Two-chain Molecule Research Articles

A Fresh Look at the Structure, Regulation, and Functions of Fodrin.

Fodrin and its erythroid cell-specific isoform spectrin are actin-associated fibrous proteins that play crucial roles in the maintenance of structural integrity in mammalian cells, which is necessary for proper cell function. Normal cell morphology is altered in diseases such as various cancers and certain neuronal disorders. Fodrin and spectrin are two-chain (αβ) molecules that are encoded by paralogous genes and share many features but also demonstrate certain differences. Fodrin (in humans, typically a heterodimer of the products of the SPTAN1 and SPTBN1 genes) is expressed in nearly all cell types and is especially abundant in neuronal tissues, whereas spectrin (in humans, a heterodimer of the products of the SPTA1 and SPTB1 genes) is expressed almost exclusively in erythrocytes. To fulfill a role in such a variety of different cell types, it was anticipated that fodrin would need to be a more versatile scaffold than spectrin. Indeed, as summarized here, domains unique to fodrin and its regulation by Ca2+, calmodulin, and a variety of posttranslational modifications (PTMs) endow fodrin with additional specific functions. However, how fodrin structural variations and misregulated PTMs may contribute to the etiology of various cancers and neurodegenerative diseases needs to be further investigated.

Single-Chain Recombinant Factor VIII for Hemophilia

Factor VIII is released from endothelial cells as a two-chain molecule that is stabilized by the von Willebrand factor. In hemophilia patients, better

Comparative in vitro and in vivo assessment of toxin neutralization by anti-tetanus toxin monoclonal antibodies

Tetanus is caused by the tetanus neurotoxin (TeNT), a 150 kDa single polypeptide molecule which is cleaved into an active two-chain molecule composed of a 50 kDa N-terminal light (L) and a 100 kDa C-terminal heavy (H) chains. Recently, extensive effort has focused on characterization of TeNT binding receptors and toxin neutralization by monoclonal antibodies (mAbs). Toxin binding inhibition and neutralization is routinely assessed either in vitro by the ganglioside GT1b binding inhibition assay or in vivo using an animal model. These two assay systems have never been compared. In the present study, we report characterization of eleven mAbs against different parts of TeNT. The toxin inhibitory and neutralization activity of the mAbs was assessed in vitro and in vivo respectively. Our data demonstrated that seven mAbs bind to fragment C of the heavy chain, two mAbs react with the light chain, one mAb recognizes both chains and one mAb reacts with neither light chain nor fragment C. Six fragment C specific mAbs were able to inhibit TeNT binding to GT1b ganglioside in vitro but three failed to neutralize the toxin in vivo. One in vitro inhibitory mAb (1F3E3) was found to synergize with the in vivo neutralizing mAbs to reduce toxin lethal activity in vivo. Sequencing of the immunoglobulin heavy and light chain variable region genes revealed that the three in vivo neutralizing mAbs were derived from a common origin. Altogether, our data suggests that fragment C specific mAbs contribute to toxin neutralization in both systems, though some of the GT1b binding inhibitory mAbs may not be able to neutralize TeNT in vivo.

Roles of CUB and LDL receptor class A domain repeats of a transmembrane serine protease matriptase in its zymogen activation

Matriptase is a type II transmembrane serine protease containing two complement proteases C1r/C1s-urchin embryonic growth factor-bone morphogenetic protein domains (CUB repeat) and four low-density lipoprotein receptor class A domains (LDLRA repeat). The single-chain zymogen of matriptase has been found to exhibit substantial protease activity, possibly causing its own activation (i.e. conversion to a disulfide-linked two-chain fully active form), although the activation seems to be mediated predominantly by two-chain molecules. Our aim was to assess the roles of CUB and LDLRA repeats in zymogen activation. Transient expression studies of soluble truncated constructs of recombinant matriptase in COS-1 cells showed that the CUB repeat had an inhibitory effect on zymogen activation, possibly because it facilitated the interaction of two-chain molecules with a matriptase inhibitor, hepatocyte growth factor activator inhibitor type-1. By contrast, the LDLRA repeat had a promoting effect on zymogen activation. The effect of the LDLRA repeat seems to reflect its ability to increase zymogen activity. The proteolytic activities were higher in pseudozymogen forms of recombinant matriptase containing the LDLRA repeat than in a pseudozymogen without the repeat. Our findings provide new insights into the roles of these non-catalytic domains in the generation of active matriptase.

