Nonidentical Polypeptide Chains Research Articles

Investigating the Role of the Alpha-C domain in Fibrin Fiber Mechanics

Fibrin fibers, which form the mesh network structure of blood clots, display a hierarchical design. The fibrin molecule is a rotationally symmetric dimer consisting of two identical subunits, each composed of three nonidentical polypeptide chains: α, β, and γ. The central region (E), where the N-termini of all 6 chains are connected via disulfide bridges, is connected to two outer, beta-sheet rich regions (labeled the βC, and γC and collectively called the D region) by a triple-helix coiled coil. The C-terminal region of the alpha chain contains two distinct parts: the connector region (amino acids 221-391 in human) and the αC domain (a392-610 in human). Within the connector region are a series of tandem amino acid repeats, leading to a largely disordered structure, while the terminal region is thought to contain beta sheet structure. The fibrin monomer polymerizes into a half-staggered protofibril via specific interactions; these protofibrils then laterally aggregate to form a fiber. We recently published a report showing that the extensibility of fibrin fibers was directly related to the length of the alpha-C connector region across various species. Chicken fibers, which have no connector region, fail at strains of 47±23%, while human fibers fail at strains of >200%. To investigate this effect further, we have begun mechanical tests of two recombinant fibrin variants, Aα251, in which the αC connector region of the human fibrin molecule has been truncated at amino acid 251, and a human/chicken hybrid fibrin molecule consisting of the amino acids 1- 197 of the human Aα chain linked to residues 199-487 of chicken Aα chain. Preliminary results indicate that Aα251 has breaking strains similar to normal human fibers (185±46%) while the human/chicken hybrid has breaking strains similar to the chicken fibers (70±35%).

The fibrinogen gamma 10034C > T polymorphism is not associated with Peripheral Arterial Disease

Conversion of fibrinogen to fibrin plays an essential role in hemostasis and results in stabilization of the fibrin clot. Fibrinogen consists of three pairs of non-identical polypeptide chains, encoded by different genes (fibrinogen alpha [FGA], fibrinogen beta [FGB] and fibrinogen gamma [FGG]). A functional single nucleotide polymorphism (SNP) in the 3' untranslated region of the FGG gene (FGG 10034C > T, rs2066865) has been associated with deep venous thrombosis and myocardial infarction. Aim of the present study was to analyze the role of this polymorphism in peripheral arterial disease (PAD). The study was designed as case-control study including 891 patients with documented PAD and 777 control subjects. FGG genotypes were determined by exonuclease (TaqMan) assays. FGG genotype frequencies were not significantly different between PAD patients (CC: 57.3%, CT: 36.7%, TT: 5.8%) and control subjects (CC: 60.9%, CT: 33.5%, TT 5.6%; p = 0.35). In a multivariate logistic regression analysis including age, sex, smoking, diabetes, arterial hypertension and hypercholesterolemia, the FGG 10034 T variant was not significantly associated with the presence of PAD (Odds ratio 1.07, 95% confidence interval 0.84 - 1.37; p = 0.60). The FGG 10034C > T polymorphism was furthermore not associated with age at onset of PAD. We conclude that the thrombophilic FGG 10034 T gene variant does not contribute to the genetic susceptibility to PAD.

