©1995 by The American Society for Biochemistry and Molecular Biology, Inc.
Secretion of Biologically Active Recombinant Fibrinogen by Yeast (*)

(Received for publication, February 9, 1995; and in revised form, July 28, 1995)

Samar N. Roy Bohdan Kudryk Colvin M. Redman (§)

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

ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
FOOTNOTES
REFERENCES

ABSTRACT

Fibrinogen (340 kDa) is a plasma protein that plays an important role in the final stages of blood clotting. Human fibrinogen is a dimer with each half-molecule composed of three different polypeptides (Aalpha, 67 kDa; Bbeta, 57 kDa; , 47 kDa). To understand the mechanism of fibrinogen chain assembly and secretion and to obtain a system capable of producing substantial amounts of fibrinogen for structure-function studies, we developed a recombinant system capable of secreting fibrinogen. An expression vector (pYES2) was constructed with individual fibrinogen chain cDNAs under the control of a Gal-1 promoter fused with mating factor Falpha1 prepro secretion signal (SS) cascade. In addition, other constructs were prepared with combinations of cDNAs encoding two chains or all three chains in tandem. Each chain was under the control of the Gal-1 promoter. These constructs were used to transform Saccharomyces cerevisiae (INVSC1; Matalpha his3-Delta1 leu2 trp1-289 ura3-52) in selective media. Single colonies from transformed yeast cells were grown in synthetic media with 4% raffinose to a density of 1 times 10^8 cells/ml and induced with 2% galactose for 16 h. Yeast cells expressing all three chains contained fibrinogen precursors and nascent fibrinogen and secreted about 30 µg/ml of fibrinogen into the culture medium. The Bbeta and chains, but not Aalpha, were glycosylated. Glycosylation of Bbeta and chains was inhibited by treatment of transformed yeast cells with tunicamycin. Intracellular Bbeta and chains, but not the Aalpha chains in secreted fibrinogen, were cleaved by endoglycosidase H. Carbohydrate analysis indicated that secreted recombinant fibrinogen contained N-linked asialo-galactosylated biantennary oligosaccharide. Recombinant fibrinogen yielded the characteristic plasmin digestion products, fragments D and E, that were immunologically indistinct from the same fragments obtained from plasma fibrinogen. The recombinant fibrinogen was shown to be biologically active in that it could form a thrombin-induced clot, which, in the presence of factor XIIIa, could undergo chain dimerization and Aalpha chain polymer formation.


INTRODUCTION

Human fibrinogen is a large plasma glycoprotein with diverse physiological functions. Its primary roles are in the final stages of blood coagulation, when it forms a fibrin clot and participates in platelet aggregation. Fibrinogen is a dimeric molecule with each half-molecule composed of three different polypeptides. The Aalpha chain has 610, the Bbeta 461, and the 411 amino acid residues. The Bbeta and chains are N-glycosylated. The six chains are connected by 29 disulfide bonds. The primary structure of fibrinogen is known, and structural studies indicate that fibrinogen is elongated and trinodal. The central E domain contains the NH(2) termini of the six polypeptide chains, and the two terminal ``D'' nodes are formed by carboxyl-terminal globular domains of the Bbeta and chains and a small (12-kDa) segment of the Aalpha chain. The COOH-terminal regions of the Aalpha chain extend beyond the globular domains of the Bbeta and chains and may fold back and contribute to the structure of the central node. Between the central and terminal domains the three chains are coiled together in an alpha-helical, rope-like manner. This alpha-helical area, which occupies about 111 amino acids in each chain, is termed the ``coiled-coil'' region and is flanked at either end by a set of interchain disulfide bonds called the ``disulfide rings.'' Unfortunately, to date, high resolution structural analysis has not been achieved since fibrinogen crystals have diffracted poorly. Nevertheless, a combination of biochemical and electron microscopy studies have reached a consensus on the general structure as described above. The structure and physiology of fibrinogen has been reviewed (1, 2, 3, 4, 5, 6, 7) .

Biologically active recombinant fibrinogen has been expressed in several mammalian cell systems(8, 9, 10, 11) , and the individual component chains of fibrinogen have been expressed in Escherichia coli(12, 13, 14) . Although recombinant fibrinogen mutants have been used for structure/function studies (15, 16, 17, 18) the procedures are hampered by the fact that prokaryotic systems do not assemble the fibrinogen chains and that transfected mammalian cells only secrete small amounts of biologically active fibrinogen. To obtain substantial quantities of biologically active fibrinogen for structure/function studies and to develop a system in which a genetic approach to understanding fibrinogen chain assembly could be undertaken, we have expressed fibrinogen in yeast.


EXPERIMENTAL PROCEDURES

Materials

The expression vector pYES2 and the yeast strain INVSC1 (Matalpha his3-Delta1 leu2 trp1-289 ura3-52) were obtained from Invitrogen, Inc. (San Diego, CA). Medium to grow the yeast in selective conditions was purchased from Bio101, Inc (La Jolla, CA). Galactose, raffinose, and tunicamycin were obtained from Sigma. Restriction enzymes, Klenow fragment, and calf intestinal phosphatase were purchased from Boehringer Mannheim. Endoglycosidase H was obtained from Genzyme Corp. (Cambridge, MA), T4 DNA ligase from New England Biolab (Beverly, MA), L-[S]methionine (1100 Ci/mmol) from DuPont NEN, and agarose AminoLink® coupling gel was purchased from Pierce. Human plasma fibrinogen was prepared by IMCO and purchased from American Diagnostics Inc. (Greenwich, CT). Other reagents used have been described previously(8, 19, 20, 21) .