An analytical approximation for the orientation-dependent excluded volume of tangent hard sphere chains of arbitrary chain length and flexibility

Onsager-like theories are commonly used to describe the phase behavior of nematic (only orientationally ordered) liquid crystals. A key ingredient in such theories is the orientation-dependent excluded volume of two molecules. Although for hard convex molecular models this is generally known in analytical form, for more realistic molecular models that incorporate intramolecular flexibility, one has to rely on approximations or on computationally expensive Monte Carlo techniques. In this work, we provide a general correlation for the excluded volume of tangent hard-sphere chains of arbitrary chain length and flexibility. The flexibility is introduced by means of the rod-coil model. The resulting correlation is of simple analytical form and accurately covers a wide range of pure component excluded volume data obtained from Monte Carlo simulations of two-chain molecules. The extension to mixtures follows naturally by applying simple combining rules for the parameters involved. The results for mixtures are also in good agreement with data from Monte Carlo simulations. We have expressed the excluded volume as a second order power series in sin (γ), where γ is the angle between the molecular axes. Such a representation is appealing since the solution of the Onsager Helmholtz energy functional usually involves an expansion of the excluded volume in Legendre coefficients. Both for pure components and mixtures, the correlation reduces to an exact expression in the limit of completely linear chains. The expression for mixtures, as derived in this work, is thereby an exact extension of the pure component result of Williamson and Jackson [Mol. Phys. 86, 819-836 (1995)].

Isolation, characterization and cDNA sequencing of acrosin from turkey spermatozoa

Acrosin (EC 3.4.21.10) is serine proteinase localized in the sperm acrosome and considered to play an essential role in fertilization. In contrast to mammalian, there are only limited data concerning avian acrosin, mostly focused on the characterization of mature enzyme. In the present study, acrosin was isolated from turkey spermatozoa using gel filtration in the presence of 4 M urea at acidic pH. N-terminal Edman sequencing allowed the identification of the first 26 N-terminal amino acids: VVGGTEALHG SWPWIVSIQNPRFAGT. This sequence was used to construct primers and obtain a cDNA sequence from the testis. The amino acid sequence deduced from the cDNA shows that turkey acrosin is initially synthesized as prepro-protein with 19-residue signal peptide. This signal sequence is followed by a 327-residue sequence corresponding to the acrosin zymogen. Turkey proacrosin does not contain a proline-rich segment at the C-terminal portion. Mature turkey acrosin is a two-chain molecule consisting of light and heavy chains and was found to be glycoprotein. The proacrosin/acrosin system exists in turkey spermatozoa and this system can be activated similarly to that of mammals. The high value of association constant strongly suggests that acrosin activity in turkeys can be controlled by a seminal plasma Kazal inhibitor under physiological conditions.

The “Hot Wires” of the Relaxin-Like Factor (Insl3)

The discovery of the total separation of receptor-binding and signal-generating regions of the relaxin-like factor (RLF) has provided an opportunity to investigate the mechanism of transmembrane signaling. The receptor-binding residues of RLF are in the B chain of the two-chain molecule and extend from the midregion of the central helix to the tryptophan in position B27. The signal initiation site resides in two residues before the N-terminal cysteine in each chain. For optimal signaling the RLF requires five L-alpha-amino acids preceding cysteine A10, whereas the B chain requires only three. The nature of the side chains of these amino acids is not critical for the signaling function. Heuristic arguments lead us to suggest that the peptide bond is the signal-generating feature of RLF and possibly of other peptide hormones.

Phenotypic Correction of Hemophilia A by Ectopic Expression of Activated Factor VII in Platelets

