Two-step selective formation of three disulfide bridges in the synthesis of the C-terminal epidermal growth factor-like domain in human blood coagulation factor IX.

The 45-residue C-terminal EGF-like domain in human blood coagulation factor IX has been synthesized by a 2-step method to form selectively 3 disulfide bridges. Four out of 6 cysteines are blocked with either trityl or 4-methyl-benzyl, and the remaining 2 cysteines are blocked with acetamidomethyl (Acm). In the first step, 4 free cysteinyl thiols are released concurrently with the removal of all protecting groups except Acm and are oxidized to form 1 of the 3 possible isomers containing 2 pairs of disulfides. In the second step, iodine is used to remove the Acm groups to yield the third disulfide bridge. This approach reduces the number of possible disulfide bridging patterns from 15 to 3. To determine the optimal protecting group strategy, 3 peptides are synthesized, each with Acm blocking 1 of the 3 pairs of cysteines involved in disulfide bridges: Cys5 to Cys16 (Cys 1-3), Cys12 to Cys26 (Cys 2-4), or Cys28 to Cys41 (Cys 5-6). Only the peptide having the Cys 2-4 pair blocked with Acm forms the desired disulfide isomer (Cys 1-3/5-6) in high yield after the first step folding, as identified by proteolytic digestion in conjunction with mass spectrometric peptide mapping. Thus, the choice of which pair of cysteines to block with Acm is critically important. In the case of EGF-like peptides, it is better to place the Acm blocking groups on one of the pairs of cysteines involved in the crossing of disulfide bonds.     

Calcium binding and putative activity of the epidermal growth factor domain of blood coagulation factor IX

The first epidermal growth factor (EGF)-like domain of human Factor IX and two chimeric analogs of this domain and EGF were synthesized unambiguously and purified to homogeneity. The synthetic EGF-like domain and its analogs showed the correct mass ions by the fission ionization mass spectrometry and similar disulfide pairings as those found in EGF, but failed to exhibit any putative EGF activity in the receptor and mitogenic assays. However, in NMR titration experiments, the EGF-like domain and one of its analogs were found to bind Ca2+ but not Mg2+. Our results therefore show that the EGF-like domain of Factor IX has the ability to bind calcium ion, shares the structural motif of EGF but does not retain the active determinants responsible for the EGF activity.     

Hydrophobic contact between the two epidermal growth factor-like domains of blood coagulation factor IX contributes to enzymatic activity

The three-dimensional structure of activated factor IX comprises multiple contacts between the two epidermal growth factor (EGF)-like domains. One of these is a salt bridge between Glu(78) and Arg(94), which is essential for binding of factor IXa to its cofactor factor VIII and for factor VIII-dependent factor X activation (Christophe, O. D., Lenting, P. J., Kolkman, J. A., Brownlee, G. G., and Mertens, K. (1998) J. Biol. Chem. 273, 222-227). We now addressed the putative hydrophobic contact at the interface between the EGF-like domains. Recombinant factor IX chimeras were constructed in which hydrophobic regions Phe(75)-Phe(77) and Lys(106)-Val(108) were replaced by the corresponding sites of factor X and factor VII. Activated factor IX/factor X chimeras were indistinguishable from normal factor IXa with respect to factor IXa enzymatic activity. In contrast, factor IXa(75-77)/factor VII displayed approximately 2-fold increased factor X activation in the presence of factor VIII, suggesting that residues 75-77 contribute to cofactor-dependent factor X activation. Activation of factor X by factor IX(106-108)/factor VII was strongly decreased, both in the absence and presence of factor VIII. Activity could be restored by simultaneous substitution of the hydrophobic sites in both EGF-like domains for factor VII residues. These data suggest that factor IXa enzymatic activity requires hydrophobic contact between the two EGF-like domains.     

