Hideko Atoda

Adenovirus Serotype 5 Hexon Mediates Liver Gene Transfer

Adenoviruses are used extensively as gene transfer agents, both experimentally and clinically. However, targeting of liver cells by adenoviruses compromises their potential efficacy. In cell culture, the adenovirus serotype 5 fiber protein engages the coxsackievirus and adenovirus receptor (CAR) to bind cells. Paradoxically, following intravascular delivery, CAR is not used for liver transduction, implicating alternate pathways. Recently, we demonstrated that coagulation factor (F)X directly binds adenovirus leading to liver infection. Here, we show that FX binds to the Ad5 hexon, not fiber, via an interaction between the FX Gla domain and hypervariable regions of the hexon surface. Binding occurs in multiple human adenovirus serotypes. Liver infection by the FX-Ad5 complex is mediated through a heparin-binding exosite in the FX serine protease domain. This study reveals an unanticipated function for hexon in mediating liver gene transfer in vivo.

Crystal structure of an anticoagulant protein in complex with the Gla domain of factor X.

The γ-carboxyglutamic acid (Gla) domain of blood coagulation factors is responsible for Ca2+-dependent phospholipid membrane binding. Factor X-binding protein (X-bp), an anticoagulant protein from snake venom, specifically binds to the Gla domain of factor X. The crystal structure of X-bp in complex with the Gla domain peptide of factor X at 2.3-Å resolution showed that the anticoagulation is based on the fact that two patches of the Gla domain essential for membrane binding are buried in the complex formation. The Gla domain thus is expected to be a new target of anticoagulant drugs, and X-bp provides a basis for designing them. This structure also provides a membrane-bound model of factor X.

Snake Venom Vascular Endothelial Growth Factors (VEGFs) Exhibit Potent Activity through Their Specific Recognition of KDR (VEGF Receptor 2)

Vascular endothelial growth factor (VEGF165) exhibits multiple effects via the activation of two distinct endothelial receptor tyrosine kinases: Flt-1 (fms-like tyrosine kinase-1) and KDR (kinase insert domain-containing receptor). KDR shows strong ligand-dependent tyrosine phosphorylation in comparison with Flt-1 and mainly mediates the mitogenic, angiogenic, and permeability-enhancing effects of VEGF165. Here we show the isolation of two VEGFs from viper venoms and the characterization of their unique biological properties. Snake venom VEGFs strongly stimulated proliferation of vascular endothelial cells in vitro. Interestingly, the maximum activities were almost twice that of VEGF165. They also induced strong hypotension on rat arterial blood pressure compared with VEGF165 in vivo. A receptor binding assay revealed that snake venom VEGFs bound to KDR-IgG with high affinity (Kd = ∼0.1 nm) as well as to VEGF165 but did not interact with Flt-1, Flt-4, or neuropilin-1 at all. Our data clearly indicate that snake venom VEGFs act through the specific activation of KDR and show potent effects. Snake venom VEGFs are a highly specific ligand to KDR and form a new group of the VEGF family.

Crystal structure of flavocetin-A, a platelet glycoprotein Ib-binding protein, reveals a novel cyclic tetramer of C-type lectin-like heterodimers.

Snake venom contains a number of the hemostatically active C-type lectin-like proteins, which affect the interaction between von Willebrand factor (vWF) and the platelet glycoprotein (GP) Ib or platelet receptor to inhibit/induce platelet activation. Flavocetin-A (FL-A) is a high-molecular mass C-type lectin-like protein (149 kDa) isolated from the habu snake venom. FL-A binds with high affinity to the platelet GP Ibalpha-subunit and functions as a strong inhibitor of vWF-dependent platelet aggregation. We have determined the X-ray crystal structure of FL-A and refined to 2.5 A resolution. This is a first elucidation of a three-dimensional structure of the platelet GP Ib-binding protein. The overall structure reveals that the molecule is a novel cyclic tetramer (alphabeta)(4) made up of four alphabeta-heterodimers related by a crystallographic 4-fold symmetry. The tetramerization is mediated by an interchain disulfide bridge between cysteine residues at the C-terminus of the alpha-subunit and at the N-terminus of the beta-subunit in the neighboring alphabeta-heterodimer. The high affinity of FL-A for the platelet GP Ib alpha-subunit could be explained by a cooperative-binding action through the multiple binding sites of the tetramer.

Characterization of a monoclonal antibody B1 that recognizes phosphorylated ser-158 in the activation peptide region of human coagulation factor IX

Blood coagulation factor IX (FIX) undergoes various post-translational modifications such as γ-carboxylation and glycosylation. Non-phosphorylated recombinant FIX has been reported to rapidly disappear from plasma, indicating that phosphorylation of FIX plays an important role in the physiological activity of this coagulation factor. In this study, we characterized the human FIX activation peptide (AP) using a monoclonal antibody that recognizes phosphorylated Ser-158 in the AP region. Murine monoclonal antibody B1 against human FIX recognized FIX with an apparent Kd value of 5 nm in the presence of Ca2+ (EC50 = 0.58 mm). B1 bound to the isolated AP of FIX and retained the Ca2+ dependence of binding to the isolated AP. The deglycosylation of AP did not affect the binding of B1 to AP, while B1 failed to bind to recombinant AP expressed in Escherichia coli. MALDI-TOF mass spectrometry showed that the m/z of plasma-derived deglycosylated AP is 82.54 Da greater than that of recombinant AP. The binding ability of B1 to AP was lost by the dephosphorylation of plasma-derived AP. B1 bound to synthetic peptide AP-(5-19), including phosphoserine-13, but not to the non-phosphorylated AP-(5-19) in the presence of Ca2+. These data provide direct evidence that Ser-13 of the plasma-derived FIX AP region (Ser-158 of FIX) is phosphorylated and that B1 recognizes the epitope, which includes Ca2+-bound phosphoserine-158. B1 should be useful in the quality control of biologically active recombinant FIX containing phosphoserine-158.