Platelets are receiving much attention as novel target cells to secrete a coagulation factor for hemophilia gene therapy. In order to extend the application of platelet-directed gene therapy, we examined whether ectopic expression of activated factor VII (FVIIa) in platelets would result in an efficient bypass therapy to induce sufficient thrombin generation on platelet surfaces in mice with hemophilia A. Transduction of bone marrow cells with a simian immunodeficiency virus (SIV)-based lentiviral vector harboring the platelet-specific GPIb alpha promoter resulted in efficient transgene expression in platelets. FVIIa antigen was expressed in platelets by this SIV system; FVII transgene products were found to localize in the cytoplasm and translocate toward the sub-membrane zone and cell surface after activation. Although FVII antigen levels in platelets did not reach the therapeutic levels seen with FVIIa infusion therapy, whole-blood coagulation, as assessed by thromboelastography, was significantly improved in mice with hemophilia A. Further, we observed correction of the bleeding phenotype in mice with hemophilia A after transplantation, even in the presence of FVIII-neutralizing antibodies. Our results demonstrate that FVIIa-expressing platelets can strengthen hemostatic function and may be useful in treating hemophilia and other inherited bleeding disorders. These findings are comparable to the proven therapeutic effects of FVIIa infusion.

Conserved Asp-137 Imparts Flexibility to Tropomyosin and Affects Function

Tropomyosin (Tm) is an alpha-helical coiled-coil that controls muscle contraction by sterically regulating the myosin-actin interaction. Tm moves between three states on F-actin as either a uniform or a non-uniform semi-flexible rod. Tm is stabilized by hydrophobic residues in the "a" and "d" positions of the heptad repeat. The highly conserved Asp-137 is unusual in that it introduces a negative charge on each chain in a position typically occupied by hydrophobic residues. The occurrence of two charged residues in the hydrophobic region is expected to destabilize the region and impart flexibility. To determine whether this region is unstable, we have substituted hydrophobic Leu for Asp-137 and studied changes in Tm susceptibility to limited proteolysis by trypsin and changes in regulation. We found that native and Tm controls that contain Asp-137 were readily cleaved at Arg-133 with t 1/2 of 5 min. In contrast, the Leu-137 mutant was not cleaved under the same conditions. Actin stabilized Tm, causing a 10-fold reduction in the rate of cleavage at Arg-133. The actin-myosin subfragment S1 ATPase activity was greater for the Leu mutant compared with controls in the absence of troponin and in the presence of troponin and Ca2+. We conclude that the highly conserved Asp-137 destabilizes the middle of Tm, resulting in a more flexible region that is important for the cooperative activation of the thin filament by myosin. We thus have shown a link between the dynamic properties of Tm and its function.

Structure of Compstatin in Complex with Complement Component C3c Reveals a New Mechanism of Complement Inhibition

Undesired complement activation is a major cause of tissue injury in various pathological conditions and contributes to several immune complex diseases. Compstatin, a 13-residue peptide, is an effective inhibitor of the activation of complement component C3 and thus blocks a central and crucial step in the complement cascade. The precise binding site on C3, the structure in the bound form, and the exact mode of action of compstatin are unknown. Here we present the crystal structure of compstatin in complex with C3c, a major proteolytic fragment of C3. The structure reveals that the compstatin-binding site is formed by the macroglobulin (MG) domains 4 and 5. This binding site is part of the structurally stable MG-ring formed by domains MG 1-6 and is far away from any other known binding site on C3. Compstatin does not alter the conformation of C3c, whereas compstatin itself undergoes a large conformational change upon binding. We propose a model in which compstatin sterically hinders the access of the substrate C3 to the convertase complexes, thus blocking complement activation and amplification. These insights are instrumental for further development of compstatin as a potential therapeutic.

Substitution of the Gla Domain in Factor X with That of Protein C Impairs Its Interaction with Factor VIIa/Tissue Factor

We previously reported that the first epidermal growth factor-like (EGF1) domain in factor X (FX) or factor IX (FIX) plays an important role in the factor VIIa/tissue factor (FVIIa/TF)-induced coagulation. To assess the role of gamma-carboxyglutamic acid (Gla) domains of FX and FIX in FVIIa/TF induced coagulation, we studied four new and two previously described replacement mutants: FX(PCGla) and FIX(PCGla) (Gla domain replaced with that of protein C), FX(PCEGF1) and FIX(PCEGF1) (EGF1 domain replaced with that of protein C), as well as FX(PCGla/EGF1) and FIX(PCGla/EGF1) (both Gla and EGF1 domains replaced with those of protein C). FVIIa/TF activation of each FX mutant and the corresponding reciprocal activation of FVII/TF by each FXa mutant were impaired. In contrast, FVIIa/TF activation of FIX(PCGla) was minimally affected, and the reciprocal activation of FVII/TF by FIXa(PCGla) was normal; however, both reactions were impaired for the FIX(PCEGF1) and FIX(PCGla/EGF1) mutants. Predictably, FXIa activation of FIX(PCEGF1) was normal, whereas it was impaired for the FIX(PCGla) and FIX(PCGla/EGF1) mutants. Molecular models reveal that alternate interactions exist for the Gla domain of protein C such that it is comparable with FIX but not FX in its binding to FVIIa/TF. Further, additional interactions exist for the EGF1 domain of FX, which are not possible for FIX. Importantly, a seven-residue insertion in the EGF1 domain of protein C prevents its interaction with FVIIa/TF. Cumulatively, our data provide a molecular framework demonstrating that the Gla and EGF1 domains of FX interact more strongly with FVIIa/TF than the corresponding domains in FIX.