Construction of Expression Vectors

Expression vectors containing fibrinogen cDNAs for single chains, for combinations of two chains, and for all three chains were inserted into multiple cloning sites at the 3`-end of the Gal-1 promoter fused to the MFalpha1 (^1)prepro secretion signal (SS)^1 cascade in pYES2 plasmid (see Fig. 1). To prepare pYES2Aalpha, pYES2Bbeta, and pYES2, full-length cDNAs were released by appropriate restriction enzymes from previously described constructs (8) and ligated to pYES2 plasmid at the 3`-end of the Gal-1-SS promoter. Other constructs, pYES2AalphaBbeta, pYES2Aalpha, pYES2Bbeta, and pYES2AalphaBbeta, were made by ligating fibrinogen chain cDNAs in tandem, each under the control of the Gal-1-SS promoter. Elution of DNA fragments from agarose gel, dephosphorylation of plasmids with calf intestinal phosphatase, fill-in reaction with Klenow fragment, and ligation were performed by standard procedures (22) and as described previously(8, 17) .


Figure 1: Expression vectors containing fibrinogen chain cDNAs. The full-length cDNAs for individual fibrinogen chains were inserted into multiple cloning sites at the 3`-end of Gal-1-SS promoter (pYES2Aalpha, pYES2Bbeta, and pYES2). In the other constructs, combinations of two chains (pYES2AalphaBbeta, pYES2Aalpha, and pYES2Bbeta) and all three chains (pYES2AalphaBbeta) were inserted in tandem. Each arrow indicates the cleavage site of the secretion signal



Transformation

Transformation of S. cerevisiae (INVSC1) with pYES2 vectors containing fibrinogen cDNAs was performed by the alkali-cation method, and the cells were plated on SC-ura plates(23) . Single colonies from each plate were grown in SC-ura medium containing 4% raffinose at 30 °C with vigorous shaking overnight and kept as a stock culture. Transformed yeast cells, with the above described constructs, were named INVSC1Aalpha, INVSC1Bbeta, INVSC1, INVSC1AalphaBbeta, INVSC1Aalpha, INVSC1Bbeta, and INVSC1AalphaBbeta.

Expression and Treatment with Tunicamycin

Stock culture was grown in 5 ml of SC-ura medium overnight at a density of 1 times 10^8cells/ml. The cells were harvested by centrifugation for 5 min at 500 times g, resuspended in SC-ura medium containing 2% galactose, and grown for an additional 16 h for induction of fibrinogen chain synthesis. The cells were again harvested, resuspended in SC-ura-Met medium containing 50 µCi/ml of L-[S]methionine, and incubated for 1 h at 30 °C. In some cases, the cells were preincubated with medium containing 10 µg/ml of tunicamycin for 1 h and then incubated with medium containing 10 µg/ml tunicamycin and L-[S]methionine for an additional hour. When determining intracellular fibrinogen, the cells were harvested, washed with phosphate-buffered saline, lysed with 0.5 ml of IP buffer (50 mM Tris-HCl, pH 7.4, 1% Triton X-100, 0.2% SDS, 150 mM NaCl, 5 mM EDTA, 10 units/ml Trasylol, 1 mM phenylmethylsulfonyl fluoride, 0.1 mM tosylphenylalanyl chloromethyl ketone, 1 µg/ml soybean-trypsin inhibitor) and 200 mg of acid-washed glass beads (0.5 mm diameter)/10^8 cells by vortexing twice for 45 s(24) . The cell lysate was diluted to 1 ml with water and centrifuged at 15,000 times g for 15 min at 4 °C. Fibrinogen was isolated by immunoprecipitation from the soluble fraction with a rabbit polyclonal antibody to human fibrinogen (Dako Corporation, Carpenteria, CA) as described elsewhere(8) .

Endoglycosidase H Treatment

INVSC1AalphaBbeta cells were metabolically labeled with L-[S]methionine for 1 h as described above. Radioactive fibrinogens were isolated from the incubation medium and from cell lysates by immunoprecipitation using polyclonal antibody to fibrinogen. The immune complex was treated with 2 mIU/ml of endoglycosidase H at 37 °C overnight, reimmunoprecipitated, and separated on SDS-PAGE as described previously (21, 25) .

Secretion of Fibrinogen

Yeast cells transformed with pYES2AalphaBbeta and grown from single colonies were inoculated in 50 ml of SC-ura medium containing 4% raffinose and grown overnight at 30 °C. The cells were then induced with 2% galactose and incubated for an additional 16 h. The culture medium was centrifuged at room temperature for 5 min at 500 times g. The pH of the medium was adjusted to 7.0 with 1 M Tris-HCl buffer, pH 8.0, and a mixture of protease inhibitors (10 units/ml Trasylol, 1 mM phenylmethylsulfonyl fluoride, 0.1 mM tosylphenylalanyl chloromethyl ketone, 1 µg/ml soybean-trypsin inhibitor, 1 µg/ml pepstatin) was added.

