Intracellular fate of fibrinogen B beta chain expressed in COS cells

Full-length fibrinogen B beta cDNA was subcloned into an expression vector, pBC12BI, and transfected into COS cells. B beta chain expression was measured by pulse-labelling cells with L-[35S]methionine, immunoprecipitating the B beta chain with antibody to fibrinogen and separating the nascent radioactive protein by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). B beta chain was expressed in transfected COS cells but was not secreted into the medium. Treatment with endoglycosidase H showed that non-secreted B beta chain contains mannose-rich carbohydrates rather than the complex form of carbohydrate which occurs in plasma fibrinogen and indicates that B beta chain is not transported to the Golgi apparatus. In transfected COS cells, antibody to fibrinogen co-immunoprecipitated B beta chain and 78 kDa immunoglobulin-binding protein (BiP) and antibody to BiP immunoprecipitated BiP and nascent B beta chains. Non-secreted B beta 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 B beta chain by itself does not contain the signal for fibrinogen secretion and that non-secreted B beta chain is associated with BiP and degraded in the rough endoplasmic reticulum.     

Identification of B beta chain domains involved in human fibrinogen assembly.

Fibrinogen chains are assembled in a stepwise manner in the rough endoplasmic reticulum prior to secretion of the final six-chain dimeric molecule. Previous studies indicated that the synthesis of B beta may be a rate-limiting factor in the assembly of human fibrinogen. To determine the domains of B beta which interact with the other two component chains of fibrinogen, deletion mutants of B beta were transiently co-expressed, together with A alpha and gamma chains, in COS cells, and fibrinogen assembly and secretion were measured. Deletion of the COOH-terminal half of the B beta chain (amino acids 208-461) did not affect assembly and secretion. Assembly of A alpha, gamma, and B beta also occurred when the first NH2-terminal 72 amino acids of B beta were deleted, but not when 93 amino acids were deleted. This indicates that the B beta domain between amino acids 73 and 93 is necessary for the assembly of the three fibrinogen chains. This domain marks the start of the alpha-helical "coiled-coil" region of fibrinogen.     

Conformational equilibria in the gamma chain COOH terminus of human fibrinogen

The conformations of the gamma chain COOH terminus of intact fibrinogen and various fragments containing this region have been compared by an immunochemical analysis. Location of a major epitope in the sequence gamma 391-405 was successfully predicted from a hydrophobicity profile. An antibody population specific for the native epitope within the gamma 391-405 segment was isolated by immunoadsorption. Between 19.2 and 22.8% of antibodies were obtained from three different antisera, indicating that this region represents one of the major epitopes of native fibrinogen. Anti-gamma 391-405(N) antibodies were used to determine the value of Kconf, the equilibrium constant for the interconversion of the non-native and native conformations of this epitope. The measurements were done using native fibrinogen, fragments D1 and DD, gamma chain, and gamma 391-405. In addition, the effect of 5 M guanidine HCl on the conformation of fragments D1 and DD, which is known to abolish their antipolymerizing activity, was studied. Radioiodinated fibrinogen was used in the determination of Kconf, CI50%, and CIs (quantitative analytical parameters calculated from competitive inhibition radioimmunoassays) by measuring the competition between 125I-fibrinogen and the fibrinogen derivatives under study for binding to the immunochemically purified antibody. The measurements indicated that the epitope is unperturbed by iodination of fibrinogen and that 38.5% of fragment D1, 8.9% of fragment DD, 3.6% of the gamma chain, and less than 0.008% of the gamma 391-405 molecules adopt in aqueous solution the native conformation within the epitope. Denaturation of fragment D1 with 5 M guanidine HCl affected only slightly the conformation of this gamma chain determinant. More significant changes in the conformation were observed when fragment DD was denatured. The results suggest that long-range interactions are necessary for the stabilization of the native structure in the region of fibrinogen that interacts with the antibody and which is in close vicinity to the polymerization site, cross-linking site, and platelet recognition site.     