Calcium-binding analysis and molecular modeling reveal echis coagulation factor IX/factor X-binding protein has the Ca-binding properties and Ca ion-independent folding of other C-type lectin-like proteins

Many biologically active heterodimeric proteins of snake venom consist of two C‐type lectin‐like subunits. One of these proteins, habu IX/X‐bp, is a Gla domain‐binding protein whose subunits both bind to a Ca2+ ion, with a total of two Ca2+‐binding sites. The molecular modeling and Ca2+‐binding analysis of echis IX/X‐bp revealed that it lacks one of two Ca2+‐binding sites, though the folding of this subunit is conserved. It is concluded that heterodimeric C‐type lectin‐like proteins function independent of Ca2+ and have essentially a similar folding to habu IX/X‐bp.

CRYSTALLIZATION AND PRELIMINARY X-RAY STUDY OF BLOOD COAGULATION FACTOR IX/FACTOR X-BINDING PROTEIN WITH ANTICOAGULANT ACTIVITY FROM HABU SNAKE VENOM

Crystals of a blood anticoagulant from the venom of the Habu snake, Trimeresurus flavoviridis, have been obtained using ammonium sulfate by the vapor diffusion method. The crystals belong to the orthorhombic space group P2(1)2(1)2 with cell dimensions a = 172 A, b = 86 A, c = 65 A, and diffract to at least 4.0 A resolution.

Structure and Functions of Coagulation Factor IX/Factor X-Binding Protein Isolated from the Venom of Trimeresurus flavoviridis

Snake venoms contain various anticoagulants that affect blood coagulation system (1, 2). The anticoagulant is classified into two categories: anticoagulant enzymes and non-enzymatic anticoagulants. The examples of the former category are phospholipases, fibrinogenolytic enzymes, protein C activators, and proteolytic enzymes that convert coagulation factors into degraded inactive forms. An interesting example of the non-enzymatic anticoagulants is an inhibitor of the activation of prothrombin. The inhibitors in this category have been first found in the venom of Agkistrodon acutus (3) and Trimeresurus gramineus (4). The anticoagulant protein isolated from A. acutus inhibits the participation of factor Xa in the prothrombinase complex formation (5, 6). In order to understand the anticoagulant mechanism we have isolated and characterized another anticoagulant protein with Mr 27,000 from the venom of the habu snake Trimeresurus flavoviridis (7). This anticoagulant protein forms a complex with either coagulation factor IX or factor X with a stoichiometry of 1 to 1 in a calcium-dependent fashion, and thereby blocks the amplification of the coagulation cascade (7). Thus, we named it IX/X-bp (factor IX/factor X-binding protein). This chapter describes the structural and functional properties of IX/X-bp from the venom of the habu snake (T. flavoviridis).

Anticoagulant Mechanism of Factor IX/factor X-binding Protein Isolated from the Venom of Trimeresurus flavoviridis

Anticoagulant mechanism of the coagulation factor IX/factor X-binding protein (IX/X-bp) isolated from the venom of Trimeresurus flavoviridis was investigated. IX/X-bp had no effect on the amidase activity of factor Xa measured with a synthetic peptide substrate Boc-Leu-Gly-Arg-pNA. Prothrombin activation by factor Xa without cofactors, such as factor Va and phospholipids, was only slightly influenced by IX/X-bp. However, prothrombin activation by factor Xa in the presence of factor Va resulted in IX/X-bp inhibiting the increase of k(cat) of thrombin formation through inhibition of interaction between factor Xa and factor Va. IX/X-bp also inhibited the decrease of K(m) for thrombin formation through interaction with phospholipids. Thus, IX/X-bp appears to act as an anticoagulant protein by inhibiting the interaction between factor Xa and its cofactors in the prothrombinase complex by binding to the Gla domain of factor Xa.

Crystallization and preliminary X-ray studies of flavocetin-A, a platelet glycoprotein Ib-binding protein from the habu snake venom

Flavocetin-A (FL-A) is a platelet glycoprotein Ib-binding protein, a high molecular mass oligomer (149 kDa) of C-type lectin-like subunits alpha and beta isolated from the habu snake venom. Purified FL-A crystallized in the tetragonal space group I4 with unit-cell dimensions a = b = 121.0, c = 63.2 A. The crystals diffract to at least 2.4 A resolution. The structure has been solved by molecular replacement using the crystal structure of factors IX/X-binding protein (PDB code 1ixx) as a search model. The asymmetric unit contains one heterodimer, showing that FL-A is a novel tetradimer (alphabeta)(4) composed of four heterodimers related by a crystallographic fourfold axis.