Isolation and Purification of Fibrinogen from the Culture Medium

Fibrinogen was isolated from the culture medium by absorption on a protamine sulfate-Sepharose 6B column (10 ml) calibrated with buffer A (50 mM Tris-HCl, pH 7.4, 5 mM -amino caproic acid, 5 mM EDTA)(11) . The column was washed with buffer A containing 0.8 M NaCl, and bound fibrinogen was eluted with 0.1 M sodium acetate, pH 4.5. The pH of eluted fibrinogen was adjusted to 7.0 with 1 M Tris-HCl, pH 8.0.

Quantitation of Secreted Fibrinogen

The amount of fibrinogen secreted in the medium was measured by competition enzyme-linked immunosorbent assay using two different fibrinogen chain-specific monoclonal antibodies, 1-8C6 (anti-Bbeta 1-21) and Fd4-7B3 (anti-fibrinogen fragment D). The specificities of the antibodies have been described(26) .

Carbohydrate Structure of Recombinant Fibrinogen

The N-linked oligosaccharide analyses of purified recombinant fibrinogen was performed by Glyko, Inc. (Novato, California) using a FACE (Fluorophore Assisted Carbohydrate Electrophoresis) N-linked oligosachharide sequencing kit. Recombinant fibrinogen, purified from the culture medium, was lyophilized and resuspended in 170 µl of water to a concentration of 2.35 mg/ml. The protein was denatured with 0.5% SDS, reduced with 1% beta-mercaptoethanol, and digested with peptide-N-glycosidase F (PNGase F) at 37 °C for 48 h. The released oligosaccharides were isolated by ethanol precipitation, labeled with fluorescent tag, and separated by electrophoresis in a 40% polyacrylamide gel. The oligosaccharide was characterized by sequential digestion with specific glycosidases and comparison of their electrophoretic mobility against standard N-linked oligosaccharides, as described in the manufacturer's protocol. Sialic acid was measured by the thiobarbituric acid assay (27) and by Glyko's monosaccharide composition kit.

Treatment with Plasmin

Purified fibrinogen (30 µg) from yeast culture medium was pretreated with digestion buffer (37 mM Tris-HCl, 150 mM NaCl, 10 mM CaCl(2), 0.1% NaN(3), pH 7.3). Digestion was initiated by the addition of 40 µl (400 units) of streptokinase (Sigma) and 25 µl (12.5 µg) of plasminogen (IMCO, Stockholm, Sweden) in 1 ml. Digestion was at 37 °C for 16 h and was terminated by the addition of 50 µl (500,000 IU) Trasylol (Miles Laboratories, Inc., Elkhart, IN).

The above digest was mixed with an equal volume of buffer A (40 mM Tris-HCl, 110 mM NaCl, 0.1% NaN(3), pH 7.5) and applied to a 2-ml column containing approximately 40 mg of agarose-conjugated (Pierce AminoLink 228) anti-fragment D monoclonal antibody (Fd4-7B3). Flow was stopped for 2 h to allow maximum binding. Nonadsorbed protein was removed by extensive washing with buffer A. Adsorbed protein was eluted with 4 ml of 3 M NaSCN in buffer A.

The above nonadsorbed fraction was applied to a 2-ml column containing approximately 40 mg of agarose-conjugated anti-fibrinogen fragment E monoclonal antibody (2N3H10). The sample was recycled several times over this column to allow maximum binding. Nonadsorbed protein was removed by extensive washing with buffer A. Adsorbed protein was eluted with 4 ml of 3 M NaSCN in buffer A.

Factor XIIIa Cross-linking

Secreted recombinant fibrinogen was treated with thrombin (6.8 NIH units/ml) with or without factor XIIIa (1.0 units/ml) to determine its ability to form a thrombin-induced clot and to cross-link. The fibrin complexes were separated by SDS-PAGE and detected by staining with Coomassie Blue and by Western immunoblots using several chain-specific antibodies: 1C2-2 (anti-fibrinogen Aalpha/fibrin alpha)(28) , Ea3 (anti-fibrinogen Bbeta/fibrin beta)(26) , T2G1 (anti-fibrin beta)(29) , and 4-2 (anti-fibrinogen /fibrin -dimer)(29) .


RESULTS

Expression of Individual Fibrinogen Chains and N-Glycosylation of Bbeta and Chains

Transformed yeast cells expressing individual fibrinogen chains (INVSCIAalpha, INVSCIBbeta, and INVSCI) and expressing all three chains (INVSCIAalphaBbeta) were metabolically labeled with L-[S]methionine in the presence and absence of tunicamycin. Intracellular radioactive fibrinogen and its precursors were isolated from the cell lysate by immunoprecipitation, separated by SDS-PAGE, and detected by autoradiography. INVSCIAalpha, INVSCIBbeta, and INVSCI expressed single fibrinogen chains that had electrophetic mobilities similar to the component chains of plasma fibrinogen.

