Cezary Marcinkiewicz

Snake venom disintegrins: novel dimeric disintegrins and structural diversification by disulphide bond engineering

We report the isolation and amino acid sequences of six novel dimeric disintegrins from the venoms of Vipera lebetina obtusa (VLO), V. berus (VB), V. ammodytes (VA), Echis ocellatus (EO) and Echis multisquamatus (EMS). Disintegrins VLO4, VB7, VA6 and EO4 displayed the RGD motif and inhibited the adhesion of K562 cells, expressing the integrin alpha5beta1 to immobilized fibronectin. A second group of dimeric disintegrins (VLO5 and EO5) had MLD and VGD motifs in their subunits and blocked the adhesion of the alpha4beta1 integrin to vascular cell adhesion molecule 1 with high selectivity. On the other hand, disintegrin EMS11 inhibited both alpha5beta1 and alpha4beta1 integrins with almost the same degree of specificity. Comparison of the amino acid sequences of the dimeric disintegrins with those of other disintegrins by multiple-sequence alignment and phylogenetic analysis, in conjunction with current biochemical and genetic data, supports the view that the different disintegrin subfamilies evolved from a common ADAM (a disintegrin and metalloproteinase-like) scaffold and that structural diversification occurred through disulphide bond engineering.

EC3, a Novel Heterodimeric Disintegrin from Echis carinatus Venom, Inhibits α4 and α5 Integrins in an RGD-independent Manner

EC3, a heterodimeric disintegrin (M r = 14,762) isolated from Echis carinatus venom is a potent antagonist of α4 integrins. Two subunits called EC3A and EC3B were isolated from reduced and alkylated EC3 by reverse-phase high performance liquid chromatography. Each subunit contained 67 residues, including 10 cysteines, and displayed a high degree of homology to each other and to other disintegrins. EC3 inhibited adhesion of cells expressing α4β1 and α4β7 integrins to natural ligands vascular cell adhesion molecule 1 (VCAM-1) and mucosal addressin cell adhesion molecule 1 (MadCAM-1) with IC50 = 6–30 nm, adhesion of K562 cells (α5β1) to fibronectin with IC50 = 150 nm, and adhesion of αIIbβ3 Chinese hamster ovary cells to fibrinogen with IC50 = 500 nm; it did not inhibit adhesion of αvβ3 Chinese hamster ovary cells to vitronectin. Ethylpyridylethylated EC3B inhibited adhesion of Jurkat cells to immobilized VCAM-1 (IC50 = 6 μm), whereas EC3A was inactive in this system. The MLDG motif appeared to be essential for activity of EC3B. Linear MLDG peptide inhibited the adhesion of Jurkat to VCAM-1 in a dose-dependent manner (IC50 = 4 mm), whereas RGDS peptide was not active at the same concentration. MLDG partially inhibited adhesion of K562 cells to fibronectin (5–10 mm) in contrast to RGDS peptide (IC50 = 3 mm), inhibiting completely at 10 mm.

Interaction of α9β1 Integrin With Thrombospondin-1 Promotes Angiogenesis

Thrombospondin-1 is a multifunctional protein interacting with several cell surface receptors including integrins. We found that it is a ligand for &agr;9&bgr;1 integrin, and has an integrin binding site within its N-terminal domain (NoC1). Interaction of thrombospondin-1 and its recombinant NoC1 domain with &agr;9&bgr;1 integrin was confirmed in ELISA and cell adhesion assays. Binding of NoC1 to cells expressing &agr;9&bgr;1 integrin activated signaling proteins such as Erk1/2 and paxillin. Blocking of this integrin by monoclonal antibody and the met-leu-asp-disintegrin inhibited dermal human microvascular endothelial cell proliferation and NoC1-induced migration of these cells. Immunohistochemical studies revealed that &agr;9&bgr;1 is expressed on microvascular endothelium in several organs including skin, lung, heart and brain. NoC1 induced neovascularization in an experimental quail chorioallantoic membrane system and Matrigel plug formation assay in mice. This proangiogenic activity of NoC1 in vivo was inhibited by &agr;9&bgr;1 inhibitors. In summary, our results revealed that &agr;9&bgr;1 integrin expressed on microvascular endothelial cells interacts with thrombospondin-1, and this interaction is involved in modulation of angiogenesis.