The N-terminal epidermal growth factor-like domain of coagulation factor IX. Probing its functions in the activation of factor IX and factor X with a monoclonal antibody

The absence or reduced activity of coagulation factor IX (FIX) causes the severe bleeding disorder hemophilia B. FIX contains an N-terminal Gla domain followed by two epidermal growth factor-like (EGF) domains and a serine protease domain. In this study, the epitope of monoclonal antibody AW, which is directed against the C-terminal part of the first EGF domain in human FIX, was defined, and the antibody was used to study interactions between the EGF domain of FIX and other coagulation proteins. Antibody AW completely blocks activation of FIX by activated factor XI, but activation by activated factor FVII-tissue factor is inhibited only slightly. The antibody also causes a marginal reduction in the apparent k(cat) for factor X both in the presence and absence of activated factor VIII. Based on these results, we produced a preliminary model of the structure of the activated factor IX-activated factor VIII-AW complex on the surface of phospholipid. The model suggests that in the Xase complex, EGF1 of activated factor IX is not involved in direct binding to activated factor VIII. Studies of the interaction of antibody AW with a mutated FIX molecule (R94D) also suggest that the Glu(78)-Arg(94) salt bridge is not important for maintaining the structure of FIX.     

The connecting segment between both epidermal growth factor-like domains in blood coagulation factor IX contributes to stimulation by factor VIIIa and its isolated A2 domain

The light chain of activated factor IX comprises multiple interactions between both epidermal growth factor-like domains that contribute to enzymatic activity and binding of factor IXa to its cofactor factor VIIIa. To investigate the association between factor IXa-specific properties and surface-exposed structure elements, chimeras were constructed in which the interconnection between the modules Leu(84)-Thr(87) and the factor IX-specific loop Asn(89)-Lys(91) were exchanged for corresponding regions of factor X and factor VII. In absence of factor VIIIa, all chimeras displayed normal enzymatic activity. In the presence of factor VIIIa, replacement of loop Asn(89)-Lys(91) resulted in a minor reduction in factor IXa activity. However, chimeras with substitutions or insertions in the spacer between the epidermal growth factor-like domains showed a major defect in response to factor VIIIa. Of these chimeras, some displayed a normal response to isolated factor VIII A2 domain as a cofactor in factor X activation. Surprisingly, chimeras containing elongated inter-domain spacers from factor X or VII displayed reduced response to both complete factor VIIIa and the isolated A2 domain. Moreover, these chimeras still displayed effective association with immobilized A2 domain as assessed by surface plasmon resonance. We conclude that both sequence and length of the junction Leu(84)-Thr(87) between both epidermal growth factor-like domains contribute to the enhancement of factor IXa enzymatic activity that occurs upon assembly with factor VIIIa.     

The structure of (xylose)2glucose-O-serine 53 found in the first epidermal growth factor-like domain of bovine blood clotting factor IX

We reported the presence of a new trisaccharide composed of two xylose and reducing terminal glucose residues linked to serine residues of bovine blood clotting factors VII and IX (Hase, S., Kawabata, S., Nishimura, H., Takeya, H., Sueyoshi, T., Miyata, T., Iwanaga, S., Takao, T., Shimonishi, Y., and Ikenaka, T. (1988) J. Biochem. (Tokyo) 104, 867-868). The present paper describes the detailed structural analysis of the trisaccharide. Glycopeptides were prepared from bovine factor IX by digestion with Pronase followed by purification by column chromatography. The trisaccharide was released from the protein by the beta-elimination reaction with hydrazine, and the reducing end of the sugar chain was tagged with 2-aminopyridine. The fluorescent pyridylamino derivative of the trisaccharide was purified by gel filtration and reversed-phase high performance liquid chromatography. The glycopeptides and pyridylamino-trisaccharide thus obtained were subjected to methylation study, 500-MHz 1H nuclear magnetic resonance spectroscopy, and periodate oxidation. Glucose and xylose belong to the D series by high performance liquid chromatography on a chiral column. From the results, the structure of the trisaccharide is proposed as: D-Xyl p alpha 1-3-D-Xyl p alpha 1-3-D-Glcp beta 1-O-Ser-53.     