Conservation of human fibrinogen conformation after cleavage of the B beta chain NH2 terminus

Human fibrinogen exposed to protease III from Crotalus atrox venom is cleaved near the NH2 terminus of the B beta chain yielding a species of Mr 325,000 (Fg325) with impaired thrombin clottability. The derivative was compared with intact fibrinogen in a number of ways to determine whether the functional defect resulted from a conformational change or from the loss of a polymerization site. NH2-terminal amino acid sequencing of isolated A alpha, B beta, and gamma chains showed that Fg325 contained intact A alpha and gamma chains, but differed from fibrinogen by the absence of the first 42 residues of the B beta chain. Fibrinopeptide A was present and was cleaved at the same rate in both fibrinogen and Fg325. The rate and extent of A alpha and gamma cross-linking by factor XIIIa was also indistinguishable. In contrast, the thrombin-catalyzed coagulation of Fg325 was 46% less in extent and 180-fold slower than observed for intact fibrinogen. A conformational comparison of Fg325 and fibrinogen was made using immunochemical and spectroscopic approaches. Antisera specific for different regions of the fibrinogen molecule were used to characterize the epitopes in Fg325. The only significant differences were found in the NH2-terminal region of the B beta chain, probed with antiserum to B beta 1-118. The conformational similarity of Fg325 and fibrinogen was confirmed by the identity of both near and far UV CD spectra of the two proteins. Structural, functional, and immunochemical results imply that cleavage of 42 NH2-terminal residues from the B beta chain is not accompanied by a measurable conformational change. The residues of this B beta chain segment, which are evidently located on the surface of the molecule, in conjunction with the NH2-terminal part of the A alpha chain appear to play an important role in the expression of a fibrin polymerization site.     

Complementary DNA sequence of lamprey fibrinogen beta chain

The cDNA sequence of the beta chain of lamprey fibrinogen has been determined. To that end, an oligonucleotide probe was synthesized that corresponded to an amino acid sequence from the carboxy-terminal region of the lamprey fibrinogen beta chain. The insert actually began with residue 3 of the fibrin beta chain; it ran through to a terminator codon following the carboxy-terminal residue at position 443 and then continued for an additional 606 nucleotides of noncoding sequence to its 3' end. The inferred amino acid sequence was verified by comparison with assorted cyanogen bromide fragments isolated from the beta-chain protein, including two carbohydrate-containing peptides that corresponded to segments containing the carbohydrate-attachment consensus sequence. Overall, the lamprey chain is 49% identical with the beta chain from human fibrinogen. This is the same degree of resemblance as was found for the lamprey and human gamma chains. Moreover, the principal regions of conservation are the same in both the beta and gamma chains. Differences and similarities in the physiological behavior of the two fibrinogens are assessed in terms of the observed amino acid replacements.     

Amino acid sequence of the beta chain of human fibrinogen.

The beta chain of human fibrinogen contains 461 amino acid residues, 15 of which are methionines. The calculated molecular weight, independent of a single carbohydrate cluster, is 52 230. In this regard, we have isolated and characterized all 16 cyanogen bromide fragments. In one case (CNI), we have concentrated on a disputed portion of a previously reported fragment. The arrangement of the cyanogen bromide peptides was deduced by the use of overlap fragments obtained from the tryptic digestion of modified and unmodified beta-chains and from digestions with staphylococcal protease, as well as by considerations involving the plasmic digestion products of fibrinogen. In one case two adjacent fragments were aligned by homology with the corresponding segments of the gamma chain. The homology of the beta chain with the gamma chain is especially strong over the course of the carboxy-terminal two-thirds of the sequence. Neither of these chains appears to be homologous with the alpha chain in these regions. With a few minor exceptions, the sequence reported in this article is in agreement with data reported by other groups in Stockholm and Munich.     

Isolation and identification of N-terminal fragments of the A-alpha chain of bovine fibrinogen

A method is suggested to obtain and purify N-end fragments of A alpha-chain from bovine fibrinogen. To identify the N-end chain fragments the fluorescent properties of tryptophan and tyrosine composing them as well as hydrolysis by ancistron H, a thrombin-like enzyme of the snake venom, have been used for the first time.     

The beta chain of chicken fibrinogen contains an atypical thrombin cleavage site

A cDNA corresponding to almost the entire coding region of the mRNA for the beta chain of chicken fibrinogen was sequenced. At the protein level, significant homology to the beta subunits of other vertebrate fibrinogens was found, with the highest degree of amino acid identity localized in the C-terminal region. In general, features conserved in the fibrinogens from other species also characterize the chicken sequence, including the cysteine motifs bordering an alpha-helical permissive region of fixed length and a single glycosylation site in the C-terminal region. However, the site of thrombin-catalyzed cleavage, which in other species consists of an Arg-Gly peptide bond, is instead an Arg-Ala bond in the chicken beta chain. The Ala was confirmed directly from a sequencing analysis of the purified beta chain of chicken fibrin. This finding may explain the observed slow clotting time of chicken fibrinogen relative to that of other species.     

Primary structure of human fibrinogen and fibrin. Structural studies on NH2-terminal part of B beta chain.