Importance of the structure of the RGD-containing loop in the disintegrins echistatin and eristostatin for recognition of αIIbβ3 and αvβ3 integrins

Echistatin and eristostatin are structurally homologous disintegrins which exhibit significant functional differences in interaction with various integrins. We hypothesized that this may reflect differences in the sequences of their RGD loops: 20CKRARGDDMDDYC32 and 23CRVARGDWNDDYC35, respectively. Mapping of eristostatin peptides obtained by proteolytic digestion suggested that it has the same alignment of SS bridges as echistatin. Synthetic echistatin D27W resembled eristostatin since it had increased platelet aggregation inhibitory activity, increased potency to block fibrinogen binding to αIIbβ3, and decreased potency to block vitronectin binding to αvβ3 as compared to wild‐type echistatin. Since eristostatin and echistatin have a similar pattern of disulfide bridges, we constructed molecular models of eristostatin based on echistatin NMR coordinates. The RGD loops of eristostatin and echistatin D27W were wider than echistatins due to the placement of tryptophan (rather than aspartic acid) immediately after the RGD sequence. We propose a hypothesis that the width and shape of the RGD loop are important ligand structural features that affect fitting of ligand to the binding pocket of αIIbβ3 and αvβ3.

Identification of sites in the cysteine-rich domain of the class P-III snake venom metalloproteinases responsible for inhibition of platelet function

Atrolysin A and jararhagin are class P‐III snake venom metalloproteinases (SVMPs) with three distinct domains: a metalloproteinase, a disintegrin‐like and a cysteine‐rich. The metalloproteinase and the disintegrin‐like domains of atrolysin A and jararhagin contain peptide sequences that interact with α2β1 integrin and inhibit the platelet responses to collagen. Recently, the recombinant cysteine‐rich domain of atrolysin A was shown to have similar effects, but the sequence(s) responsible for this is unknown. In this report, we demonstrate two complete peptide sequences from the homologous cysteine‐rich domains of atrolysin A and jararhagin that inhibit both platelet aggregation by collagen and adhesion of α2‐expressing K562 cells to this protein. In addition, the peptide effects on platelets do not seem to involve an inhibition of GPVI. These results identify, for the first time, sites in the cysteine‐rich domain of SVMPs that inhibit cell responses to collagen and reveal the complexity of the potential biological effects of these enzymes with multifunctional domains.

Augmentation of integrin-mediated mechanotransduction by hyaluronic acid

Changes in tissue and organ stiffness occur during development and are frequently symptoms of disease. Many cell types respond to the stiffness of substrates and neighboring cells in vitro and most cell types increase adherent area on stiffer substrates that are coated with ligands for integrins or cadherins. In vivo cells engage their extracellular matrix (ECM) by multiple mechanosensitive adhesion complexes and other surface receptors that potentially modify the mechanical signals transduced at the cell/ECM interface. Here we show that hyaluronic acid (also called hyaluronan or HA), a soft polymeric glycosaminoglycan matrix component prominent in embryonic tissue and upregulated during multiple pathologic states, augments or overrides mechanical signaling by some classes of integrins to produce a cellular phenotype otherwise observed only on very rigid substrates. The spread morphology of cells on soft HA-fibronectin coated substrates, characterized by formation of large actin bundles resembling stress fibers and large focal adhesions resembles that of cells on rigid substrates, but is activated by different signals and does not require or cause activation of the transcriptional regulator YAP. The fact that HA production is tightly regulated during development and injury and frequently upregulated in cancers characterized by uncontrolled growth and cell movement suggests that the interaction of signaling between HA receptors and specific integrins might be an important element in mechanical control of development and homeostasis.

Disulphide-bond pattern and molecular modelling of the dimeric disintegrin EMF-10, a potent and selective integrin alpha5beta1 antagonist from Eristocophis macmahoni venom.

The disulphide-bond pattern of the heterodimeric disintegrin EMF-10, a potent and selective integrin alpha(5)beta(1) antagonist from Eristocophis macmahoni venom, was established by combination of amino-acid analysis, N-terminal sequencing and collision-induced dissociation by nanoelectrospray ionization quadrupole ion-trap MS of fragments isolated by reversed-phase HPLC after degradation of EMF-10 with oxalic acid. Each EMF-10 subunit contains four intrachain disulphide bonds. Two interchain cystine residues join the EMF-10 polypeptides. The intrachain linkages are conserved in monomeric disintegrins. A molecular model of EMF-10 was built using averaged NMR co-ordinates of flavoridin as a template. The active hairpin loops of the EMF-10 subunits occupy opposite locations at the ends of an elongated disulphide-bond ladder. In the EMF-10 model the N-terminal polypeptide of EMF-10B is close to the RGD-loop of the EMF-10A subunit, suggesting that the N-terminal region of the B-subunit could potentially influence the biological activity of the A-subunit.