Role of the N-terminal epidermal growth factor-like domain of factor X/Xa

The functional importance of the N-terminal epidermal growth factor-like domain (EGF-N) of factor X/Xa (FX/Xa) was investigated by constructing an FX mutant in which the exon coding for EGF-N was deleted from FX cDNA. Following expression and purification to homogeneity, the mutant was characterized with respect to its ability to function as a zymogen for either the factor VIIa-tissue factor complex or the factor IXa-factor VIIIa complex and then to function as an enzyme in the prothrombinase complex to catalyze the conversion of prothrombin to thrombin. It was discovered that EGF-N is essential for the recognition and efficient activation of FX by both activators in the presence of the cofactors. On the other hand, the FXa mutant interacted with factor Va with a normal apparent dissociation constant and activated prothrombin with approximately 3-fold lower catalytic efficiency in the prothrombinase complex. Surprisingly, the mutant activated prothrombin with approximately 12-fold better catalytic efficiency than wild-type FXa in the absence of factor Va. The mutant was inactive in both prothrombin time and activated partial thromboplastin time assays; however, it exhibited a similar specific activity in a one-stage FXa clotting assay. These results suggest that EGF-N of FX is required for the cofactor-dependent zymogen activation by both physiological activators, but it plays no apparent role in FXa recognition of the cofactor in the prothrombinase complex.     

The N-terminal epidermal growth factor-like domain in factor IX and factor X represents an important recognition motif for binding to tissue factor.

Factors VII, IX, and X play key roles in blood coagulation. Each protein contains an N-terminal gamma-carboxyglutamic acid domain, followed by EGF1 and EGF2 domains, and the C-terminal serine protease domain. Protein C has similar domain structure and functions as an anticoagulant. During physiologic clotting, the factor VIIa-tissue factor (FVIIa*TF) complex activates both factor IX (FIX) and factor X (FX). FVIIa represents the enzyme, and TF represents the membrane-bound cofactor for this reaction. The substrates FIX and FX may utilize multiple domains in binding to the FVIIa*TF complex. To investigate the role of the EGF1 domain in this context, we expressed wild type FIX (FIX(WT)), FIX(Q50P), FIX(PCEGF1) (EGF1 domain replaced with that of protein C), FIX(DeltaEGF1) (EGF1 domain deleted), FX(WT), and FX(PCEGF1). Complexes of FVIIa with TF as well as with soluble TF (sTF) lacking the transmembrane region were prepared, and activations of WT and mutant proteins were monitored by SDS-PAGE and by enzyme assays. FVIIa*TF or FVIIa*sTF activated each mutant significantly more slowly than the FIX(WT) or FX(WT). Importantly, in ligand blot assays, FIX(WT) and FX(WT) bound to sTF, whereas mutants did not; however, all mutants and WT proteins bound to FVIIa. Further experiments revealed that the affinity of the mutants for sTF was reduced 3-10-fold and that the synthetic EGF1 domain (of FIX) inhibited FIX binding to sTF with K(i) of approximately 60 microm. Notably, each FIXa or FXa mutant activated FVII and bound to antithrombin, normally indicating correct folding of each protein. In additional experiments, FIXa with or without FVIIIa activated FX(WT) and FX(PCEGF1) normally, which is interpreted to mean that the EGF1 domain of FX does not play a significant role in its interaction with FVIIIa. Cumulatively, our data reveal that substrates FIX and FX in addition to interacting with FVIIa (enzyme) interact with TF (cofactor) using, in part, the EGF1 domain.     

The epidermal growth factor-like domains of factor IX. Effect on blood clotting and endothelial cell binding of a fragment containing the epidermal growth factor-like domains linked to the gamma-carboxyglutamic acid region

