acids inhibited dimer formation, and equal amounts of half-molecule and dimer were secreted. This suggested the involvement of some of the amino-terminal amino acid residues of the Bbeta chain, other than cysteine, in dimer formation.

In this report we test other Bbeta deletion mutants and also amino-terminal Aalpha and deletion mutants and demonstrate that, in addition to the cysteine at position 65, sequences within the first 72 amino acid residues of Bbeta participate in dimer formation as do amino-terminal residues in the Aalpha and chains. The results further suggest that a linear amino acid sequence within the Bbeta amino-terminal region may not be responsible for linking the two half-molecules, since BbetaDelta1-8 had little or no effect on dimer formation; BbetaDelta9-72 slightly inhibited dimer formation, but BbetaDelta1-72 significantly interrupted dimer formation.

BbetaDelta9-72 is the same as a naturally occurring mutant fibrinogen, termed Fibrinogen New York 1, which was identified as a heterozygous congenital dysfibrinogenemia with thrombotic tendency. In the patient, Fibrinogen New York 1 was present, in plasma, as a dimer. In the recombinant system small amounts of half-molecules are also secreted when BbetaDelta9-72 was co-expressed with normal Aalpha and chains. If some half-molecules were present in the plasma of Fibrinogen New York, they could remain undetected because plasma fibrinogen was not analyzed in nonreduced SDS-PAGE gels. Alternatively, in vivo, the half-molecule may not be secreted by hepatocytes or may be quickly cleared from the circulation.

Clearly the major link between the two half-molecules is the amino-terminal disulfide bonds(19, 30) . However, our current results indicate that noncovalent interactions in all three chains also participate. For example, COS cells co-expressing Aalpha28s,36s, 8s,9s, and normal Bbeta chains are capable of assembling and secreting equal amounts of dimeric fibrinogen and half-molecules. By contrast, cells that co-expressed AalphaDelta1-41, Delta1-15, and normal Bbeta chains only assembled and secreted half-molecules. This indicates that besides cysteines at Aalpha 28 and 36 and 8 and 9, noncovalent interactions of other residues in the regions of AalphaDelta1-41 and Delta1-15 participate in dimer formation. Further analysis showed that COS cells co-expressing AalphaDelta1-41, 8s,9s, and normal Bbeta chains secreted both dimer and half-molecules, while COS cells co-expressing Aalpha28s,36s, Delta1-15, and normal Bbeta chains only secreted half-molecules, indicating that noncovalent interactions of Delta1-15 residues may play a more important role than that of Aalpha chain in holding the two half-molecules together.

A structural feature of the three fibrinogen chains is that each chain contains about 111 hydrophobic amino acids, which intertwine to form an alpha-helical coiled-coil region. The coiled-coil region is flanked by a pair of cysteine (Cys-X-X-X-Cys) residues, which form interchain disulfide bridges, termed disulfide rings. Theoretical considerations suggested that the coiled-coil domain and the disulfide ring may play a key role in fibrinogen assembly(4) . Our early studies demonstrated that the first 207 amino acids of the Bbeta chain, which contains the amino-terminal residues and an intact coiled-coil domain with disulfide rings, can form a dimeric structure when co-expressed with normal Aalpha and chains(17) . The present data show that removal of the first 72 amino acids of the Bbeta chain results in assembly of the three chains but that dimer formation is inhibited. This indicates that amino acid residues 73-207 of the Bbeta chain are sufficient for assembly of half-molecules, while a subset of amino acid residues 1-72 of Bbeta chain are necessary for dimer formation. Our data also demonstrate that deletion of an amino-terminal segment that contains the cysteine residues that are part of the disulfide rings completely eliminates dimer formation. This is consistent with our earlier studies, which showed that disruption of the disulfide rings by substituting cysteine residues with serine abolished dimer formation.


FOOTNOTES

*
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 by hereby marked ``advertisement'' in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

Portions of this study were presented at the annual meeting of the American Society of Biochemistry and Molecular Biology (May 1994).

§
To whom correspondence should be addressed: New York Blood Center, 310 E. 67 St., New York, NY 10021. Tel.: 212-570-3059; Fax: 212-879-0243.

(^1)
The abbreviations used are: PAGE, polyacrylamide gel electrophoresis; Neo, neomycin phosphotransferase.