Diabetes mellitus as a prothrombotic condition

Diabetes mellitus (DM) is characterized by fasting hyperglycaemia and a high risk of atherothrombotic disorders affecting the coronary, cerebral and peripheral arterial trees. The risk of myocardial infarction (MI) is 3-5 fold higher in Type 2 DM and a DM subject with no history of MI has the same risk as a non-DM subject with a past history of MI. In total around 70% of deaths are vascular with poorer outcomes to both acute events and cardiological interventions. It was proposed that clustering of vascular risk factors (hyperinsulinaemia, dysglycaemia, dyslipidaemia and hypertension) around insulin resistance (IR) accounted for the increase in risk with Type 2 DM. The importance of this became apparent with the recognition that risk clustering occurs in normoglycaemic and impaired glucose tolerance (IGT) subjects with IR, in total around 25% of the population in addition to long-standing Type 1 subjects with renal disease. Evidence indicates that thrombotic risk clustering also occurs in association with IR, suppression of fibrinolysis due to elevated concentrations of the fibrinolytic inhibitor, plasminogen activator inhibitor-1 (PAI-1) is invariable with IR and there is evidence that this is regulated by the effects of triglyceride on the PAI-1 gene promoter. Other studies indicated that prothrombotic risk (coagulation factors VII, XII and fibrinogen) also associates with the IR syndrome. The development of endothelial cell dysfunction with suppression of nitric oxide and prostacyclin synthesis, combined with platelet resistance to the anti-aggregatory effects of these hormones leads to loss of control over platelet activation. In addition, hyperglycaemia and glycation have marked effects on fibrin structure function, generating a clot which has a denser structure, resistant to fibrinolysis. The combination of increased circulating coagulation zymogens, inhibition of fibrinolysis, changes in fibrin structure/function and alterations in platelet reactivity creates a thrombotic risk clustering which underpins the development of cardiovascular disease.

The sialic acid content of fibrinogen decreases during pregnancy and increases in response to fibrate therapy

Fibrinogen is a central protein in blood coagulation and preserves the integrity of the haemovascular system by polymerising to form clots at sites of injury. Human fibrinogen is a 340-kDa glycoprotein, which is synthesized in the liver, and consists of three pairs of non-identical polypeptide chains: Aa, Bh and g [1]. The carbohydrate side-chains, which are N-linked to Asn residue 364 on the Bh chain and Asn residue 52 on the g chain, are biantennary and terminate with sialic acid [2]. Therefore, one fibrinogen molecule could have a maximum of eight sialic acid residues; however, the molecule is partially desialylated in vivo, with on average only 6 mol of sialic acid per mole of fibrinogen [2].

Isolation and characterization of a convulxin-like protein from Crotalus durissus collilineatus venom.

A convulxin (Cvx)-like protein was isolated from Crotalus durissus collilineatus venom by a combination of molecular exclusion and reversed-phase HPLC chromatographies. The molecular mass of the Cvx-like protein in the absence and presence of DTT was 78 kDa and 12-13 kDa, respectively. The Cvx-like protein consisted of two nonidentical polypeptide chains (alpha and beta). The N-terminal amino-acid sequences of the alpha and beta subunits were GLHCPSDWYAYDGHCYKIFNEEMNWED and GFCCPSHWSSYSRYCYKFFSQEMNWEDAEK, respectively, with both subunits having a high content of Glu, Ser, Cys, and Asp. The Cvx-like protein showed high homology with other venom C-type lectins, but had low hemagglutinating activity on intact and trypsinized erythrocytes. The Cvx-like protein stimulated insulin receptor phosphorylation and potentiated insulin secretion from isolated islets in the presence of sub- (2.8 mM) or supra-physiological (16.7 mM) glucose concentrations. These results suggest that the increase in insulin secretion induced by Cvx-like protein may be mediated by a protein tyrosine kinase-dependent pathway and may involve other membrane receptors, such as GP VI or Scr family proteins.

The dysfibrinogenaemias.

The dysfibrinogenaemias.

Structural studies of the glycopeptides of B-chain of cinnamomin--a type II ribosome-inactivating protein by nuclear magnetic resonance.