The binding of factor IX to cultured bovine endothelial cells was characterized using isolated domains of bovine factor IX. An NH2-terminal fragment that consists of the gamma-carboxyglutamic acid (Gla) region linked to the two epidermal growth factor (EGF)-like domains bound to the endothelial cells with the same affinity as intact factor IX, indicating that the serine protease part of factor IX is not involved in binding. This fragment also inhibited the factor IXa beta'-induced clotting of plasma at a concentration that would suggest a competition for phospholipid binding sites. However, after proteolytic removal of the Gla region from the fragment, the two EGF-like domains inhibited clotting almost as effectively, suggesting a direct interaction between this part of the molecule and the cofactor, factor VIIIa. Using affinity-purified Fab fragments against the Gla region, the EGF-like domains, and the serine protease part, it was observed that the serine protease part of the molecule undergoes a large conformational change upon activation, whereas the Gla region and the EGF-like domains appear to be unaffected. All three classes of Fab fragments were equally efficient as inhibitors of the factor IXa beta'-induced clotting reaction. Part of factor Va and factor VIIIa have significant sequence homology to a lectin. We therefore investigated the effect on in vitro clotting of the recently identified unique disaccharide Xyl alpha 1-3Glc, that is O-linked to a serine residue in the NH2-terminal EGF-like domain of human factor IX (Hase, S., Nishimura, H., Kawabata, S.-I., Iwanaga, S., and Ikenaka, T. (1990) J. Biol. Chem. 265, 1858-1861). However, no effect on blood clotting was observed in the assay system used. Our results are compatible with a model in which the serine protease part provides the specificity of the binding of factor IXa to factor VIIIa-phospholipid, but that the EGF-like domain(s) also contributes to the interaction of the enzyme with its cofactor.     

Conformation of the transmembrane domain of the epidermal growth factor receptor

The transmembrane domain of growth factor receptors, such as the epidermal growth factor receptor (EGFR) and the relatedc-erbB-2/neu oncogene protein, has been implicated in the process of receptor dimerization and mitogenic signal transduction, and hence in cellular transformation and oncogenesis. Amino acid substitutions in the transmembrane domain of thec-erbB-2/neu protein that cause a transforming effect may exert this effect through a conformational change from a bend conformation to an-helical structure in this region of the protein, but similar amino acid substitutions at homologous positions in the transmembrane domain of the EGFR (e.g., ValGlu at position 627) fail to have a transforming effect. To examine whether this failure may be due to structural effects, we have used conformational energy analysis to determine the preferred three-dimensional structures for the nonapeptide sequence of the transmembrane domain of the EGFR from residues 623–631 with Val or Glu at position 627. The global minimum energy conformations of both nonapeptides were found to be non--helical with bends at positions 624–625 and 627–628. The failure of the ValGlu substitution to produce a conformational change to an-helix in this region may be responsible for its lack of transforming effect. However, the presence of higher energy-helical conformations for the nonapeptide from the normal EGFR may provide an explanation for the presence of a transforming effect from overexpression of the EGFR.     

Real-time immuno-polymerase chain reaction in a 384-well format: Detection of vascular endothelial growth factor and epidermal growth factor-like domain 7

Immuno-polymerase chain reaction (immuno-PCR) combines the specificity of antibodies with the amplification power of PCR to detect low levels of proteins. Here, we describe the development of a 384-well immuno-PCR method that uses streptavidin coated on a PCR plate to capture complexes of biotinylated capture antibody, antigen, and DNA-labeled detection antibody. Unbound molecules are removed by a wash step using a standard plate washer. Antibody–DNA molecules in bound complexes are then detected directly on the plate using real-time PCR. Circulating human vascular endothelial growth factor concentrations measured by this method correlated with measurements obtained from enzyme-linked immunosorbent assay (ELISA). Using this method, we developed an assay for human epidermal growth factor-like domain 7 (EGFL7), an extracellular matrix-bound angiogenic factor. EGFL7 is expressed at a higher level in certain cancers, although endogenous EGFL7 concentrations have not been reported. Our 384-well EGFL7 immuno-PCR assay can detect 0.51 pM EGFL7 in plasma, approximately 16-fold more sensitive than the ELISA, utilizing the same antibodies. This assay detected EGFL7 in lysates of non-small-cell lung cancer and hepatocellular carcinoma cell lines and also hepatocellular carcinoma, breast cancer, and ovarian cancer tissues. This 384-well immuno-PCR method can be used to develop high-throughput biomarker assays.     