The cyanogen bromide fragment, N-DSK, containing the NH2-terminal portions of the three chains of fibrinogen, was found to exist in dimeric and polymeric forms. These different forms gave rise to identical chain fragments on reduction and alkylation. The B beta chain of N-DSK from fibrinogen and the beta chain of N-DSK from fibrin were isolated and characterized. The B beta chain fragment has a blocked NH2-terminal residue, and fibrinopeptide B is released on digestion with thrombin. The beta chain fragment has glycine as NH2-terminal residue. The molecular weight of the B beta chain fragment is 12200 as determined by ultracentrifugal analysis. Gel electrophoresis in sodium dodecyl sulphate gave the molecular weights of 14000 and 13000 for the B beta chain and beta chain fragments, respectively. The NH2-terminal B beta chain fragment consists of 118 amino acid residues and the beta chain fragment of 104 residues. The amino acid sequence of beta chain fragment is identical to B beta chain fragment except for the fibrinopeptide B portion. The isolation of a B beta-related fragment (B beta +), with a molecular weight of 30000, is also reported. The presence of B beta + was explained on the basis of incomplete cleavage at the Met-118 residue during treatment with cyanogen bromide. Some functional aspects of the B beta chain fragment are discussed.     

A 130-kDa protein on endothelial cells binds to amino acids 15-42 of the B beta chain of fibrinogen.

Factors which stimulate the release of von Willebrand factor (vWf) from endothelial cell Weibel-Palade bodies and which induce the expression of the leukocyte-binding adhesion molecule P-selectin (PADGEM, GMP-140, CD62) on the endothelial cell surface remain incompletely characterized. Fibrin but not fibrinogen is a potent stimulus for the release of stored von Willebrand factor from endothelial cells. Removal of fibrinopeptides A and B from fibrinogen occurs during the formation of fibrin, and the removal of fibrinopeptide B is a requirement for fibrin to induce vWf secretion. The cleavage of fibrinopeptide A by reptilase enzyme forms a fibrin gel yet it is incapable of stimulating Weibel-Palade body degranulation. As a consequence of removing fibrinopeptide B, B beta 15-42 becomes the new NH2 terminus of the beta chain of fibrin. We have shown that the peptide B beta 15-42 in solution inhibits the release of vWf stimulated by fibrin. In addition, B beta 15-42 coupled to ovalbumin supports the binding and spreading of endothelial cells, while a scrambled form of this peptide coupled to the same carrier does not. We investigated whether these determinants near the amino terminus of the beta chain of fibrin bind to a specific protein on the surface of endothelial cells. A 130-kDa protein was isolated from surface-labeled human umbilical vein endothelial cells by specific binding to B beta 15-42 immobilized on Sepharose. This glycoprotein was eluted with the B beta 15-42 peptide in solution but not with the scrambled form of this peptide. The fibrin-derived peptides B beta 19-26 and B beta 37-56-cysteine were also incapable of eluting the 130-kDa protein bound to immobilized B beta 15-42 as were the arginine-glycine-aspartic acid-serine RGDS tetrapeptide and EDTA. The 130-kDa protein is recognized neither by antibodies to the known integrins found on endothelial cells nor by antibodies to CD31 (endoCAM, PECAM-1), a member of the immunoglobulin family of receptors found on endothelial cells. The beta chain of fibrin thus contains a sequence near its amino terminus which specifically binds to what is likely a novel endothelial cell surface protein. This glycoprotein may promote endothelial cell adhesion to fibrin during the wound healing process and is a candidate for a receptor involved in fibrin-mediated release of Weibel-Palade bodies from endothelial cells.     

Atomistic simulations of the effects of polyglutamine chain length and solvent quality on conformational equilibria and spontaneous homodimerization

Aggregation of expanded polyglutamine tracts is associated with nine different neurodegenerative diseases, including Huntington's disease. Experiments and computer simulations have demonstrated that monomeric forms of polyglutamine molecules sample heterogeneous sets of collapsed structures in water. The current work focuses on a mechanistic characterization of polyglutamine homodimerization as a function of chain length and temperature. These studies were carried out using molecular simulations based on a recently developed continuum solvation model that was designed for studying conformational and binding equilibria of intrinsically disordered molecules such as polyglutamine systems. The main results are as follows: Polyglutamine molecules form disordered, collapsed globules in aqueous solution. These molecules spontaneously associate at conditions approaching those of typical in vitro experiments for chains of length N ≥ 15. The spontaneity of these homotypic associations increases with increasing chain length and decreases with increasing temperature. Similar and generic driving forces govern both collapse and spontaneous homodimerization of polyglutamine in aqueous milieus. Collapse and dimerization maximize self-interactions and reduce the interface between polyglutamine molecules and the surrounding solvent. Other than these generic considerations, there do not appear to be any specific structural requirements for either chain collapse or chain dimerization; that is, both collapse and dimerization are nonspecific in that disordered globules form disordered dimers. In fact, it is shown that the driving force for intermolecular associations is governed by spontaneous conformational fluctuations within monomeric polyglutamine. These results suggest that polyglutamine aggregation is unlikely to follow a homogeneous nucleation mechanism with the monomer as the critical nucleus. Instead, the results support the formation of disordered, non-β-sheet-like soluble molten oligomers as early intermediates—a proposal that is congruent with recent experimental data.     