In human plasma fibrinogen the Bbeta and chains, but not Aalpha, are N-glycosylated. To determine if correct N-glycosylation occurred the transformed cells expressing single chains were incubated in the absence or presence of tunicamycin, which prohibits N-glycosylation. Tunicamycin had no effect on Aalpha chain, but Bbeta and had faster electrophoretic mobilities, indicating that they lacked N-linked carbohydrates (Fig. 2A).


Figure 2: N-Glycosylation of Bbeta and 32 chains. Transformed yeast cells, expressing individual chains and all three fibrinogen chains, were metabolically labeled with L-[S]methionine in the presence or absence of tunicamycin. Fibrinogen chains were isolated from the cell lysate by immunoprecipitation and separated by 7.5% SDS-PAGE. An autoradiogram is shown. Panel A, expression of individual fibrinogen chains (reduced samples). Panel B, expression of all three fibrinogen chains (nonreduced samples). The relative positions of molecular size markers are shown on the left of each autoradiogram. The locations of fibrinogen (Fbg), its intermediates, and free chains are shown on the right.



INVSCIAalphaBbeta expressed all three fibrinogen chains, and analysis of intracellular fibrinogen complexes on nonreduced SDS-PAGE demonstrated the presence of several fibrinogen precursors. Free Aalpha, Bbeta, and , two chain complexes (Bbeta- and Aalpha-), a three-chain half-molecule, and dimeric fibrinogen accumulated intracellularly (Fig. 2B). The intracellular complexes were characterized by their estimated molecular weights, based on SDS-PAGE, and the radioactive bands were excised and re-electrophoresed in reduced conditions to determine the chain compositions (data not shown).

On treatment with tunicamycin the Aalpha chain expressed by INVSCIAalphaBbeta had a mobility similar to Aalpha chains from untreated cells, but both Bbeta and chains had faster electrophoretic mobilities. Tunicamycin also affected the electrophoretic mobilities of the higher molecular weight complexes. In tunicamycin-treated cells the two-chain and three-chain fibrinogen complexes were not as distinct as the corresponding complexes from untreated cells (Fig. 2B).

Endoglycosidase H Treatment of Intracellular and Secreted Fibrinogen

Nascent glycoproteins present in the endoplasmic reticulum contain mannose-rich carbohydrate side chains, which are later trimmed and further processed in the trans Golgi compartment before secretion occurs. The mannose-rich oligosaccharides, but not the fully processed side chains, are cleaved from glycoproteins by endoglycosidase H. To determine the carbohydrate nature of intracellular and secreted fibrinogen, INVSCIAalpha, INVSCIBbeta, and INVSCI cells were metabolically labeled with L-[S]methionine, and intracellular and secreted fibrinogen were treated with endoglycosidase H. Analysis on reduced SDS-PAGE showed that intracellular Bbeta and chains, but not Aalpha, were cleaved by endoglycosidase H. By contrast the secreted fibrinogen chains were not affected by endoglycosidase H, which is to be expected if the N-linked carbohydrates were fully processed (Fig. 3).


Figure 3: Endoglycosidase H treatment of intracellular and secreted fibrinogen. INVSC1AalphaBbeta cells were metabolically labeled with L-[S]methionine, and fibrinogen was immunoprecipitated from the cell lysate and from the medium. The isolated fibrinogen was treated with endoglycosidase H, and the component chains were separated by 7.5% SDS-PAGE. An autoradiogram is shown. The relative positions of molecular size markers are indicated on the left, and those of component chains on the right.



Carbohydrate Structure and Composition

Digestion with peptide-N-glycosidase F for 48 h at 37 °C released two oligosaccharides (Fig. 4, panelA, lane3, asterisks) in the region of the gel where N-linked sugars migrate. A third band that migrated similarly to band 3 of the partial wheat starch digest (Fig. 4, panelA, lane1) could be maltotetraose (Fig. 4, panelA, lane4) or some other glucose polymer. A similar contaminant was noted in the blank sample (Fig. 4, panelA, lane2). In two other digestions with peptide-N-glycosidase F the uppermost band was not noted, and only one band (marked by an asterisk) migrated in the position of N-linked oligosaccharides (panelB, lane5 and panelC, lane4). All digestions showed the presence of maltotetraose or glucose polymer contamination and, at the bottom of the gel, of unincorporated fluorophore.


Figure 4: Carbohydrate analysis of purified recombinant fibrinogen. Panel A, lane1, glucose polymers obtained from partial wheat starch digest. The bands are numbered on the leftside. Lane2, blank sample; lane3, peptide-N-glycosidase F digest of recombinant fibrinogen. Two N-linked oligosaccharides are marked by asterisks. Lane4, 100 pmol of maltotetraose. Panel B, lane1, partial wheat starch digest; lane2, peptide-N-glycosidase F-released oligosaccharide (starting material) treated with neuraminidase III; lane3, starting material digested with endoglycosidase H; lane4, partial digestion with beta-galactosidase; lane5, starting material obtained by peptide-N-glycosidase F treatment; lane6, maltotetraose standard. Panel C, lane1, partial wheat starch digest; lane2, complete digestion of starting material with beta-galactosidase; lane3, digestion with combination of beta-galactosidase and hexosaminidase III; lane4, starting material obtained with peptide-N-glycosidase F treatment; lane5, partial wheat starch digest.