Calcium binding to the isolated beta-hydroxyaspartic acid-containing epidermal growth factor-like domain of bovine factor X

Coagulation factor X is a vitamin K-dependent protein composed of discrete domains or modules. A proteolytically modified derivative of factor X that lacks the NH2-terminal gamma-carboxyglutamic acid (Gla)-containing region retains one Ca2+ binding site. To localize this Gla-independent Ca2+ binding site and to facilitate future studies aimed at elucidating structure-function relationship in the factor X molecule, we have devised a method to isolate the first beta-hydroxyaspartic acid (Hya)-containing epidermal growth factor (EGF)-like domain from proteolytic digests of bovine factor X performed under strictly controlled conditions. The EGF-like domain, corresponding to residues 45-86 in bovine factor X, was obtained in more than 50% recovery, and was at least 98% homogeneous as judged by NH2-terminal sequence analysis. Ca2+ binding to the isolated EGF-like domain was studied by 1H NMR spectroscopy. On binding of Ca2+ to the domain the resonances from Tyr-68 centered at 6.8 ppm were affected. The Ca2+ concentration dependence of the chemical shift was used to calculate the Ca2+ binding constant, resulting in a K alpha of 4 X 10(3) M-1 at pH 8.5 and 1 X 10(3) M-1 at pH 7.4, the higher value presumably reflecting an increase in negative surface charge due to deprotonation of a histidine residue with a pK alpha of 7.4. The NMR spectra gave no evidence of a conformational change in the EGF-like domain between pH 6 and 8.5.     

Sequence-specific 1H NMR assignments, secondary structure, and location of the calcium binding site in the first epidermal growth factor like domain of blood coagulation factor IX.

Factor IX is a blood clotting protein that contains three regions, including a gamma-carboxyglutamic acid (Gla) domain, two tandemly connected epidermal growth factor like (EGF-like) domains, and a serine protease region. The protein exhibits a high-affinity calcium binding site in the first EGF-like domain, in addition to calcium binding in the Gla domain. The first EGF-like domain, factor IX (45-87), has been synthesized. Sequence-specific resonance assignment of the peptide has been made by using 2D NMR techniques, and its secondary structure has been determined. The protein is found to have two antiparallel beta-sheets, and preliminary distance geometry calculations indicate that the protein has two domains, separated by Trp28, with the overall structure being similar to that of EGF. An NMR investigation of the calcium-bound first EGF-like domain indicates the presence and location of a calcium binding site involving residues on both strands of one of the beta-sheets as well as the N-terminal region of the peptide. These results suggest that calcium binding in the first EGF-like domain could induce long-range (possibly interdomain) conformational changes in factor IX, rather than causing structural alterations in the EGF-like domain itself.     

Effects of gamma-carboxyglutamic acid and epidermal growth factor-like modules of factor IX on factor X activation. Studies using proteolytic fragments of bovine factor IX.

Factor IX is a vitamin K-dependent zymogen of a serine protease. The NH2-terminal half of the molecule consists of a Ca(2+)-binding gamma-carboxyglutamic acid (Gla)-containing module and two modules homologous to the epidermal growth factor (EGF) precursor. To elucidate the role of these non-catalytic modules of factor IXa beta in factor X activation, we have isolated and characterized fragments of bovine factor IX, containing one or both of the EGF-like modules as well as these modules linked to the Gla module. The fragments were used as inhibitors of factor IXa beta-mediated factor X activation in a plasma clotting system and in systems with purified components of the Xase complex. Fragments consisting of either the two EGF-like modules of factor IX linked together or the NH2-terminal EGF-like module alone were found to inhibit factor Xa generation both in the presence and absence of the cofactor, factor VIIIa. Moreover, a fragment consisting of the corresponding modules of factor X had a similar effect. We therefore propose that factor IXa beta and factor X interact directly through their EGF-like modules on or in the vicinity of a phospholipid surface. We have also found that the isolated Gla module of factor IX inhibits the formation of factor Xa both in the presence and absence of phospholipid but not in the absence of factor VIIIa. Our results are compatible with a model of the Xase complex, in which both the serine protease part and the Gla module of factor IXa beta interact with factor VIIIa.     

New Allyl Ester Linker and Solid-phase Block Synthesis of the Serglycin Core Region

The prototype glycopeptidyl fragments of serglycin, a proteoglycan with the characteristic peptide sequence of repeating L-seryl-L-glycine, were synthesized by a convergent method involving block condensation on a solid support. In order to facilitate detachment of the protected glycopeptides from the resin, a new allyl ester type of linker, which is cleavable by Pd(0)-catalysis, was designed and used in combination with the commercial acid-labile Sieber amide resin for the solid-phase synthesis. Glycopeptide blocks consisting of [O-(2,3,4-tri-O-acetyl-D-xylosyl)-L-seryl-L-glycine]n (n=1-8) were produced in good yields. Block condensation in a solution was also successful to synthesize up to the hexadecapeptide (n=8).     

