Characterization of myo-inositol hexakisphosphate deposits from larval Echinococcus granulosus Cecilia Casaravilla1, Charles Brearley2, Silvia Soule´ 3, Carolina Fontana3, Nicola´ s Veiga4, Marı´a I. Bessio3, Fernando Ferreira3, Carlos Kremer4 and Alvaro Dı´az1
1 Ca´ tedra de Inmunologı´a, Facultad de Quı´mica ⁄ Ciencias, Universidad de la Repu´ blica, Montevideo, Uruguay 2 School of Biological Sciences, University of East Anglia, Norwich, UK 3 Laboratorio de Carbohidratos y Glicoconjugados, Departamento de Quı´mica Orga´ nica, Facultad de Quı´mica ⁄ Facultad de Ciencias ⁄ Facultad de Medicina, Universidad de la Repu´ blica, Montevideo, Uruguay 4 Ca´ tedra de Quı´mica Inorga´ nica, Departamento Estrella Campos, Facultad de Quı´mica, Universidad de la Repu´ blica, Montevideo, Uruguay
Keywords calcium; inositol hexakisphosphate; inositol pentakisphosphate; magnesium; phytic acid
Correspondence A. Dı´az, Ca´ tedra de Inmunologı´a, Instituto de Higiene, Avda, Alfredo Navarro 3051, piso 2. CP 11600, Montevideo, Uruguay Fax: +5982 487 43 20 Tel: +5982 487 43 20 E-mail: adiaz@fq.edu.uy
(Received 24 March 2006, revised 16 May 2006, accepted 18 May 2006)
doi:10.1111/j.1742-4658.2006.05328.x
The abundant metabolite myo-inositol hexakisphosphate (InsP6) can form vesicular deposits with cations, a widespread phenomenon in plants also found in the cestode parasite, Echinococcus granulosus. In this organism, the deposits are exocytosed, accumulating in a host-exposed sheath of extracellular matrix termed the laminated layer. The formation and mobil- ization of InsP6 deposits, which involve precipitation and solubilization reactions, respectively, cannot yet be rationalized in quantitative chemical terms, as the solids involved have not been formally described. We report such a description for the InsP6 deposits from E. granulosus, purified as the solid residue left by mild alkaline digestion of the principal mucin compo- nent of the laminated layer. The deposits are largely composed of the compound Ca5H2LÆ16H2O (L representing fully deprotonated InsP6), and additionally contain Mg2+ (6–9% molar ratio with respect to Ca2+), but not K+. Calculations employing recently available chemical constants show that the precipitation of Ca5H2LÆ16H2O is predicted by thermodynamics in secretory vesicle-like conditions. The deposits appear to be similar to microcrystalline solids when analysed under the electron microscope; we estimate that each crystal comprises around 200 InsP6 molecules. We calcu- late that the deposits increase, by three orders of magnitude, the surface area available for adsorption of host proteins, a salient ability of the lamin- ated layer. The major inositol phosphate in the deposits, other than InsP6, is myo-inositol (1,2,4,5,6) pentakisphosphate, or its enantiomer, inositol (2,3,4,5,6) pentakisphosphate. The compound appears to be a subproduct of the intracellular pathways leading to the synthesis and vesicular accumu- lation of InsP6, rather than arising from extracellular hydrolysis of InsP6.
phytates). The most abundant of these contain mag- nesium, potassium and calcium [1]. In developing Ara- bidopsis seeds, phytates containing Mg2+ ⁄ K+ ⁄ Ca2+, Mn2+ and Zn2+ are located in distinct vesicular com- partments [5]. In spite of extensive studies [6,7], no
myo-Inositol hexakisphosphate (InsP6) is a ubiquitous compound in eukaryotic cells [1–3]. In animal systems, it generally has a cytosolic and a nuclear distribution [4]. In addition, in plant storage tissues it forms insol- (often called uble deposits with inorganic cations
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Abbreviations al, attolitres; GL, germinal layer; HCW, hydatid cyst wall; InsP6, myo-inositol hexakisphosphate; InsP5, myo-inositol pentakisphosphate; LL, laminated layer.
C. Casaravilla et al. Echinococcus granulosus InsP6 deposits
stoichiometry has yet, to our knowledge, been reported for a plant phytate. This means that the quantitative tools of chemistry cannot be applied to the biological processes of phytate deposit formation and mobiliza- tion.
