Mary Ann McLane

Structural Requirements of Echistatin for the Recognition of αvβ3 and α5β1Integrins

There are key differences between the amino acid residues of the RGD loops and the C termini of echistatin, a potent antagonist of αIIbβ3, αvβ3 and α5β1, and eristostatin, a similar disintegrin selectively inhibiting αIIbβ3. In order to identify echistatin motifs required for selective recognition of αvβ3 and α5β1integrins, we expressed recombinant echistatin, eristostatin, and 15 hybrid molecules. We tested them for their ability to inhibit adhesion of different cell lines to fibronectin and von Willebrand factor and to express ligand-induced binding site epitope. The results showed that Asp27 and Met28 support recognition of both αvβ3 and α5β1. Replacement of Met28 with Asn completely abolished echistatins ability to recognize each of the integrins, while replacement of Met28 with Leu selectively decreased echistatins ability to recognize α5β1only. Eristostatin in which C-terminal WNG sequence was substituted with HKGPAT exhibited new activity with α5β1, which was 10–20-fold higher than that of wild type eristostatin. A hypothesis is proposed that the C terminus of echistatin interacts with separate sites on β1 and β3 integrin molecules.

The disulphide bond pattern of bitistatin, a disintegrin isolated from the venom of the viper Bitis arietans

The disulphide bond pattern of the long disintegrin bitistatin (83 amino acids, 14 cysteines) was established using structural information gathered by amino acid analysis, N‐terminal sequencing, and molecular mass determination of fragments isolated by reversed‐phase HPLC after polypeptide degradation with trypsin and oxalic acid. A computer program was used to calculate all possible combinations of disulphide‐bonded peptides matching the mass spectrometric data, and the output was filtered using compositional and sequence data. Disulphide bonds between cysteines 16–34, 18–29, 28–51, 42–48, 47–72, and 60–79 are conserved in medium‐long disintegrins flavoridin and kistrin (70 amino acids, 12 cysteines), and the two cysteine residues at positions 5 and 24 found in bitistatin but not in other disintegrin molecules are disulphide‐bridged. This linkage creates an extra, large loop, which, depending on whether the NMR structure of flavoridin or kistrin is used for modelling the structure of bitistatin, lies opposite or nearly parallel, respectively, to the biologically active RGD‐containing loop.

Inhibition of lung tumor colonization and cell migration with the disintegrin crotatroxin 2 isolated from the venom of Crotalus atrox.

Disintegrins are low molecular weight proteins (4-15 kDa) with an RGD binding region at their binding loop. Disintegrin and disintegrin-like proteins are found in the venom of four families of snakes: Atractaspididae, Elapidae, Viperidae, and Colubridae. This report describes the biological activity of a disintegrin, crotatroxin 2, isolated by a three-step chromatography procedure from the venom of the Western diamondback rattlesnake (Crotalus atrox). The intact molecular mass for crotatroxin 2 was 7.384 kDa and 71 amino acids. Crotatroxin 2 inhibited human whole blood platelet aggregation with an IC(50) of 17.5 nM, inhibited cell (66.3p) migration by 63%, and inhibited experimental lung tumor colonization in BALB/c mice at 1000 microg/kg. Our data suggest that while crotatroxin 2 inhibits platelet aggregation, cancer cell migration, and lung tumor colonization, it is done via different integrins.

Integrin αIIbβ3:ligand interactions are linked to binding-site remodeling

This study tested the hypothesis that high‐affinity binding of macromolecular ligands to the αIIbβ3 integrin is tightly coupled to binding‐site remodeling, an induced‐fit process that shifts a conformational equilibrium from a resting toward an open receptor. Interactions between αIIbβ3 and two model ligands—echistatin, a 6‐kDa recombinant protein with an RGD integrin‐targeting sequence, and fibrinogens γ‐module, a 30‐kDa recombinant protein with a KQAGDV integrin binding site—were measured by sedimentation velocity, fluorescence anisotropy, and a solid‐phase binding assay, and modeled by molecular graphics. Studying echistatin variants (R24A, R24K, D26A, D26E, D27W, D27F), we found that electrostatic contacts with charged residues at the αIIb/β3 interface, rather than nonpolar contacts, perturb the conformation of the resting integrin. Aspartate 26, which interacts with the nearby MIDAS cation, was essential for binding, as D26A and D26E were inactive. In contrast, R24K was fully and R24A partly active, indicating that the positively charged arginine 24 contributes to, but is not required for, integrin recognition. Moreover, we demonstrated that priming—i.e., ectodomain conformational changes and oligomerization induced by incubation at 35°C with the ligand‐mimetic peptide cHarGD—promotes complex formation with fibrinogens γ‐module. We also observed that the γ‐modules flexible carboxy terminus was not required for αIIbβ3 integrin binding. Our studies differentiate priming ligands, which bind to the resting receptor and perturb its conformation, from regulated ligands, where binding‐site remodeling must first occur. Echistatins binding energy is sufficient to rearrange the subunit interface, but regulated ligands like fibrinogen must rely on priming to overcome conformational barriers.

New Insights on Disintegrin-Receptor Interactions: Eristostatin and Melanoma Cells

Viper venom disintegrins have been used frequently to study the cellular receptors which characterize various types of cells, including platelets, endothelial cells and cancer cells. While the majority of such analyses have pointed to involvement of integrin receptors αvβ3, α5β1 or αIIbβ3, this may not always be so. Eristostatin, from Eristocophis macmahoni, is a potent inhibitor of ADP-induced platelet aggregation as well as of human and murine melanoma metastases in mouse model systems. This disintegrin requires an RGDW motif, as well as an intact C-terminus, in order to interact with both platelets and four different types of melanoma cells. Eristostatin causes nonmetastatic SBc12 melanoma cells to show higher susceptibility to specific killing by NK-like TALL-104 cells. While it is known that eristostatin binds to αIIbβ3 on platelets, the receptor with which eristostatin binds to the melanoma cells has not yet been identified.