Unfolding, Aggregation, and Seeded Amyloid Formation of Lysine-58-Cleaved β2-Microglobulin

Beta(2)-microglobulin (beta(2)m) is the amyloidogenic protein in dialysis-related amyloidosis, but the mechanisms underlying beta(2)m fibrillogenesis in vivo are largely unknown. We study a structural variant of beta(2)m that has been linked to cancer and inflammation and may be present in the circulation of dialysis patients. This beta(2)m variant, DeltaK58-beta(2)m, is a disulfide-linked two-chain molecule consisting of amino acid residues 1-57 and 59-99 of intact beta(2)m, and we here demonstrate and characterize its decreased conformational stability as compared to wild-type (wt) beta(2)m. Using amide hydrogen/deuterium exchange monitored by mass spectrometry, we show that DeltaK58-beta(2)m has increased unfolding rates compared to wt-beta(2)m and that unfolding is highly temperature dependent. The unfolding rate is 1 order of magnitude faster in DeltaK58-beta(2)m than in wt-beta(2)m, and at 37 degrees C the half-time for unfolding is more than 170-fold faster than at 15 degrees C. Conformational changes are also reflected by a very prominent Congo red binding of DeltaK58-beta(2)m at 37 degrees C, by the evolution of thioflavin T fluorescence, and by changes in intrinsic fluorescence. After a few days at 37 degrees C, in contrast to wt-beta(2)m, DeltaK58-beta(2)m forms well-defined high molecular weight aggregates that are detected by size-exclusion chromatography. Atomic force microscopy after seeding with amyloid-beta(2)m fibrils under conditions that induce minimal fibrillation in wt-beta(2)m shows extensive amyloid fibrillation in DeltaK58-beta(2)m samples. The results highlight the instability and amyloidogenicity under near physiological conditions of a slightly modified beta(2)m variant generated by limited proteolysis and illustrate stages of amyloid formation from early conformational variants to overt fibrillation.

Factor V Activation and Inactivation by Venom Proteases

Blood coagulation factor V is a single-chain glycoprotein with M_r = 330,000 which plays an important role in the procoagulant and anticoagulant pathways. Thrombin activates factor V into factor Va, a two-chain molecule which is composed of a heavy (M_r = 105,000) and a light chain (M_r = 71,000/74,000). Factor Va accelerates factor Xa-catalysed prothrombin activation more than 1,000-fold and under physiological conditions the cofactor activity of factor Va in prothrombin activation is down-regulated by activated protein C. Factor V can also be activated by a wide variety of snake venoms (e.g. from Vipera species, Naja naja oxiana, Bothrops atrox) and by proteases present in the bristles of a South American caterpillar (Lonomia achelous). Some venoms, notably of Vipera lebetina turanica and Lonomia achelous, contain proteases that are able to inactivate factor V or factor Va. Venom factor V activators are excellent tools in studying the structure-function relationship of factor V(a) and they are also used in diagnostic tests for quantification of plasma factor V levels and for the screening of defects in the protein C pathway. In this review, the structural and functional properties of animal venom factor V activators and inactivators is described.

