α2β1 Integrin Is Not Recognized by Rhodocytin but Is the Specific, High Affinity Target of Rhodocetin, an RGD-independent Disintegrin and Potent Inhibitor of Cell Adhesion to Collagen

Abstract

and the ¶Institut fü r Physikalische Chemie, Schlossplatz 7, Universitä t Mü nster, 48149 Mü nster, GermanyWe have recombinantly expressed a soluble form of human ␣ 2 ␤ 1 integrin that lacks the membrane-anchoring transmembrane domains as well as the cytoplasmic tails of both integrin subunits. This soluble ␣ 2 ␤ 1 integrin binds to its collagen ligands the same way as the wildtype ␣ 2 ␤ 1 integrin. Furthermore, like the wild-type form, it can be activated by manganese ions and an integrinactivating antibody. However, it does not bind to rhodocytin, a postulated agonist of ␣ 2 ␤ 1 integrin from the snake venom of Calloselasma rhodostoma, which elicits platelet aggregation. Taking advantage of the recombinantly expressed, soluble ␣ 2 ␤ 1 integrin, an inhibition assay was established in which samples can be tested for their capability to inhibit binding of soluble ␣ 2 ␤ 1 integrin to immobilized collagen. Thus, by scrutinizing the C. rhodostoma snake venom in this protein-protein interaction assay, we found a component of the snake venom that inhibits the interaction of soluble ␣ 2 ␤ 1 integrin to type I collagen efficiently. N-terminal sequences identified this inhibitor as rhodocetin, a recently published antagonist of collagen-induced platelet aggregation. We could demonstrate that its inhibitory effect bases on its strong and specific binding to ␣ 2 ␤ 1 integrin, proving that rhodocetin is a disintegrin. Standing apart from the growing group of RGD-dependent snake venom disintegrins, rhodocetin interacts with ␣ 2 ␤ 1 integrin in an RGD-independent manner. Furthermore, its native conformation, which is stabilized by disulfide bridges, is indispensibly required for its inhibitory activity. Rhodocetin does not contain any major collagenous structure despite its high affinity to ␣ 2 ␤ 1 integrin, which binds to collagenous molecules much more avidly than to noncollagenous ligands, such as laminin. Blocking ␣ 2 ␤ 1 integrin as the major collagen receptor on platelets, rhodocetin is responsible for hampering collagen-induced, ␣ 2 ␤ 1 integrin-mediated platelet activation, leading to hemorrhages and bleeding disorders of the snakebite victim. Moreover, having a widespread tissue distribution, ␣ 2 ␤ 1 integrin also mediates cell adhesion, spreading, and migration. We showed that rhodocetin is able to inhibit ␣ 2 ␤ 1 integrin-mediated adhesion of fibrosarcoma cells to type I collagen completely.Integrins are cell adhesion molecules that consist of two noncovalently associated subunits, ␣ and ␤ (for review see Refs. 1 and 2). The subfamily of integrins sharing the ␤ 1 subunit are well known receptors for extracellular matrix molecules, such as collagens, laminins, and fibronectin. The subfamily of ␤ 3 subunit containing cytoadhesins comprise the platelet integrin, ␣ IIb ␤ 3 which binds fibrinogen/fibrin (3) and the vitronectin receptor ␣ V ␤ 3 . The latter ones, along with several ␤ 1 integrin, such as the fibronectin receptor ␣ 5 ␤ 1 integrin, recognize a linear arginyl-glycyl-aspartyl sequence within their respective ...

Plumieribetin, a Fish Lectin Homologous to Mannose-binding B-type Lectins, Inhibits the Collagen-binding α1β1 Integrin

“…It is abundantly expressed on hepatocytes and vascular smooth muscle cells. At the cellular level, plumieribetin failed to block attachment of these cell types to the collagen IV fragment CB3, even when the compensating ␣2␤1 integrin was blocked by rhodocetin (16,31). However, plumieribetin weakened the interaction of both cell types and resulted in rounding up of the otherwise flat cells and in rearrangement of the actin cytoskeleton.…”

“…13, the production of collagen I is described in Ref. 16, and the production of laminin-332 from the supernatant of SCC25 cells is described in Ref. 28.…”

“…Inhibition ELISA-The inhibition ELISA with soluble integrins was performed as previously described (16). Briefly, 10 g/ml CB3, 6 g/ml laminin-332, and laminin-111 in 50 mM Tris/HCl, pH 7.4, 150 mM NaCl 2 mM MgCl 2 (TBS/Mg) buffer and 10 g/ml collagen I in 0.1 M acetic acid were coated onto multiwell plate at 4°C overnight.…”

“…Materials-The soluble human integrins, ␣1␤1, ␣2␤1, ␣3␤1, and ␣7␤1, were produced in transfected Schneider S2 cells and isolated as previously described (16,27). Soluble ␣1␤1 integrin was deglycosylated by incubation of the integrin with recombinant protein-N-glycosidase F (Roche Applied Science) in TBS, 2 pH 7.4, supplemented with 2 mM MgCl 2 for 13 h at 21°C.…”

“…Several C-type lectins have been identified as versatile toxins against platelet receptors (15). Among them, rhodocetin from the Malayan pit viper (Calloselasma rhodostoma) was characterized as a specific ␣2␤1 integrin antagonist (16), which is able to block ␣2␤1 integrin-mediated migration and infiltration of tumor cells (17). C-type lectins are one subclass of lectins that are characterized by a huge structural and functional variability.…”