REFERENCES

  1. Blomback, B., and Blomback, M. (1972) Ann. N. Y. Acad. Sci. 202, 77-79 [Medline] [Order article via Infotrieve]
  2. Henschen, A., Lottspeich, F., Kehl, M., and Southan, C. (1983) Ann. N. Y. Acad. Sci. 498, 28-43 [Medline] [Order article via Infotrieve]
  3. Doolittle, R. F. (1984) Annu. Rev. Biochem. 53, 195-229 [CrossRef][Medline] [Order article via Infotrieve]
  4. Doolittle, R. F., Goldbaum, D. M., and Doolittle, L. R. (1978) J. Mol. Biol. 120, 311-325 [Medline] [Order article via Infotrieve]
  5. Fu, Y., Weissbach, L., Plant, P. W., Oddoux, C., Cao, Y., Liang, J., Roy, S. N., Redman, C. M., and Grieninger, G., (1992) Biochemistry 31, 11968-11972 [Medline] [Order article via Infotrieve]
  6. Blomback, B., Hessel, B., and Hogg, D. (1976) Thromb. Res. 8, 639-658 [Medline] [Order article via Infotrieve]
  7. Hoeprich, P. D., and Doolittle, R. F. (1983) Biochemistry 22, 2049-2055 [Medline] [Order article via Infotrieve]
  8. Henschen, A. (1964) Arkiv. Kemi 22, 355-373
  9. Blomback, B., Blomback, M., Henschen, A., Hessel, B., Iwanaga, S., and Woods, K. R. (1968) Nature 218, 130-134 [Medline] [Order article via Infotrieve]
  10. Iwanaga, S., Wallen, P., Grondahl, N. J., Henschen, A., and Blomback, B. (1967) Biochim. Biophys. Acta 147, 606-609 [Medline] [Order article via Infotrieve]
  11. Bouma, H., Takagi, T., and Doolittle, R. F. Thromb. Res. 13, 557-562
  12. Hall, C. E., and Slayter, H. S. (1959) J. Biophys. Biochem. Cytol. 5, 11-16 [Abstract/Free Full Text]
  13. Fowler, W. E., and Erickson, H. P. (1979) J. Mol. Biol. 134, 241-249 [Medline] [Order article via Infotrieve]
  14. Weisel, J. W., Phillips, G. N., Jr., and Cohen, C. (1981) Nature 289, 263-267 [Medline] [Order article via Infotrieve]
  15. Mosesson, M. W., Hainfeld, J., Wall, J., and Haschemeyer, R. H. (1981) J. Mol. Biol. 153, 695-718 [Medline] [Order article via Infotrieve]
  16. Rao, S. P. S., Poojary, M. D., Elliott, B. W., Melanson, L. A., Oriel, B., and Cohen, C. (1991) J. Mol. Biol. 222, 89-98 [Medline] [Order article via Infotrieve]
  17. Zhang, J. Z., and Redman, C. M. (1992) J. Biol. Chem. 267, 21727-21732 [Abstract/Free Full Text]
  18. Zhang, J. Z., Kudryk, B., and Redman, C. M. (1993) J. Biol. Chem. 268, 11278-11282 [Abstract/Free Full Text]
  19. Zhang, J. Z., and Redman, C. M. (1994) J. Biol. Chem. 269, 652-658 [Abstract/Free Full Text]
  20. Kaufman, R., Davies, M., Wasley, L., and Michnik, D. (1991) Nucleic Acids Res. 19, 4485-4490 [Abstract]
  21. Yu, S., Sher, B., Kudryk, B., and Redman, C. M. (1984) J. Biol. Chem. 259, 10574-10581 [Abstract/Free Full Text]
  22. Danishefsky, K., Hartwig, R., Banerjee, D., and Redman, C. M. (1990) Biochim. Biophys. Acta 1048, 202-208 [Medline] [Order article via Infotrieve]
  23. Roy, S. N., Mukhopadhyay, G., and Redman, C. M. (1990) J. Biol. Chem. 265, 6389-6393 [Abstract/Free Full Text]
  24. Roy, S. N., Mukhopadhyay, G., and Redman, C. M. (1990) J. Biol. Chem. 265, 6389-6393 [Abstract/Free Full Text]
  25. Kunkel, T. A. (1985) Proc. Natl. Acad. Sci. U. S. A. 82, 488-492 [Abstract]
  26. Zoller, M. J., and Smith, M. (1982) Nucleic Acids Res. 10, 6487-6500 [Abstract]
  27. Sanger, F., Nicklen, S., and Coulson, A. R. (1977) Proc. Natl. Acad. Sci. U. S. A. 74, 5463-5467 [Abstract]
  28. Cullen, B. R. (1987) Methods Enzymol. 152, 684-704 [Medline] [Order article via Infotrieve]
  29. Liu, C. Y., Koehn, J. A., and Morgan, F. J. (1985) J. Biol. Chem. 260, 4390-4396 [Abstract]
  30. Huang, S., Cao, Z., and Davie, E. W. (1993) Biochem. Biophys. Res. Commun. 190, 488-495 [CrossRef][Medline] [Order article via Infotrieve]

©1996 by The American Society for Biochemistry and Molecular Biology, Inc.