Construction and characterization of a recombinant chimeric plasminogen activator consisting of a fibrin peptide and a low molecular mass single-chain urokinase

A recombinant chimeric plasminogen activator (fβ/scuPA-32k), with a fibrin β-chain peptide (comprising Gly15 through Arg 42) linked to the N-terminal of a low molecular mass (32 kDa) single-chain urokinase (scuPA-32k, comprising Leu144 through Leu 411) via a 50 amino acid linker sequence, was produced by expression the corresponding chimeric cDNA in Escherichia coli cells. After refolding in vitro, the chimeric protein was purified to homogeneity by zinc chelate-Sepharose chromatography, Sephacryl S200 chromatography and benzamidine-Sepharose chromatography in sequence. The apparent molecular mass was 36 kDa shown by SDS-PAGE analysis. The special activity was 87 000 IU/mg detected by fibrin plate determination. Fβ/scuPA-32k could directly activate plasminogen following Michaelis-Menten kinetics with K m = 0.52 μM and k 2 = 0.0024 s -1. Mediated by plasmin, the single-chain molecule could be converted to the active two-chain molecule. The chimeric protein had 3.3 times higher fibrin affinity than scuPA-32k in the fibrin concentration of 3.2 mg/mL, while the chimeric protein inhibited the fibrin clotting and platelet aggregation. Fβ/scuPA-32k showed a higher thrombolytic potency in vitro plasma clot lysis than scuPA-32k and depleted less fibrinogen in plasma. These results showed that the chimeric protein had not only higher fibrinolytic activity but also anti-thrombus activity. Further evaluation of the thrombolytic potential in appropriate animal models is required.

Characterization of a recombinant chimeric plasminogen activator with enhanced fibrin binding

A recombinant chimeric plasminogen activator (GHRP-scu-PA-32K), consisting of the tetrapeptide Gly-His-Arg-Pro fused to the N-terminus of the low-molecular single-chain urokinase-type plasminogen activator (Leu144–Leu411), was produced by expression in CHO cells. The stable expression cell line was selected for large-scale expression. The product was purified by antibody–Sepharose affinity chromatography with a recovery of 67%. The apparent molecular weight of purified GHRP-scu-PA-32K was 33 kDa according to SDS–PAGE. Its specific activity was 150 000 IU/mg protein according to fibrin plate determination. The conversion of single-chain to two-chain molecules mediated by plasmin was comparable for GHRP-scu-PA-32K ( K m=4.9 μM, k 2=0.35 s −1) and scu-PA-32K. The activation of plasminogen by GHRP-scu-PA-32K ( K m=1.02 μM, k 2=0.0028 s −1) was also similar to that of scu-PA-32K. The fibrin binding of GHRP-scu-PA-32K was 2.5 times higher than that of scu-PA-32K at a fibrin concentration of 3.2 mg/ml. In contrast to scu-PA-32K in vitro 125I-fibrin-labeled plasma clot lysis, GHRP-scu-PA had a higher thrombolytic potency, whereas it depleted less fibrinogen in plasma. These results show that GHRP-scu-PA-32K as expected is a potential thrombolytic agent.

Synthesis and conformational analysis of the insulin-like 4 gene product.

Insulin-like 4 (INSL-4) is a protein expressed in the early placenta. Its primary structure is insulin-like with reference to the distribution of cysteine residues and the single chain pro-form. Insulin-like 4 was generated by solid-phase peptide synthesis of the two chains followed by the sequential synthesis of the three disulfide bonds. Two disulfide isomers were produced, one with an insulin-like disulfide bonding pattern and the other with a reversed chain orientation. The CD spectra of the two disulfide isomers were indistinguishable without any features produced by periodic structures. In addition, the hydrodynamic properties of the two isomers were identical which implied a very open structure of the disulfide-bonded two-chain molecules. It appears that insulin-likeness cannot be defined solely on the basis of the primary structure of cDNA.

Influence of Contact System Deficiencies during Cardiopulmonary Bypass

SummaryAs a result of the expanding indications for cardiopulmonary bypass (CPB) there is a growing number of patients undergoing this procedure who are at high surgical risk, and increasing efforts to investigate the mechanisms involved in CPB complications are warranted. Blood contact with foreign surfaces triggers systemic inflammatory response and may induce coagulation (1), this is believed to be responsible of most of the unwanted effects associated with CPB. During this procedure, contact phase activation occurs (2), involving factor XII (FXII), prekallikrein (PK) and high molecular weight kininogen (HK). FXII binds to negatively charged surfaces undergoing autoactivation with cleavage of Arg353-Val354 to form a two-chain molecule of 80 kD, i.e. activated FXII (FXIIa, also known as FXIIa). FXIIa can cleave PK to kallikrein which in turn cleaves FXII to produce FXIIa and a factor XII fragment of 28-30 kD (FXIIf, also known as FXIIa) and cleaves HK to HKa generating the nonapeptide bradykinin. FXIIa can cleave both FXI and PK to active enzymes, while FXIIf (which cannot bind to surfaces) cleaves PK (2) and the first component of complement (C1) (3). These mechanisms of activation differ from those initiating the coagulation cascade during in vivo haemostasis through the tissue factor pathway as a result of tissue injury (4).