Integrin α9β1 is a receptor for nerve growth factor and other neurotrophins

The integrin α9β1 is a multifunctional receptor that interacts with a variety of ligands including vascular cell adhesion molecule 1, tenascin C and osteopontin. We found that this integrin is a receptor for nerve growth factor (NGF) and two other neurotrophins, brain-derived neurotrophic factor and NT3, using a cell adhesion assay with the α9SW480 cell line. Interaction of α9β1 with NGF was confirmed in an ELISA assay by direct binding to purified integrin. α9β1 integrin binds to neurotrophins in a manner similar to another common neurotrophin receptor, p75NTR (NGFR), although α9β1 activity is correlated with induction of pro-survival and pro-proliferative signaling cascades. This property of α9β1 resembles the interaction of NGF with a high affinity receptor, TrkA, however, this integrin shows a low affinity for NGF. NGF induces chemotaxis of cells expressing α9β1 and their proliferation. Moreover, α9β1 integrin is a signaling receptor for NGF, which activates the MAPK (Erk1/2) pathway. The α9β1-dependent chemotactic ability of NGF appears to result from the activation of paxillin.

Role of multiple β1 integrins in cell adhesion to the disintegrin domains of ADAMs 2 and 3

Abstract ADAM disintegrin domains can support integrin-mediated cell adhesion. However, the profile of which integrins are employed for adhesion to a given disintegrin domain remains unclear. For example, we suggested that the disintegrin domains of mouse sperm ADAMs 2 and 3 can interact with the α6β1 integrin on mouse eggs. Others concluded that these disintegrin domains interact instead with the α9β1 integrin. To address these differing results, we first studied adhesion of mouse F9 embryonal carcinoma cells and human G361 melanoma cells to the disintegrin domains of mouse ADAMs 2 and 3. Both cell lines express α6β1 and α9β1 integrins at their surfaces. Antibodies to the α6 integrin subunit inhibited adhesion of both cell lines. An antibody that recognizes human α9 integrin inhibited adhesion of G361 cells. VLO5, a snake disintegrin that antagonizes α4β1 and α9β1 integrins, potently inhibited adhesion of both cell lines. We next explored expression of the α9 integrin subunit in mouse eggs. In contrast to our ability to detect α6β1, we were unable to convincingly detect α9β1 integrin on the surface of mouse eggs. Moreover, treatment of mouse eggs with 250 nm VLO5, which is 250 fold over its ∼IC50 for inhibition of somatic cell adhesion, had minimal effect on sperm-egg binding or fusion. We did detect α9 integrin protein on epithelial cells of the oviduct. Additional studies showed that antibodies to the α6 and α7 integrins additively inhibited adhesion of mouse trophoblast stem cells and that an antibody to the α4 integrin inhibited adhesion of MOLT-3 cells to these disintegrin domains: Our data suggest that multiple integrins (on the same cell) can participate in adhesion to a given ADAM disintegrin domain and that interactions between ADAMs and integrins may be important for sperm transit through the oviduct.

Compression stiffening of brain and its effect on mechanosensing by glioma cells

Many cell types, including neurons, astrocytes and other cells of the central nervous system respond to changes in extracellular matrix or substrate viscoelasticity, and increased tissue stiffness is a hallmark of several disease states including fibrosis and some types of cancers. Whether the malignant tissue in brain, an organ that lacks the protein-based filamentous extracellular matrix of other organs, exhibits the same macroscopic stiffening characteristic of breast, colon, pancreatic, and other tumors is not known. In this study we show that glioma cells like normal astrocytes, respond strongly in vitro to substrate stiffness in the range of 100 to 2000 Pa, but that macroscopic (mm to cm) tissue samples isolated from human glioma tumors have elastic moduli on the order of 200 Pa that are indistinguishable from those of normal brain. However, both normal brain and glioma tissues increase their shear elastic moduli under modest uniaxial compression, and glioma tissue stiffens more strongly under compression than does normal brain. These findings suggest that local tissue stiffness has the potential to alter glial cell function, and that stiffness changes in brain tumors might arise not from increased deposition or crosslinking of collagen-rich extracellular matrix but from pressure gradients that form within the tumors in vivo.