Cinnamomin is a plant type II ribosome-inactivating protein (RIP) isolated from the seeds of Cinnamomum camphora. It consists of two nonidentical polypeptide chains (A- and B-chain) held together through one disulfide linkage. Its A- and B-chain contain 0.3% and 3.9% sugars respectively. The B-chain of cinnamomin was digested by pronase E and then the liberated glycopeptides were separated from non-glycopeptides by gel filtration chromatography on a Bio-Gel P-4 column. Three crude glycopeptides were obtained by continuing chromatography over anion-exchange resin (AG1-X2) in the buffer of 2% pyridine-acetic acid (pH 8.3) with a polygradient elution system. Through further purification by the gel filtration chromatography and HPLC, three major glycopeptides, GP1, GP2 and GP3 were obtained. Mainly by two-dimensional Nuclear Magnetic Resonance (NMR) including TOCSY, DQF-COSY, NOESY, HMQC and HMBC, their primary structures were analyzed as: Man\balpha1,3Man\balpha1,6(Man\balpha1,3)(Xyl\bbeta1,2)Man\bbeta1,4GlcNAc\bbeta1,4GlcNAc\bbeta1-(Gly-)Asn-Asn-Thr(GP1), Man\balpha1,6(Man\balpha1,3)(Xyl\bbeta1,2)Man\bbeta1,4GlcNAc\bbeta1,4(Fuc\balpha1,3)GlcNAc\bbeta1-Asn-Ala-Thr(GP2),Man\balpha1,6(Man\balpha1,3)Man\balpha1,6(Man\balpha 1,2 Man\balpha1,3)Man\bbeta1,4GlcNAc\bbeta1,4GlcNAc\bbeta1-(Ala-)Asn-Gly-Thr(GP3).

Fibrinogen

Fibrinogen is a blood-borne glycoprotein comprised of three pairs of nonidentical polypeptide chains. Following vascular injury, fibrinogen is cleaved by thrombin to form fibrin which is the most abundant component of blood clots. As well as controlling blood loss at sites of tissue damage, other properties of fibrinogen have recently been discovered. For example, various cleavage products of fibrinogen and fibrin, released during coagulation and fibrinolysis, respectively, regulate cell adhesion and spreading, display vasoconstrictor and chemotactic activities, and are mitogens for several cell types including fibroblasts, endothelial and smooth muscle cells. Current research aims to define the bioactive fibrinogen molecule moieties and cellular receptors involved in these processes. Future studies may provide us with new opportunities to develop agents which are useful in promoting tissue repair or conversely in inhibiting fibrosis in inflammatory and fibroproliferative diseases where endothelial cell damage or chronic leakage of blood proteins is a feature.

Purification, characterization, crystallization and preliminary X-ray diffraction of acuthrombin-B, a thrombin-like enzyme from Agkistrodon acutus venom.

Acuthrombin-B, a thrombin-like enzyme from Agkistrodon acutus venom, has been isolated and purified to homogeneity by ion-exchange chromatography on DEAE-Sepharose, gel filtration on Sephacryl S-100 and fast performance liquid chromatography on DEAE-8HR. The protease is an acid protein (pI 6.0) consisting of two non-identical polypeptide chains (14.4 and 16 kDa) and there is no disulfide bond between the subunits. Its molecular weight is 27 kDa as estimated by gel filtration on Sephacryl S-100. The protease has arginine-esterase activity and hydrolyzes synthetic substrates such as p-toluenesulfonyl arginine methyl ester and alpha-N-benzoyl-L-arginine amide ethyl ester, and shows clotting activity with human fibrinogen, rabbit citrated plasma and human citrated plasma in vitro. The specific activity with human fibrinogen was estimated to be 230 NIH units mg-1. The protease is considered as a serine-type protease and contains metal ion(s) to some extent, as indicated by the fact that its clotting and arginine-esterase activities could be completely inhibited by PMSF and partially inhibited by the chelating agent EDTA, while the thrombin inhibitor heparin had no effect on its clotting activity towards rabbit citrated plasma. Acuthrombin-B crystals with a resolution limit of 2.06 A were obtained by conventional hanging-drop vapour diffusion. The crystals belong to space group P21 with unit-cell parameters a = 34.97, b = 53.58, c = 67.88 A, beta = 98.89 degrees and contain one molecule per asymmetric unit.

The human complement C8G gene, a member of the lipocalin gene family: polymorphisms and mapping to chromosome 9q34.3.