Scratching below the Surface: Wound Healing and Alanine Mutagenesis Provide Unique Insights into Interactions between Eristostatin, Platelets and Melanoma Cells

To study the molecular mechanism of the disintegrin eristostatin, cellular functional studies were performed using ten recombinant alanine mutants. ADP-induced platelet aggregation revealed critical contributions of seven residues within the ‘RGD loop’ (R24, R27, G28, N31) and C-terminus (W47, N48, G49) of this disintegrin. Using an in vitro scratch wound healing assay, four human melanoma cell lines yielded similar results when exposed to wildtype eristostatin. All eristostatin-treated cells healed less of the wounded area than control conditions. This phenomenon was reproduced when using fibronectin as the matrix. C8161 cells showed significant delay in wound closure with the N-terminal mutant P4A but not with R24A or G28A. Evidence from our laboratory and others suggests neither alpha IIb, alpha 4 nor alpha 5 integrins are directly involved in eristostatin’s interactions. Eristostatin did not affect the number of melanoma cells in culture after 24 h or the development of apoptosis. However, phosphorylation studies performed after these melanoma cells were exposed to eristostatin revealed changes in several tyrosine phosphorylated molecules.

Integrin antagonists affect growth and pathfinding of ventral motor nerves in the trunk of embryonic zebrafish

Integrins are thought to be important receptors for extracellular matrix (ECM) components on growing axons. Ventral motor axons in the trunk of embryonic zebrafish grow in a midsegmental pathway through an environment rich in ECM components. To test the role of integrins in this process, integrin antagonists (the disintegrin echistatin in native and recombinant form, as well as the Arg-Gly-Asp-Ser peptide) were injected into embryos just prior to axon outgrowth at 14-16 h postfertilization (hpf). All integrin antagonists affected growth of ventral motor nerves in a similar way and native echistatin was most effective. At 24 hpf, when only the three primary motor axons per trunk hemisegment had grown out, 80% (16 of 20) of the embryos analyzed had abnormal motor nerves after injection of native echistatin, corresponding to 19% (91 of 480) of all nerves. At 33 hpf, when secondary motor axons were present in the pathway, 100% of the embryos were affected (24 of 24), with 20% of all nerves analyzed (196 of 960) being abnormal. Phenotypes comprised abnormal branching (64% of all abnormal nerves) and truncations (36% of all abnormal nerves) of ventral motor nerves at 24 hpf and mostly branching of the nerves at 33 hpf (94% of all abnormal nerves). Caudal branches were at least twice as frequent as rostral branches. Surrounding trunk tissue and a number of other axon fascicles were apparently not affected by the injections. Thus integrin function contributes to both growth and pathfinding of axons in ventral motor nerves in the trunk of zebrafish in vivo.

Cloning, expression, and characterization of a bi-functional disintegrin/alkaline phosphatase hybrid protein.

Integrins are transmembrane heterodimeric glycoproteins responsible for cellular communication; therefore, they play an essential role in many physiological events. Viper snake venoms contain integrin antagonists called disintegrins which bind and inhibit integrin function. They present a loop containing an RGD motif responsible for integrin binding. The engineering of disintegrins fused to a reporter enzyme will be an interesting approach to build integrin markers. Even more, the disintegrin scaffold could be used to present other protein binding motifs. In this work, we have obtained alkaline phosphatase (APv) tagged eristostatin (Er) by cloning and expressing eristostatin DNA into the pLIP6-GN vector. Eristostatin, a 49 residue disintegrin, binds selectively to alphaIIbbeta3 integrin, inhibiting its binding to fibrinogen. The resulting fusion protein Er/APv was identified by SDS-PAGE and by Western blotting using both anti-Er and anti-AP antibodies. This fusion protein showed enzymatic AP activity similar to that of wild APv and its potential use for an alphaIIbbeta3 integrin assay was tested in a one-step dot blot using immobilized cells incubated with the marker and developed by AP substrate. Er/APv showed selectivity towards platelets and alphaIIbbeta3 integrin transfected cells and reacted with the same region as unlabeled Er, as analyzed in competition assays. Our data present a novel tool, Er/APv, with potential use as molecular marker in processes where the alphaIIbbeta3 integrin is involved.

A simple, novel and robust test to diagnose type I Glanzmann thrombasthenia.

Glanzmann thrombasthenia (GT) is an inherited bleeding disorder due to either absence or dysfunction of fibrinogen binding receptors, i.e either GPIIb (GPIIβ)[1][1] or GPIIIa (GPIIIα)[2][2] on platelet membrane. The complete fibrinogen receptor, i.e. GPIIβGPIII which binds fibrinogen on activated

MONOMERIC AND DIMERIC DISINTEGRINS: PLATELET ACTIVE AGENTS FROM VIPER VENOM

When the term “disintegrin” was first coined in 1990, it described a family of naturally occurring proteins with low molecular weights and highly conserved sequences, both in their cysteine arrangements and adhesive Arg-Gly-Asp (RGD) motifs. Another common characteristic was the inhibitory potential these proteins demonstrated in interacting with cell-surface integrin receptors. Measurement of the effect by disintegrins on the interaction between the platelet receptor α IIbβ 3 and its ligand, fibrinogen, has become a hallmark assay for comparing the activities of members of this increasingly diverse family discovered in the past two decades. This review focuses on the inhibitory profiles, based on platelet function, of the monomeric and heterodimeric disintegrins described to date, as well as the informative contributions of disintegrin mutations in our understanding of the structure–function relationships between ligand and α IIbβ 3. The challenge of naming future examples of these proteins is also addressed.