Based on the relative migration of standard oligosaccharides, as compared with the bands obtained from partial digestion of wheat starch (data not shown), the main N-linked oligosaccharide (marked by an asterisk) obtained by peptide-N-glycosidase F digestion is consistent with it being an asialo-galactosylated biantennary oligosaccharide. Subsequent experiments confirmed the structure. Digestion with neuraminidase III, (panelB, lane2) had no effect on the major band, indicating lack of sialic acid, and treatment with endoglycosidase H (panelB, lane3) also did not affect the mobility of the N-linked oligosaccharide, indicating, as was shown in a previous experiment (Fig. 3), that secreted recombinant fibrinogen glycoprotein is not of the high mannose type. Partial digestion with beta-galactosidase (panelB, lane4) showed the starting material and the appearance of two lower bands. This suggests that at least two galactose monomers were cleaved.

Further confirmation of the oligosaccharide structure was obtained by complete digestion with beta-galactosidase (panelC, lane2), which demonstrates removal of approximately two galactose units. Treatment of the starting material with a combination of beta-galactosidase and a hexosaminidase (hexosaminidase III) (panelC, lane3) indicated complete removal of galactose and GlcNAc from the nonreducing end of the starting oligosaccharide. Taken together these results are consistent with the recombinant fibrinogen being a glycoprotein that is not of the high mannose type but contains an asialo-galactosylated biantennary oligosaccharide.

The absence of sialic acid was confirmed by assaying the recombinant yeast fibrinogen by the thiobarbituric acid method(27) . Plasma fibrinogen, used as a control, yielded about 1.25 µmol of sialic acid/µmol of fibrinogen, but sialic acid was not detected in yeast fibrinogen. Also a monosaccharide composition assay, performed by Glyko Inc., failed to detect sialic acid (data not shown). It was not possible, however (because of a high background of glucose, which was also present in the blank sample and is possibly due to contamination from the dialysis membranes), to accurately determine the molar concentration of the sugars (data not shown).

Secretion of Fibrinogen

After induction with galactose for 16 h the media of INVSCIAalphaBbeta cells was collected and neutralized, and the amount of fibrinogen was determined. Quantitation was performed using two different monoclonal antibodies with specificities to different domains of fibrinogen. One of the monoclonal antibodies (1-8C6) reacted with Bbeta chain at amino acid residues 1-21, and the other (Fd4-7Bc) recognizes a plasmin digest fragment of fibrinogen (fragment D). Using both antibodies, human plasma fibrinogen and recombinant fibrinogen gave identical curves (Fig. 5). Transformed cells (10^8 cells/ml) secreted 25-30 µg/ml after 16 h of induction with galactose.


Figure 5: Standard curves used to quantitate fibrinogen in yeast culture medium. Fibrinogen in the culture medium (CCM) was determined by an enzyme-linked immunosorbent assay-based competitive assay using two different antibodies. A, anti-Bbeta 1-42; B, anti-fragment D.



In some cases the secreted fibrinogen was isolated from the incubation medium by affinity chromatography using protamine sulfate conjugated to Sepharose. Fibrinogen was the principal protein product present in the incubation medium although there was a large amount of low molecular weight material, which did not bind to the protamine-sulfate column and which absorbed at 280 nm (Fig. 6A). Analysis of the component fibrinogen chains by SDS-PAGE indicated that they had similar electrophoretic mobilities as human plasma fibrinogen (Fig. 6B) and that only Bbeta and , and not Aalpha, reacted with periodic acid-Schiff stain.


Figure 6: Analysis of purified fibrinogen secreted by INVSC1AalphaBbeta cells. Secreted fibrinogen produced by yeast cells was purified by adsorption on a protamine sulfate-Sepharose column. The chains were separated by 7.5% SDS-PAGE and stained with Coomassie Blue or with periodic acid-Schiff stain. Panel A, typical elution profile of fibrinogen from protamine sulfate coupled to Sepharose 6B. The fibrinogen peak (Fbg) was the only protein eluted at pH 4.5. Panel B, plasma fibrinogen and yeast recombinant fibrinogen were reduced and separated by SDS-PAGE and stained with periodic acid-Schiff base (lanes1 and 2) and with Coomassie Blue (lanes3 and 4). Lanes1 and 3, human plasma fibrinogen (Imco); lanes2 and 4, recombinant yeast fibrinogen.