Complement component C8 is a plasma glycoprotein consisting of three nonidentical polypeptide chains (alpha, beta, gamma) which are encoded by three separate genes (C8A, C8B, C8G). The gamma chain whose functional role remains undefined is not related to any other complement protein but is a member of the lipocalins, a family of proteins that bind small hydrophobic ligands. The present report describes the first known polymorphisms for the human C8G gene, namely one polymorphic site in exon 1 (207T/G) and two polymorphic sites in intron 1 (213 + 37G --> A; 213 + 65del3). Specific typing can be performed using simple polymerase chain reaction-based assays. C8G genotyping in eight CEPH reference families demonstrated that C8G is closely linked to a series of marker loci located in the most telomeric region of chromosome 9q. Multipoint analysis placed C8G with 1000:1 support distal to D9S207. C8G is thus located at 9q34.3. Remarkably, this chromosomal region contains at least four other lipocalin genes.

Biochemistry and measurement of fibrinogen.

Fibrinogen is a large heterogeneous family of closely related molecules consisting of three pairs of non-identical polypeptide chains: two A alpha-, two B beta- and two gamma-chains, held together by disulphide bridges. The heterogeneity of fibrinogen is due to heterogeneities in all three chains. Four main types of assay are used to determine fibrinogen:clotting rate (Clauss), clottable protein, precipitation and immunological assays. Heterogeneities may differ from person to person and may affect the apparent fibrinogen concentrations in different assays. A further complicating factor was, until recently, the lack of an international fibrinogen standard. The ratio of Clauss:enzyme immunoassay (EIA) for high + low molecular weight fibrinogen decreases during therapy for acute myocardial infarction and increases again after thrombolytic therapy to above normal values. Furthermore, high molecular weight fibrinogen tends to clot more easily than low molecular weight fibrinogen. This suggests that high molecular weight fibrinogen might be associated with increased thrombotic risk. Fibrinogen assessed by a functional assay (Clauss) alone is strongly associated with ischaemic heart disease. Although not proven, it is conceivable that a fibrinogen with a Clauss:EIA ratio of > 1 has an even stronger association in epidemiological studies.

Purification and Characterization of Salicylhydroxamic Acid Reductase from Rat Liver

Salicylhydroxamic acid reductase, which catalyzes the reduction of salicylhydroxamic acid to salicylamide, was purified from rat liver cytosol. The purification procedure consisted of fractionation with ammonium sulfate, chromatography with Phenyl-Toyopearl 650, DEAE-cellulose, hydroxyapatite, and Sephadex G-200, and chromatofocusing with PBE94. The molecular weight of the enzyme was estimated to be about 140,000 by Sephadex G-200 gel filtration and 152,000 by polyacrylamide gel electrophoresis. The enzyme was dissociated into two different subunits with estimated molecular weights of 41,000 and 32,000, respectively, by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. These facts suggested that the enzyme is a heterotetramer consisting of two pairs of two nonidentical polypeptide chains. The K m value of the enzyme for salicylhydroxamic acid was estimated to be 91.5 or 88.7 μM in the presence of NADH or NADPH, respectively. The isoelectric point of the enzyme is pH 5.4. The enzyme was highly specific for salicylhydroxamic acid, but it also showed some activity with other hydroxamic acids such as nicotinohydroxamic acid and N-hydroxy-2-acetylaminofluorene. The enzyme activity was inhibited by allopurinol, oxipurinol, dicumarol, menadione, p-chloromercuribenzoic acid, sodium arsenite, potassium cyanide, cupric sulfate, and disulfiram, but little inhibition was observed with oxygen.

New Trends in Understanding the Regulation of Red Cell Membrane Skeleton Organization

Publisher Summary This chapter discusses the regulation of red cell membrane skeleton organization. The principal associations responsible for maintaining the integrity of the erythrocyte-membrane skeleton have been known and characterized for some time. The membrane skeleton is an interconnected network of proteins whose major constituents are spectrin, F-actin, band 4.1, and band 4.9. The spectrin molecules that are composed of two nonidentical polypeptide chains (α and β) exist principally in the form of α 2 β 2 tetramers. The protein-denoted band 4.1 plays a central role in membrane skeletal organization. The best understood role of this multifunctional protein is to promote or strengthen the association between spectrin and F-actin, one of the key skeletal interconnections. The proteins of the membrane skeleton that form a flexible two-dimensional lattice laminating the cytoplasmic membrane surface are responsible for the remarkable flexibility and resiliency of the red-cell membrane and for maintaining the shape of the normal red-blood cell. Abnormally shaped or fragile red cells that are characteristic of several types of inherited hemolytic anemias may, in some cases be the result of a genetic defect of one of the major membrane skeletal proteins.