The effect of thrombomodulin on the cleavage of fibrinogen and fibrinogen fragments by thrombin

Thrombomodulin acts as a linear competitive inhibitor of thrombin with respect to the substrate fibrinogen. In the present study the effect of thrombomodulin on the activity of thrombin with fragments of the A alpha and B beta chain of fibrinogen has been examined. The cleavage of fibrinopeptide A from the N-terminal disulphide knot, fragment 1-44 and fragment 1-51 of the A alpha chain was inhibited by thrombomodulin. The average value for the inhibition constant obtained with these substrates was 0.83 +/- 0.09 nM, which was in good agreement with the values obtained previously for the inhibition of thrombin by thrombomodulin with native fibrinogen as the substrate [Hofsteenge, J., Taguchi, H. & Stone, S. R. (1986) Biochem. J. 237, 243-251]. In contrast, the cleavage of fibrinopeptide A from fragment 1-23 and fragment 1-29 of the A alpha chain was not affected by thrombomodulin. Although the cleavage of the B beta chain in the intact fibrinogen molecule was inhibited by thrombomodulin [Hofsteenge, J., Taguchi, H. & Stone, S. R. (1986) Biochem. J. 237, 243-251], the release of fibrinopeptide B from the N-terminal disulphide knot and the N-terminal 118-residue fragment of the B beta chain was not inhibited by thrombomodulin. In addition, we determined the second-order rate constants of cleavage of these substrates using human thrombin. Fragments of the A alpha chain whose cleavage was inhibited by thrombomodulin were found to have values for kcat/Km that were within one order of magnitude of that for the native fibrinogen, whereas those for A alpha chain fragments whose cleavage was not inhibited by thrombomodulin were found to be more than two orders of magnitudes lower. From these results we conclude that only a relatively small portion of the A alpha chain of the fibrinogen molecule is responsible for the specific binding to thrombin that is affected by thrombomodulin. Moreover, residues 30-44 of the A alpha chain play an important role in this thrombin-fibrinogen interaction.     

Immunochemical determination of human lactoferrin

The immunochemically pure preparation of lactoferrin was isolated from human colostrum and used for the immunization of animals with a view of obtaining antiserum, and also as a reference preparation for the determination of the content of lactoferrin in the standard. The monospecific antiserum to lactoferrin, obtained as the result of this procedure, was used for the determination of the content of lactoferrin in samples of human milk by the method of radial immunodiffusion. Through the content of lactoferrin in human milk showed considerable fluctuations, its level essentially decreased on the second week of lactation. In cases of the microbial contamination of milk the tendency towards an increase in the content of lactoferrin was observed irrespective of the time of lactation.     

Immunochemical studies of fragments of bovine serum albumin.

Antigenic properties of 14 fragments of bovine albumin were measured using antisera to albumin and to two of its fragments. All seven fragments larger than 21,000 daltons formed immune precipitates. Although immune precipitates were not formed with smaller fragments, inhibition tests indicated the presence of antigenic sites on several of these fragments as well. The results predict the occurrence of six or more antigenic determinants and allow assignment of their positions in the parent molecule. These sites are distributed along the entire protein chain, with the sites of greatest antibody affinity situated in the COOH-terminal region. Evidence is presented that some sites are homologous, reacting with the same populations of antibodies, and that other sites are unique, binding to an exclusive population of antibodies.     

