Biochimica et Biophysica Acta (BBA) - Gene Structure and Expression
Regular paperIntracellular fate of fibrinogen Bβ chain expressed in COS cells
Abstract
Full-length fibrinogen Bβ cDNA was subcloned into an expression vector, pBC12BI, and transfected into COS cells. Bβ chain expression was measured by pulse-labelling cells with l-[35S]methionine, immunoprecipitating the Bβ chain with antibody to fibrinogen and separating the nascent radioactive protein by sodium dodecyl sulfate-pholyacrylamine gel electrophoresis (SDS-PAGE). Bβ chain was expressed in transfected COS cells but was not secreted into the medium. Treatment with endoglycosidase H showed that non-secreted Bβ chain contains mannose-rich carbohydrates rather than the complex form of carbohydrate which occurs in plasma fibrinogen and indicates that Bβ chain is not transported to the Golgi apparatus. In transfected COS cells, anitibody to fibrinogen co-immunoprecipitated Bβ chain and 78 kDa immunoglobulin-binding protein (BiP) and antibody to BiP immunoprecipitated BiP and nascent Bβ chains. Non-secreted Bβ chain was degraded intracellularly with a half-life of 5 h by enzymes which were not affected by incubating transfected cells with NH4Cl, which indicates a non-lysosomal pathway of degradation. These studies indicate that Bβ chain by itself does not contain the signal for fibrinogen secretion and that non-secreted Bβ chain is associated with BiP and degraded in the rough endoplasmic reticulum.
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Differential degradation of the three fibrinogen chains by proteasomes. Involvement of Sec61p and cytosolic Hsp70
2001, Archives of Biochemistry and BiophysicsHepG2 cells, which synthesize and secrete fibrinogen, accumulate surplus Aα and γ chains. The nonsecreted fibrinogen chains are degraded both by proteasomes and lysosomes, with unassembled chains primarily degraded by proteasomes and an Aα–γ complex by lysosomes. To further determine the mechanisms by which unassembled fibrinogen chains are degraded, and to explain the pools of Aα and γ chains that occur in HepG2 cells, the association of fibrinogen chains with Sec61β, a component of the translocon, and with a cytosol chaperone, Hsp70, was studied in both HepG2 cells and COS cells expressing single fibrinogen chains. Retrotranslocation from the lumen of the endoplasmic reticulum was shown by treatment with MG132, a proteasome inhibitor. MG132 caused glycosylated Bβ to accumulate on Sec61β in COS cells expressing Bβ and acted similarly with all three fibrinogen chains in HepG2 cells. In HepG2 cells, Bβ was associated with Sec61β ahead of Aα and γ chains, suggesting that pools of Aα and γ chains may be caused by unequal rates of retrotranslocation. In COS cells, retrotranslocation into the cytoplasm was demonstrated by the ATP-sensitive association of ubiquitinylated Aα, Bβ, and γ chains bound to Hsp70. More Aα and γ than Bβ accumulated on Hsp70 of HepG2 cells, consistent with more rapid degradation of Bβ. Overexpression of Hsp70 in HepG2 cells resulted in decreased secretion, but not synthesis, of fibrinogen. Decreased secretion may be due to enhanced degradation of unassembled fibrinogen chains, indicating that proteolysis by proteasomes might regulate both the intracellular pools of fibrinogen chains and fibrinogen secretion.
Endoplasmic Reticulum-Associated Protein degradation: An Unconventional Route to a Familiar Fate
1998, Advances in Molecular and Cell BiologyThis chapter discusses the early studies and general characteristics of endoplasmic reticulum associated protein degradation (ERAD), and reviews the recent advances towards investigating the molecular mechanisms of this essential cellular process. A quality-control process exists to ensure that proteins inserted into the endoplasmic reticulum (ER) are correctly folded, processed, and completely assembled before they exit this compartment for further transport through the secretory pathway. Most proteins that fail to pass this checkpoint are degraded. The process by which these aberrant and unassembled secretory proteins are removed from the ER is referred to as ERAD. A protein-degradation event associated with the ER can selectively remove polypeptides from the secretory pathway. Although the mechanism of ERAD has been obscure, recent reports using both yeast and mammalian cells indicate that ERAD substrates are first targeted to the cytoplasm for degradation. Proteolysis is then catalyzed in the cytosol by the proteasome. Thus, ERAD is comprised of at least three distinct events: (1) recognition of a polypeptide for degradation, (2) efflux of this substrate from the ER to the cytosol, and (3) degradation by the proteasome.
Fibrinogen assembly and secretion: Role of intrachain disulfide loops
1996, Journal of Biological ChemistryHuman 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.
Assembly and secretion of fibrinogen: Involvement of amino-terminal domains in dimer formation
1996, Journal of Biological ChemistryFibrinogen is a dimer with each half-molecule composed of three different chains (Aα, Bβ, γ). 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 Bβ 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 Bβ 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 Aα chain or the first 15 residues of the γ chain, which correspond to BβΔ1-72, did not affect chain assembly and secretion. However, co-expression of both AαΔ1-41 and γΔ1-15 with normal Bβ, 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.
Secretion of biologically active recombinant fibrinogen by yeast
1995, Journal of Biological ChemistryFibrinogen (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 (Aα, 67 kDa; Bβ, 57 kDa; γ, 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 Fα1 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; Matα his3-Δ1 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 × 108 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 Bβ and γ chains, but not Aα, were glycosylated. Glycosylation of Bβ and γ chains was inhibited by treatment of transformed yeast cells with tunicamycin. Intracellular Bβ and γ chains, but not the Aα 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 γ chain dimerization and Aα chain polymer formation.
ORF3 protein of hepatitis E virus interacts with the Bβ chain of fibrinogen resulting in decreased fibrinogen secretion from HuH-7 cells
2009, Journal of General Virology