An unusual bovine pancreatic protein exhibiting pH-dependent globule-fibril transformation and unique amino acid sequence.

An unusual hitherto unreported protein, extracted in acid from fresh bovine pancreas, has been purified and characterized biochemically. It precipitates in the neutral pH range in the form of uniform double-helical threads, each strand of which is smooth and of uniform diameter, about 7-8 nm. The threads dissolve to a nonviscous solution below pH 3.6 and above pH 9.4, and they reconstitute reversibly in the pH range in between. The monomer in acid has an apparent molecular weight of 17,800 and consists of two disulfide-linked nonidentical polypeptide chains of different lengths. It is rich in aromatic amino acids, particularly tryptophan. There is no significant content of carbohydrate, fatty acid, or bound phosphate. The amino acid sequences of the first NH2-terminal 48 residues of the A chain and 35 residues of the B chain appear to be unique, differing from all other reported animal proteins, including those of the pancreas. Thus far, a function has not been found.

Characterization of the third component of pig complement and its utilization in a C3b receptor study.

The third component of the pig complement system (C3) was isolated in hemolytically active form and characterized. The C3 component is a beta-globulin with the molecular weight of 191,000 and is composed of 2 non-identical polypeptide chains of Mr 112,000 and 74,000. The isolated C3 can be used for the detection of the C3b receptor on the membranes of heterologous peritoneal macrophages.

The isolation and characterization of a new elastase inhibitor, pre-alpha 2-elastase inhibitor, of the horse.

A new and probably unique elastase inhibitor of horse serum was identified, purified to homogeneity and called pre-alpha 2-elastase inhibitor of the horse. Electrophoretically it migrated immediately in front of the alpha 2 position. Its molecular weight was 188 000 by pore limit polyacrylamide gel electrophoresis and 225 000 by Sephadex G-200 gel filtration. The inhibitor was composed of at least two non-identical polypeptide chains of Mr 68 400 and 87 600. A banding pattern of restricted heterogeneity focused between pH 4.9 and 5.2 was revealed by isoelectric focusing. Of 13 animal, microbial and plant proteinases, horse pre-alpha 2-elastase inhibitor inhibited only pancreatic elastase and trypsin efficiently. Chymotrypsin was inhibited only in traces. No analogy between the elastase inhibitor and the known human serum inhibitors could be found with respect to immunological and biochemical criteria.

Xenopus fibrinogen. Characterization of subunits and hormonal regulation of biosynthesis.

In this paper we describe the purification and characterization of Xenopus plasma fibrinogen and the hormonal factors which regulate synthesis and secretion of fibrinogen in liver parenchymal cells in primary culture. As in other vertebrate species, Xenopus fibrinogen is composed of three nonidentical polypeptide chains, A alpha, B beta, and gamma. In contrast to mammalian fibrinogens, the B beta chain of Xenopus fibrinogen has a higher apparent molecular weight than the A alpha chain. The gamma chain has the lowest molecular weight in the frog protein, as in that of other species. The relatively large size of the frog B beta chain results from the unusually large size of the NH2-terminal B fibrinopeptide, which is released by thrombin cleavage of fibrinogen. Hormonal regulation of fibrinogen biosynthesis was examined using a primary cell culture system. Purified Xenopus liver parenchymal cells, maintained for several weeks in a defined culture medium, gradually decrease the synthesis and secretion of fibrinogen. Sustained production of this protein is dependent upon the addition of a glucocorticoid, dexamethasone, to the culture medium. Fibrinogen production is suppressed if an estrogen, estradiol-17 beta, is added to the culture medium together with dexamethasone and triiodothyronine. The Xenopus system provides new insight into the structure of fibrinogen, the evolution of this protein, and the hormonal factors which regulate its synthesis.