Combined proteomic and biochemical analyses redefine the consensus sequence requirement for epidermal growth factor-like domain hydroxylation

Epidermal growth factor-like domains (EGFDs) have important functions in cell–cell signaling. Both secreted and cell surface human EGFDs are subject to extensive modifications, including aspartate and asparagine residue C3-hydroxylations catalyzed by the 2-oxoglutarate oxygenase aspartate/asparagine-β-hydroxylase (AspH). Although genetic studies show AspH is important in human biology, studies on its physiological roles have been limited by incomplete knowledge of its substrates. Here, we redefine the consensus sequence requirements for AspH-catalyzed EGFD hydroxylation based on combined analysis of proteomic mass spectrometric data and mass spectrometry–based assays with isolated AspH and peptide substrates. We provide cellular and biochemical evidence that the preferred site of EGFD hydroxylation is embedded within a disulfide-bridged macrocycle formed of 10 amino acid residues. This definition enabled the identification of previously unassigned hydroxylation sites in three EGFDs of human fibulins as AspH substrates. A non-EGFD containing protein, lymphocyte antigen-6/plasminogen activator urokinase receptor domain containing protein 6B (LYPD6B) was shown to be a substrate for isolated AspH, but we did not observe evidence for LYPD6B hydroxylation in cells. AspH-catalyzed hydroxylation of fibulins is of particular interest given their important roles in extracellular matrix dynamics. In conclusion, these results lead to a revision of the consensus substrate requirements for AspH and expand the range of observed and potential AspH-catalyzed hydroxylation in cells, which will enable future study of the biological roles of AspH.     

Identification of a basic region on tissue factor that interacts with the first epidermal growth factor-like domain of factor X

Tissue factor (TF) facilitates the recognition and rapid activation of factor X (fX) by factor VIIa (fVIIa) in the extrinsic Xase pathway. TF makes extensive interactions with both light and heavy chains of fVIIa; however, with the exception of a basic recognition site for the Gla domain of fX, no other interactive site on TF for the substrate has been identified. Structural and modeling data have predicted that a basic region of TF comprised of residues Asn-199, Arg-200, and Lys-201 is located at a proper height on the membrane surface to interact with either the C-terminus of the Gla domain or the EGF-1 domain of fX. To investigate this possibility, we prepared the Ala substitution mutants of these residues and evaluated their ability to function as cofactors for fVIIa in the activation of wild-type fX and its two mutants which lack either the Gla domain (GD-fX) or both the Gla and EGF-1 domains (E2-fX). All three TF mutants exhibited normal cofactor activity in the amidolytic activity assays, but the cofactor activity of Arg-200 and Lys-201 mutants in fVIIa activation of both fX and GD-fX, but not E2-fX, was impaired approximately 3-fold. Further kinetic analysis revealed that kcat values with both TF mutants are impaired with no change in Km. These results suggest that both Arg-200 and Lys-201 of TF interact with EGF-1 of fX to facilitate the optimal docking of the substrate into the catalytic groove of the protease in the activation complex.     

The interface between the EGF2 domain and the protease domain in blood coagulation factor IX contributes to factor VIII binding and factor X activation