Polymerization site in the beta chain of fibrin: mapping of the B beta 1-55 sequence

The formation of a fibrin clot occurs through binding of putative complementary sites, called fibrin polymerization sites, located in the NH2- and COOH-terminal domains of fibrin monomer molecules. In this study, we have investigated the structure of the NH2-terminal fibrin polymerization site by using fibrinogen-derived peptides and fragments. Fibrinogen was digested with Crotalus atrox protease III, to two major molecular species: a Mr 325,000 derivative (Fg325) and a peptide of Mr 5000. The peptide and its thrombin-cleavage product were purified by ion-exchange and reverse-phase HPLC; the authenticity of the B beta 1-42 and beta 15-42 peptides, respectively, was confirmed by amino acid sequencing. Since Fg325 had decreased thrombin coagulability, we addressed the question of whether the peptide B beta 1-42 contained a fibrin polymerization site. In order to identify and map the site, the peptides B beta 1-42 and beta 15-42 were tested for their ability to inhibit fibrin monomer polymerization. In addition the following peptides prepared by chemical synthesis were also tested: beta 15-18, beta 15-26, beta 24-42, beta 40-54, beta 50-55, and alpha 17-19-Pro. While B beta 1-42 had no inhibitory activity, the peptide devoid of fibrinopeptide B, beta 15-42, was a strong inhibitor. The peptides beta 15-18, beta 15-26, and beta 15-42 decreased the rate of fibrin polymerization by 50% at a molar excess of the peptide to fibrin monomer of 500, 430, and 50, respectively. The peptides beta 24-42, beta 40-54, and beta 50-55 were inactive.(ABSTRACT TRUNCATED AT 250 WORDS)     

Specific binding of integrin alpha v beta 3 to the fibrinogen gamma and alpha E chain C-terminal domains

Integrin alpha v beta 3, a widely distributed fibrinogen receptor, recognizes the RGD572-574 motif in the alpha chain of human fibrinogen. However, this motif is not conserved in other species, nor is it required for alpha v beta 3-mediated fibrin clot retraction, suggesting that fibrinogen may have other alpha v beta 3 binding sites. Fibrinogen has conserved C-terminal domains in its alpha (E variant), beta, and gamma chains (designated alpha EC, beta C, and gamma C, respectively), but their function in cell adhesion is not known, except that alpha IIb beta 3, a platelet fibrinogen receptor, binds to the gamma C HHLGGAKQAGDV400-411 sequence. Here we used mammalian cells expressing recombinant alpha v beta 3 to show that recombinant alpha EC and gamma C domains expressed in bacteria specifically bind to alpha v beta 3. Interaction between alpha v beta 3 and gamma C or alpha EC is blocked by LM609, a function-blocking anti-alpha v beta 3 mAb, and by RGD peptides. alpha v beta 3 does not require the HHLGGAKQAGDV400-411 sequence of gamma C for binding, and alpha EC does not have such a sequence, indicating that the alpha v beta 3 binding sites are distinct from those of alpha IIb beta 3. A small fragment of gamma C (residues 148-226) supports alpha v beta 3 adhesion, suggesting that an alpha v beta 3 binding site is located within the gamma chain 148-226 region. We have reported that the CYDMKTTC sequence of beta 3 is responsible for the ligand specificity of alpha v beta 3. gamma C and alpha EC do not bind to wild-type alpha v beta 1, but do bind to the alpha v beta 1 mutant (alpha v beta 1-3-1), in which the CYDMKTTC sequence of beta 3 is substituted for the corresponding beta 1 sequence CTSEQNC. This suggests that gamma C and alpha EC contain determinants for fibrinogen's specificity to alpha v beta 3. These results suggest that fibrinogen has potentially significant novel alpha v beta 3 binding sites in gamma C and alpha EC.     

Immunochemical determination of the cellular content of polypeptide chain release factor RF3 in Escherichia coli

Polypeptide chain termination in Escherchia coli is known to require two codon specific release factors. RF1 and RF2. A third factor, RF3, has been described to stimulate the termination. Earlier investigations have estimated the cellular content of factors RF1 and RF2. Two different immunological techniques for measuring the amount of RF3 per cell in crude E coli cell extracts are reported here, using a sensitive immunoblotting method and a sandwich assay by ELISA. Monoclonal murine antibodies and polyclonal rabbit antibodies were raised against extensively purified recombinant E coli RF3. The immunoblotting involves a specific monoclonal antibody (mAb), biotinylated second antibody and finally radioactive iodinated streptavidin. In the sandwich assay polyclonal antibodies are immobilised on a polystyrene surface before addition of crude cell extract: a specific mAb serves as primary antibody and an HRP-labelled anti-mouse Ig as secondary antibody. Both methods are accurate and rapid to perform. The number of RF3 molecules per cell in exponentially growing E coli cells was found to vary considerably according to the K12 strain examined and depended on the culture medium (from 20 to 500 molecules per cell), faster growth being positively correlated with the number of RF3 molecules per cell.