Clotting Properties of Recombinant Fibrinogen

Secreted recombinant fibrinogen, purified from the incubation medium by affinity chromatography on a protamine-Sepharose sulfate column, was treated with thrombin or with thrombin and factor XIIIa. The recombinant fibrinogen formed a visible fibrin clot, which was solubilized in an SDS-containing buffer with dithiothreitol. The solubilized proteins from the fibrin clot were separated by SDS-PAGE and analyzed by Western immunoblots using two different monoclonal antibodies. One of the monoclonal antibodies reacted with the chain of fibrin(ogen) and dimer from fibrin, and the other reacted with the beta fibrin chain. The results are shown in Fig. 7. As controls, reduced plasma fibrinogen and untreated recombinant fibrinogen are shown in lanes2 and 3. The Aalpha chain of plasma and recombinant fibrinogen, as is often the case, was partially degraded (panelA). On treatment with thrombin, in the absence of factor XIIIa, a similar pattern to the control samples was noted. Removal of fibrinopeptides A and B was not expected to show a marked difference in electrophoretic mobility of the alpha and beta chains as compared with Aalpha and Bbeta. Western blots with antibody to the beta chain (panel B, lane4) and to chain (panelC, lane4) also showed that these chains are present in the fibrin clots. On treatment with factor XIIIa, the chain was not detected by Coomassie Blue staining (panelA, lane5) and was markedly reduced as determined by Western blot with antibody to chain. There was a concomitant appearance of dimer (panelC, lane5). As a control it was noted that factor XIIIa had no effect on the beta chain (panelB, lane5). Taken together these results demonstrate that recombinant fibrinogen forms a thrombin-induced clot and undergoes factor XIIIa-catalyzed cross-linking.


Figure 7: Clotting properties of recombinant yeast fibrinogen. Purified recombinant fibrinogen secreted by yeast cells was incubated with thrombin or thrombin + factor XIIIa at 37 °C for 4 h. After clotting, each sample was solubilized in a dithiothreitol- and SDS-containing buffer separated by SDS-PAGE (5-15% gradient gel), and Western blot analyses were performed with two monoclonal antibodies. One monoclonal antibody reacts with fibrin beta chain, and the other reacts with fibrinogen chain and fibrin -dimer. Panel A, stained with Coomassie blue; panel B, immunoblot reacted with fibrin beta-chain antibody (T2G1); panel C, immunoblot reacted with fibrinogen chain/fibrin -dimer antibody). Lane1, molecular size markers; lane2, plasma fibrinogen; lane3, yeast fibrinogen; lane4, non-cross-linked fibrin prepared from yeast fibrinogen; lane5, factor XIIIa-cross-linked yeast fibrinogen. The position of cross-linked chain dimer is shown.



Structure of Recombinant Fibrinogen Determined by Plasmin Digestion

Plasmin digestion of plasma fibrinogen yields well defined fragments that are dependent on the structural integrity of fibrinogen and the correct assembly of its component chains. To determine whether recombinant fibrinogen, on treatment with plasmin, yields fragments D and E, which are derived from the terminal and central domains of dimeric fibrinogen, purified recombinant fibrinogen was digested with plasmin. The digest was subsequently fractionated by affinity chromatography using antibodies specific to fragments D and E. Adsorbed proteins were further characterized by SDS-PAGE and Western immunoblots. Recombinant fibrinogen, like plasma fibrinogen, yielded fragments D and E (Fig. 8), indicating that they have similar structures.


Figure 8: Plasmin digestion of recombinant fibrinogen. Purified fibrinogen from yeast culture medium was digested with plasmin. The digested material was divided into two parts. The first part was adsorbed with antibody to fragment D, and the second part was adsorbed with antibody to fragment E coupled to Agarose columns. The bound materials from these two affinity columns were eluted and run on SDS-PAGE followed by Coomassie Blue staining and Western blot analyses. Panel A, protein stain; panel B, immunoblot reacted with anti-fragment D; panel C, immunoblot reacted with anti-fragment E. Lane1, plasmin digest of yeast fibrinogen; lane2, material absorbed by anti-fragment D; lane3, material absorbed by anti-fragment E.




DISCUSSION

Human fibrinogen has been expressed in a number of different recombinant systems(8, 9, 10, 11) . Although these procedures usually only produce small amounts of secreted fibrinogen they can be scaled up, using cells in suspension and roller bottles, to yield sufficient quantities to study structure/function relationships. The yeast system offers an advantage in that it is more easily adaptable to express and secrete milligram quantities of fibrinogen. The fibrinogen expressed in yeast is biologically active in that it forms a thrombin-induced clot and undergoes factor XIIIa cross-linking. Carbohydrate processing, composition, and sequence was determined by several different methods. Periodic acid-Schiff staining of the separated chains, treatment of transformed yeast cells with tunicamycin, and endoglycosidase H digestion of intracellular and secreted fibrinogen showed that only Bbeta and chains are glycosylated. In addition, tunicamycin and endoglycosidase H treatment suggest that initial N-linked glycosylation of recombinant fibrinogen occurs in a manner similar to that in hepatocytes. Tunicamycin treatment only affected the processing of Bbeta and chains, and digestion with endoglycosidase H indicated that mannose-rich fibrinogen precursors are present in the ER and are processed before secretion occurs. Carbohydrate analysis demonstrated that recombinant fibrinogen, unlike plasma fibrinogen, does not contain terminal sialic acid but otherwise may be similar in composition and sequence to plasma fibrinogen(30) . These results are in keeping with the synthesis of N-linked glycans by yeast. The early stages of N-glycosylation in yeast and animal systems are similar, but further oligosaccharide processing, which occurs in the Golgi, differs. In yeast, mannose-rich oligosaccharides are usually formed, although galactose and N-acetylglucosamine residues may be added(31) . Our studies indicate that recombinant yeast fibrinogen is not of the high mannose variety and is similar but not identical to that of plasma fibrinogen, since it lacks terminal sialic acid.