The light chain of activated factor IX (FIXa) is involved in a number of functional properties, including FIXa enzymatic activity. This suggests the existence of a functional link between the FIXa light chain and the catalytic domain. The FIXa structure includes a few putative interactions between EGF2 and the protease domain. The role thereof has been addressed in this study. Recombinant FIX variants FIX-N92A, FIX-N92H, FIX-Y295A, and FIX-F299A were produced in 293 cells. After activation, the purified mutants were analyzed for a variety of functional parameters. None of these substitutions had a major effect on the interaction with antithrombin or the cleavage of the chromogenic substrate CH(3)SO(2)-d-CHG-Gly-Arg-p-nitroanilide. All FIXa mutants, however, exhibited a reduced level of factor X (FX) activation. Defective proteolytic activity occurred both in the absence and in the presence of activated factor VIII (FVIIIa). All mutants also exhibited a reduced level of FX activation in the absence of phospholipids. This suggests that putative interdomain contacts involving residues Asn(92), Tyr(295), and Phe(299) affect reactivity toward FX. Detailed kinetic studies in the presence of phospholipids and FVIIIa revealed substrate inhibition, particularly for mutants FIXa-N92A and FIXa-N92H. Surface plasmon resonance demonstrated that the same replacements weaken the association with the isolated factor VIII (FVIII) A2 domain and the FVIII light chain. This implies a defect in the formation of the FX-activating complex that is membrane-independent. We conclude that contacts between EGF2 and the protease domain of FIXa are crucial for FIXa enzymatic activity and for the assembly of the FX-activating complex.     

Synthesis and characterization of novel fluorogenic substrates of coagulation factor XIII-A

Further development of our recently published Glu(pNA)-containing peptides (Anal. Biochem. 428 (2012) 73–80) provided new fluorogenic substrates for the activated blood coagulation factor XIII. A first series was designed by incorporation of Glu(AMC) at the penultimate position from the N terminus. For the best derivative H-Tyr-Glu(AMC)-Val-Lys-Val-Ile-NH₂, a moderate k_cat/K_m value of 34 s⁻¹ M⁻¹ was determined, which is more than 100-fold reduced compared with the previously reported Glu(pNA) substrates. Furthermore, two fluorescence resonance energy transfer (FRET) substrates were prepared by incorporation of an N-methyl-anthraniloyl fluorophore and a 2,4-dinitrophenyl quencher. Both substrates were excellently cleaved by FXIII-A₂^∗, which is generated from its zymogen by activation of thrombin in the presence of calcium ions. In the absence and presence of H-Gly-ethyl ester, k_cat/K_m values of 8010 and 8660 s⁻¹ M⁻¹, respectively, were found for the conversion of H-Lys(N(Me)Abz)-Glu(NH-(CH₂)₄-NH-Dnp)-Val-Lys-Val-Ile-Gly-NH₂ (substrate 8). These values are more than 200-fold improved compared with the Glu(AMC) substrates. Substrate 8 is suitable for the measurement of FXIII-A₂^∗ activities in plasma samples as well as for in vitro measurements. Furthermore, it was used for the determination of the inhibitory potency of a newly synthesized chloromethyl ketone derivative, Cbz-Phe-Glu(CMK)-Val-Lys-Val-Ile-Gly-NH₂, which was found to be a potent irreversible inhibitor of FXIII-A₂^∗.     

Synthesis of cyclic analogues of loop 4 of nerve growth factor

Cyclic peptides cyclo(-Gly-Asp-Glu-Lys-), cyclo(-Gly-Gly-Asp-Glu-Lys-) and cyclo(-Gly-Gly-Gly-Asp-Glu-Lys-) were synthesized as models of theβ-turn of nerve growth factor loop 4. The corresponding protected linear precursors were obtained in 52–83% yields by the solid-phase method with the use of the Fmoc/Bu ^t strategy and a chlorotrityl anchor group. The cyclization was carried out with benzotriazolyloxytris(dimethylamino)phosphonium (BOP) hexafluorophosphate, N-[(1H-benzotriazole-1-yl)-(dimethylamino)methylene]-N-methylmetanaminium-N-oxide (HBTU) hexafluorophosphate, and diphenylphosphorylazide (DPPA) at a dilution of 10^?3 M. The distribution of reaction products was studied for each cyclopeptide in dependence on the type of the coupling agent. The use of DPPA was shown to completely inhibit the formation of cyclodimers in the synthesis of five-and six-membered cyclopeptides; however, in the case of a four-membered peptide, an additional tenfold dilution of the reaction mixture was necessary to achieve the effect. The identification of several byproducts during the synthesis showed that the elongation of the polypeptide chain using the BOP reagent can be complicated by substantial racemization, and the cleavage of the chlorotrityl anchor group by 0.5% TFA in dichloromethane proceeds with insufficient selectivity and is accompanied by the premature Boc deblocking of the lysine side function.