Immunoreactive forms of erythrocyte spectrin and ankyrin in brain.

Polypeptides immunologically related to erythrocyte spectrin and ankyrin have been detected in brain. The cross-reacting proteins include soluble as well as membrane-associated forms. A class of soluble cross-reacting polypeptides have been identified as high molecular mass microtubule-associated proteins (MAPS). MAP1, a group of polypeptides of molecular mass ca . 370 kDa contains a component that cross-reacts with anti-ankyrin IgG. MAP2, a polypeptide of molecular mass 300 kDa cross-reacts with anti-spectrin IgG, with the shared antigenic sites localized to the α chain of spectrin. The functional basis for structural homology between MAP1 and ankyrin may involve association with tubulin, since erythrocyte ankyrin binds to microtubules polymerized from pure brain tubulin. Spectrin did not associate with microtubules, but does have in common with MAP2 the ability to bind to actin (Brenner & Korn 1979; Sattilaro et al . 1981) and the shape of a flexible rod as visualized by rotary shadowing (Shotton et al . 1979; Voter & Erickson 1981). Immunoreactive forms of spectrin and ankyrin are also present in membrane fractions. A homologue of spectrin which constitutes 3% of the total membrane protein has been purified from low ionic strength extracts of membranes. This protein contains two non-identical polypeptide chains of molecular masses of 260 and 265 kDa, binds to F-actin, and displaces binding of erythrocyte spectrin to erythrocyte membranes. The brain protein pas been visualized by rotary shadowing as an extended rod-like molecule 195 nm in length. These studies indicate that the organization of proteins in the membrane-cytoskeleton complex of erythrocytes has direct relevance to other types of cells, and suggest the existence of families of proteins related to spectrin and ankyrin.

F-actin-binding and cross-linking properties of porcine brain fodrin, a spectrin-related molecule.

The axonally transported high molecular weight protein fodrin, known to be present in the cortical cytoplasm of neurones and other cells, has been purified to homogeneity and several of its biochemical properties have been characterized. Fodrin is an F-actin-binding and cross-linking protein inducing actin gels. It is composed of two nonidentical polypeptide chains (Mr = 240,000 and 235,000) which form a tetrameric complex of a molecular weight close to 930,000. The similarity of fodrin with tetrameric erythrocyte spectrin is directly shown by rotary shadowed molecules both alone and in interaction with F-actin. The gelation and cross-linking activity of fodrin is influenced both by ionic strength and pH in a manner similar to other cross-linking factors. These results strengthen previous concepts concerning the existence of spectrin-related molecules in nonerythroid cells and point to a possible related function in the submembranous microfilament organization in nonmuscle cells.

Evidence for two dissimilar polypeptide chains in the beta 2 subunit of hexosaminidase.

The major isoenzymes of human hexosaminidase have the structures alpha beta 2 (hex A) and 2 beta 2 (hex B). In this study, we present evidence that the beta 2 subunit of hex B and hex BA (the form of hex B derived from hex A) is composed of two nonidentical polypeptide chains. We have called these chains beta a and beta b. They have similar molecular weights (25,000) but have pI values that differ by 1 unit. We have used a two-dimensional analytical gel electrophoresis method in combination with peptide mapping to compare the primary sequence structure of the two beta chains. In this method, the polypeptide chains of hex B or hex BA were first separated by isoelectric focusing in 8.5 M urea. The separated chains were subjected to partial proleolytic digestion in the stacking gel of a second NaDodSO4/polyacrylamide gel with subsequent separation of peptides by electrophoresis into the second gel. Partial digestion by protease V8 or papain showed that the beta a and beta b species have distinct primary structures, neither of which was similar to that of the alpha chain. On the basis of these results, we suggest that the beta 2 subunit of hexosaminidase has the structure of beta a beta b. The possibility that the distinct beta chains are encoded by a single gene is discussed in the light of genetic and other data.