Journal of Cellular Biochemistry 93:1272–1281 (2004) Unique Precipitation and Exocytosis of a Calcium Salt of myo-Inositol Hexakisphosphate in Larval Echinococcus granulosus Florencia Irigoı ´n, 1 Cecilia Casaravilla, 1 Francisco Iborra, 2 Robert B. Sim, 3 Fernando Ferreira, 4 and Alvaro Dı ´az 1 * 1 Ca ´tedra de Inmunologı ´a, Facultad de Quı ´mica/Ciencias, Universidad de la Repu ´blica, Uruguay 2 MRC Molecular Haematology Unit, The Weatherall Institute of Molecular Medicine, University of Oxford, United Kingdom 3 MRC Immunochemistry Unit, Department of Biochemistry, University of Oxford, United Kingdom 4 Laboratorio de Carbohidratos y Glicoconjugados, Facultad de Quı ´mica/Medicina, Universidad de la Repu ´ blica, Uruguay Abstract The ubiquitous intracellular molecule myo-inositol hexakisphosphate (IP 6 ) is present extracellularly in the hydatid cyst wall (HCW) of the parasitic cestode Echinococcus granulosus. This study shows that extracellular IP 6 is present as its solid calcium salt, in the form of deposits that are observed, at the ultrastructural level, as naturally electron dense granules some tens of nanometers in diameter. The presence of a calcium salt of IP 6 in these structures was determined by two different electron microscopy techniques: (i) the analysis of the spatial distribution of phosphorus and calcium in the outer, acellular layer of the HCW (the laminated layer, LL) through electron energy loss spectroscopy, and (ii) the observation, by transmission electron microscopy, of HCW that were selectively depleted of IP 6 by treatment with EGTA or phytase, an enzyme that catalyses the dephosphorylation of IP 6 . The deposits of the IP 6 -Ca(II) salt are also observed inside membrane vesicles in cells of the germinal layer (the inner, cellular layer of the HCW), indicating that IP 6 precipitates with calcium within a cellular vesicular compartment and is then secreted to the LL. Thus, much as in plants (that produce vesicular IP 6 deposits), the existence of transporters for IP 6 or its precursors in internal membranes is needed to explain the compound’s cellular localisation in E. granulosus. J. Cell. Biochem. 93: 1272 – 1281, 2004. ß 2004 Wiley-Liss, Inc. Key words: calcium; phytic acid; inositol polyphosphates; cestode; hydatidosis myo-Inositol hexakisphosphate (IP 6 ) is an ubiquitous intracellular molecule in eukar- yotes, thought to have a cytosolic and nuclear distribution [Irvine and Schell, 2001; Shears, 2001; Raboy, 2003]. In a previous article, we reported that in the larva of the parasitic cestode Echinococcus granulosus, IP 6 is loca- lised extracellularly [Irigoı ´n et al., 2002]. This larva has the form of a fluid-filled cyst, limited by a two-layered structure named the hydatid cyst wall (HCW). The outer layer of the HCW, in direct contact with the host tissue, is the laminated layer (LL), an acellular meshwork composed mainly of glycoconjugates [Kilejian et al., 1962; Kilejian and Schwabe, 1971]. The LL, attaining up to 2 mm thickness, is a con- spicuous structure found only in the members of the genus Echinococcus. It is synthesised by the underlying, cellular layer of the HCW, named germinal layer. The LL provides the cyst with mechanical support and protects the parasite cells from direct contact with the potentially ß 2004 Wiley-Liss, Inc. Abbreviations used: IP 6 , myo-inositol hexakisphosphate; HCW, hydatid cyst wall; LL, laminated layer; PEI, polyethyleneimine; TCA, trichloroacetic acid; EELS, elec- tron energy loss spectroscopy; TEM, transmission electron microscopy; ER, endoplasmic reticulum. Florencia Irigoı ´n’s present address is Center for Free Radical and Biomedical Research, Departamento de Bio- quı ´mica, Facultad de Medicina, Universidad de la Repu ´ b- lica, Uruguay. *Correspondence to: Alvaro Dı ´az, Ca ´ tedra de Inmunologı ´a, Instituto de Higiene, Avda. Alfredo Navarro 3051, piso 2. CP 11600, Montevideo, Uruguay. E-mail: adiaz@fq.edu.uy Received 2 April 2004; Accepted 4 June 2004 DOI 10.1002/jcb.20262