Biological activity of recombinant fibrinogen was shown by its ability to form a thrombin-induced clot and to undergo factor XIIIa-catalyzed cross-linking of fibrin chains. In addition the response of recombinant fibrinogen to thrombin and factor Xllla shows that recombinant fibrinogen has a structure similar to that of plasma fibrinogen. Cleavage of fibrinopeptides A and B by thrombin, polymerization, and correct alignment of alpha and chains for participation in factor XIIIa-catalyzed cross-linking, requires proper chain assembly and folding. Further evidence that recombinant fibrinogen has a structure similar to plasma fibrinogen was obtained by determining that fragments D and E are produced when recombinant fibrinogen is digested with plasmin. Fragments D and E are characteristic products when fibrin(ogen) is treated with plasmin and can only be obtained if the fibrinogen chains are organized in the correct configuration.

Fibrinogen chains are assembled in a series of stepwise reactions in which single chains are linked into two-chain complexes, followed by the addition of a third chain to form half-molecules, which are subsequently joined to produce dimeric fibrinogen(19, 20, 21, 32, 33, 34) . In the yeast recombinant system, the same intermediates that accumulate in HepG2 cells are noted. Free Aalpha, Bbeta, and chains, two-chain complexes (Aalphabullet and Bbetabullet), and half-molecules as well as dimeric fibrinogen accumulated in transformed yeast cells. This suggests that the sequence of chain assembly in yeast is similar to that in mammalian cells and that this recombinant system will be useful for studying the mechanisms of fibrinogen chain assembly and folding. Folding and assembly probably involve several chaperones present in the endoplasmic reticulum, and the yeast system allows the use of a genetic approach to studying this process. Yeast mutants, defective in secretory factors or chaperones, can be prepared and used to analyze chain assembly, folding, and secretion(35, 36, 37, 38, 39, 40) .

Knowledge gained from congenital dysfibrinogens, from analyzing evolutionary conserved domains, and from biochemical and structural determinations has led to assignment of specific domains as important in the functional properties of fibrinogen. However, many of these assignments were reached by inference and have not been unambiguously elucidated. It is obvious that the recombinant systems provide the opportunity to mutate specific domains and study functional modifications. The yeast system should prove useful in this regard since it produces relatively large amounts of secreted fibrinogen that is biologically active.


FOOTNOTES

*
These studies were supported by National Institutes of Health Grant HL37457. Portions of this study were presented in September 1994 at the 16th International Congress of Biochemistry and Molecular Biology, New Delhi, India and in December 1994 at the 34th Annual Meeting of the American Society for Cell Biology, San Francisco, CA. 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.

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

(^1)
The abbreviations used are: MFalpha1, mating factor alpha1; SS, prepro secretion signal; IP buffer, immunoprecipitation buffer; PAGE, polyacrylamide gel electrophoresis.