Two-chain high molecular weight kininogen induces endothelial cell apoptosis and inhibits angiogenesis: partial activity within domain 5.

We previously reported that the binding of two-chain high molecular weight kininogen (HKa) to endothelial cells may occur through interactions with endothelial urokinase receptors. Since the binding of urokinase to urokinase receptors activates signaling responses and may stimulate mitogenesis, we assessed the effect of HKa binding on endothelial cell proliferation. Unexpectedly, HKa inhibited proliferation in response to several growth factors, with 50% inhibition caused by approximately 10 nM HKa. This activity was Zn(2+) dependent and not shared by either single-chain high molecular weight kininogen (HK) or low molecular weight kininogen. HKa selectively inhibited the proliferation of human umbilical vein and dermal microvascular endothelial cells, but did not affect that of umbilical vein or human aortic smooth muscle cells, trophoblasts, fibroblasts, or carcinoma cells. Inhibition of endothelial proliferation by HKa was associated with endothelial cell apoptosis and unaffected by antibodies that block the binding of HK or HKa to any of their known endothelial receptors. Recombinant HK domain 5 displayed activity similar to that of HKa. In vivo, HKa inhibited neovascularization of subcutaneously implanted Matrigel plugs, as well as rat corneal angiogenesis. These results demonstrate that HKa is a novel inhibitor of angiogenesis, whose activity is dependent on the unique conformation of the two-chain molecule.

Urokinase‐derived peptides regulate vascular smooth muscle contractionin vitroandin vivo

We examined the effect of urokinase (uPA) and its fragments on vascular smooth muscle cell contraction. Single-chain uPA inhibits phenylepherine (PE) -induced contraction of rat aortic rings, whereas two-chain uPA exerts the opposite effect. Two independent epitopes mediating these opposing activities were identified. A6, a capped peptide corresponding to amino acids 136-143 (KPSSPPEE) of uPA, increased the EC(50) of PE-induced vascular contraction sevenfold by inhibiting the release of calcium from intracellular stores. A6 activity was abolished by deleting the carboxyl-terminal Glu or by mutating the Ser corresponding to position 138 in uPA to Glu. A single-chain uPA variant lacking amino acids 136-143 did not induce vasorelaxation. A second epitope within the kringle of uPA potentiated PE-induced vasoconstriction. This epitope was exposed when single-chain uPA was converted to a two-chain molecule by plasmin. The isolated uPA kringle augmented vasoconstriction, whereas uPA variant lacking the kringle had no procontractile activity. These studies reveal previously undescribed vasoactive domains within urokinase and its naturally derived fragments.

Urokinase-derived peptides regulate vascular smooth muscle contraction in vitro and in vivo.

We examined the effect of urokinase (uPA) and its fragments on vascular smooth muscle cell contraction. Single-chain uPA inhibits phenylepherine (PE) -induced contraction of rat aortic rings, whereas two-chain uPA exerts the opposite effect. Two independent epitopes mediating these opposing activities were identified. A6, a capped peptide corresponding to amino acids 136-143 (KPSSPPEE) of uPA, increased the EC(50) of PE-induced vascular contraction sevenfold by inhibiting the release of calcium from intracellular stores. A6 activity was abolished by deleting the carboxyl-terminal Glu or by mutating the Ser corresponding to position 138 in uPA to Glu. A single-chain uPA variant lacking amino acids 136-143 did not induce vasorelaxation. A second epitope within the kringle of uPA potentiated PE-induced vasoconstriction. This epitope was exposed when single-chain uPA was converted to a two-chain molecule by plasmin. The isolated uPA kringle augmented vasoconstriction, whereas uPA variant lacking the kringle had no procontractile activity. These studies reveal previously undescribed vasoactive domains within urokinase and its naturally derived fragments.