Interaction of lipopolysaccharide with anticoagulant protein tachyplesin from hemocytes of horseshoe crab

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In humans, lipopolysaccharide (LPS) released during infection by Gram-negative bacteria can cause the severe pathological changes associated with septic shock. In the USA,septic shock is responcible for about 100,000 deaths annually and no specific drugs are available. LPS is the principal component of the outer leaflet of the outer membrane of Gram-negative bacteria. Lipid A,the membrane anchor of LPS,consists of a central phosphodisaccharide unit that is attached to up to seven fatty acid chains and it prossesses most of the biological activities of LPS.The toxicity in humans arises from the interaction of LPS or Lipid A with membrane-bound receptors or serum proteins, leading to an increase in the pro-inflammatory mediators (e.g.tomor necrosis factor, interleukin-1 and interleukin-6).Horseshoe crab (Tachypleus tridentatus) are ancient arachnids that possess a primitive circulatory system, the hemolymph, containing only one kind of cell, the hemocyte. Exposure of hemocytes to bacterial endotoxins (LPS) results in the activation of an intracellular coagulation cascade, a defense against microbial invasion. The system consists of several proteins, including tachyplesin, tachycitin and tachystatin that may inhibit the cascade. These are small basic proteins, which bind and neutralize LPS and have strong anti-bacterial effects on the growth of Gram-negative bacteria.In this study, we studied the interaction of tachyplesin with synthetic lipid A and found that negative charges of phsphate groups of lippid A interact with positive charges of tachyplesin. The amphipathic loop was proposed as an important motif of LPS binding site and may be used in the design of molecules with therapeutic properties against septic shock.distance geometry calculation by using X-PLOR.In the structure of tachycitin, we found the chitinbinding motif like hevein. The main chain structure of tachystatin was similar to that of Ca-channel blocker omega-conotoxin.