REFERENCES

  1. Blomback, B., and Blomback, M. (1972) Ann. N. Y. Acad. Sci. 202,77-79 [Medline] [Order article via Infotrieve]
  2. Doolittle, R. F. (1984) Annu. Rev. Biochem. 53,195-229 [CrossRef][Medline] [Order article via Infotrieve]
  3. Henschen, A., Lottspeich, F., Kehl, M., and Southan, C. (1983) Ann. N. Y. Acad. Sci. 408,28-43 [Medline] [Order article via Infotrieve]
  4. Mosesson, M. W. (1992) Semin. Hematol. 29,177-188 [Medline] [Order article via Infotrieve]
  5. Hantgan, R., Francis, C. W., and Marder, V. J. (1994) Hemostasis and Thrombosis: Basic Principles and Clinical Practice (Colman, R., Hirsch, J., Marder, V., and Salzman, E., eds) pp. 277-300, J. B. Lippincott Co., Philadelphia
  6. Crabtree, G. (1987) The Molecular Biology of Blood Diseases (Stamatoyannopoulus, G., Nienhuis, A., Leder, P., and Majerus, P., eds) pp. 631-661, W. B. Saunders Co., Philadelphia
  7. Weisel, J. W., Stauffacher, C. V., Bullitt, E., and Cohen, C. (1985) Science 230,1388-1391 [Medline] [Order article via Infotrieve]
  8. Roy, S. N., Procyk, R., Kudryk, B., and Redman, C. M. (1991) J. Biol. Chem. 266,4758-4763 [Abstract/Free Full Text]
  9. Hartwig, R., and Danishefsky, K. J. (1991) J. Biol. Chem. 266,6578-6585 [Abstract/Free Full Text]
  10. Farrell, D. H., Mulvihill, E. R., Huang, S., Chung, D. W., and Davie, E. W. (1991) Biochemistry 30,9414-9420 [Medline] [Order article via Infotrieve]
  11. Binnie, C. G., Hettasch, J. M., Strickland, E., and Lord, S. T. (1993) Biochemistry 32,107-113 [Medline] [Order article via Infotrieve]
  12. Bolyard, M. G., and Lord, S. T. (1988) Gene (Amst.) 66,183-192 [Medline] [Order article via Infotrieve]
  13. Bolyard, M. G., and Lord, S. T. (1989) Blood 73,1202-1206 [Abstract]
  14. Lord, S. T., and Fowlkes, D. M. (1989) Blood 73,166-171 [Abstract]
  15. Bolyard, M., and Lord, S. T. (1991) Biochem. Biophys. Res. Commun. 174,853-860 [Medline] [Order article via Infotrieve]
  16. Binnie, C. G., and Lord, S. T,. (1993) Blood 81,3186-3192 [Medline] [Order article via Infotrieve]
  17. Farrell, D. H., Thiagarajan, P., Chung, D., and Davie, E. W. (1992) Proc. Natl. Acad. Sci. U. S. A. 89,10729-10732 [Abstract]
  18. Farrell, D. H., and Thiagarajan, P. (1994) J. Biol. Chem. 269,226-231 [Abstract/Free Full Text]
  19. Yu, S., Sher, B., Kudryk, B., and Redman, C. M. (1983) J. Biol. Chem. 258,13407-13410 [Abstract/Free Full Text]
  20. Yu, S., Sher, B., Kudryk, B., and Redman, C. M. (1984) J. Biol. Chem. 259,10574-10581 [Abstract/Free Full Text]
  21. Roy, S., Yu, S., Banerjee, D., Overton, O., Mukhopadhyay, G., Oddoux, C., Grieninger, G., and Redman, C. (1992) J. Biol. Chem. 267,23151-23158 [Abstract/Free Full Text]
  22. Sambrook, J., Fritsch, E. F., and Maniatis, T. (1989) Molecular Cloning: A Laboratory Manual , 2nd Ed., Cold Spring Harbor Laboratory, Cold Spring Harbor, NY
  23. Rose, M. D., Winston, F., and Hieter, P. (1990) Methods in Yeast Genetics: A Laboratory Course Manual , pp. 39-52, Cold Spring Harbor Laboratory, Cold Spring Harbor, NY
  24. Franzusoff, J. R., and Schekman, R. (1991) Methods Enzymol. 194,662-674 [Medline] [Order article via Infotrieve]
  25. Danishefsky, K., Hartwig, R., Banerjee, D., and Redman, C. (1990) Biochim. Biophys. Acta 1048,202-208 [Medline] [Order article via Infotrieve]
  26. Kudryk, B., Grossman, Z. D., McAfee, J. G., and Rosebrough, S. F. (1989) Monoclonal Antibodies as Probes for Fibrin(ogen) Proteolysis: Monoclonal Antibodies in Immunoscintigraphy (Chatal, J. F., ed) pp. 365-398, CRC Press, Inc., Boca Raton, FL
  27. Warren, L. (1963) Methods Enzymol. 6,463-465
  28. Procyk, R., Bishop, P. D., and Kudryk, B. (1993) Thromb. Res. 71,127-138 [Medline] [Order article via Infotrieve]
  29. Procyk, R., Medved, L., Engelke, K. J., Kudryk, B., and Blomback, B. (1992) Biochemistry 31,2273-2278 [Medline] [Order article via Infotrieve]
  30. Townsend, R. R., Hilliker, E., Li, Y.-T., Laine, R. A., Bell, W. R., and Lee, Y. C. (1982) J. Biol. Chem. 257,9704-9710 [Abstract/Free Full Text]
  31. Kukuruzinska, M. A., Bergh, M. L. E., and Jackson, B. J. (1987) Annu. Rev. Biochem. 56,915-944 [CrossRef][Medline] [Order article via Infotrieve]
  32. Kudryk, B., Okada, M., Redman, C. M., and Blomback, B. (1982) Eur. J. Biochem. 125,673-682 [Medline] [Order article via Infotrieve]
  33. Huang, S., Mulvihill, E. R., Farrell, D. H., Chung, D. W., and Davie, E. W. (1993) J. Biol. Chem. 26,8919-8926
  34. Zhang, J. Z., and Redman, C. M. (1994) J. Biol. Chem. 269,652-658 [Abstract/Free Full Text]
  35. Normington, K., Kohno, K., Kozutsumi, Y., Gething, M.-J., and Sambrook, J. (1989) Cell 57,1223-1236 [Medline] [Order article via Infotrieve]
  36. Rose, M. D., Misra, L. M., and Vogel, J. P. (1989) Cell 57,1211-1221 [Medline] [Order article via Infotrieve]
  37. Tachibana, C., and Stevens, T. H. (1992) Mol. Cell. Biol. 12,4601-4611 [Abstract]
  38. d'Enfert, C. L., Wuestehube, J., Lila, T., and Schekman, R. (1991) J. Cell Biol. 114,663-670 [Abstract]
  39. Franzusoff, A., Redding, K., Crossby, J., Fuller, R. S., and Schekman, R. (1991) J. Cell Biol. 112,27-37 [Abstract]
  40. Wilson, D. W., Whiteheart, S. W., Wiedmann, M., Brunner, M., and Rothman, J. E. (1992) J. Cell Biol. 117,531-538 [Abstract]

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