Our recent work has shown that solid InsP6 deposits are also formed in an animal system, namely the larva the parasitic cestode Echinococcus granulosus. In of this system, InsP6 reaches a vesicular compartment and precipitates mainly with Ca2+, the resulting solid then being exocytosed and accumulating in major amounts in the extracellular medium [8,9].
(discussed below,
The extracellular structure in which InsP6 accumu- lates in E. granulosus is an unusual one, being the so-called laminated layer (LL), a mm-thick outer layer that protects the bladder-like parasite larva (termed hydatid cyst) from attack by host cells. The LL is syn- thesized by the thin, underlying germinal layer (GL) of the parasite, which makes contact with the LL through an outer syncitial tegument [10]. Across the genus Echi- nococcus, the fundamental component of the LL is a meshwork of carbohydrate-rich fibrils [11] probably made up from mucin-like molecules [12]. In addition, the E. granulosus LL uniquely contains the ‘electron- dense granules’ [13] that we have shown to be InsP6 deposits [9]. These are observed to occur individually within vesicles in the GL tegumental cells, displaying a defined 41-nm size and subspherical shape [13]. After exocytosis onto the LL, the granules seem to associate with the fibrils and to cluster together, losing individu- ality [9].
The E. granulosus LL is an ultrastructurally simple, acellular, assembly, its only detectable components being the mucin-rich fibrils and the InsP6-rich granules [13]. This fact, together with the massive scale in which InsP6 accumulates, makes working with the InsP6 deposits in this system relatively straightforward. In this article we describe the purification and comprehensive characterization of these deposits, supported by recently available chemical information on InsP6 solids [13a]. We define chemically the major solid constituent of the deposits and show that its formation is predicted from thermodynamic constants under the relevant biological conditions.
purify the InsP6 deposits after treatment of pulverized hydatid cyst walls (HCW) with dissociating agents were unsuccessful. Meanwhile, it was observed that the alkaline hydrolysis used to free the LL glycans from their putative mucin cores solubilized the mater- ial with the exception of a solid that dissolved in the presence of EDTA or strong acid, characteristics expected of calcium InsP6. In addition, the available information on the chemistry of InsP6 solids [13a] indicated that preservation of these compounds under alkaline conditions was to be expected. Starting with the pulverized HCW, the alkaline treatment resulted in increasing losses of mass from the solid phase, reach- ing a plateau corresponding to (cid:1) 80% solubilization towards 48 h of treatment (Fig. 1A). The remaining the supernatant, contained InsP6 in solid, but not amounts similar to those in the starting material its 1H-NMR spectrum (after (Fig. 1B). Furthermore, solubilization by use of Dowex resin in proton form) showed strong signals corresponding only to InsP6 less-abundant myo-inositol and to an accompanying, (InsP5) and pentakisphosphate shown in Fig. 3). In agreement, when the solid was dissolved in EDTA and run on SDS ⁄ PAGE, only trace amounts of protein were detectable (estimated on the basis of Coommassie Blue staining at less than 0.5% by mass of solid). While we considered the pos- sibility that parasite proteins nucleating calcium InsP6 precipitation were present within the deposits, MS peptide fingerprinting detected only host-derived proteins. The LL carbohydrates were found to be in the supernatant, the residue being basically devoid of sugars (Fig. 1A). In agreement with solubilization of the sugars being caused by release from mucins, the kinetics of mass loss from the solid phase were paralleled by an increase in the absorbance (A) at 240 nm (Fig. 1A); this parameter is usually employed to monitor the formation of derivatives of 2-amino propenoic and 2-amino buten-2-oic acids, the prod- ucts of b-elimination of O-glycosylated serine and threonine residues abundant in mucins [14]. Thus, mild alkaline hydrolysis solubilizes the LL mucins selec- tively, while leaving in the insoluble phase a material that contains InsP6 as the only abundant organic molecule.
Results
The major component of the purified InsP6 deposits has the stoichiometry Ca5H2LÆ16H2O (L being deprotonated InsP6)
Mild alkaline treatment of the E. granulosus cyst wall hydrolyses and solubilizes the putative mucins, while leaving InsP6 in the solid phase
The carbohydrate-rich fibril meshwork of the LL is singularly difficult to solubilize. Therefore, attempts to
The results described above made it plausible that the deposits were formed by a single major compound and could thus be assigned a defined stoichiometry. As we
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similar to synthetic calcium InsP6 that had been subjec- ted in parallel to alkaline treatment, washing and dry- ing. Consistent with their very low protein content, the E. granulosus deposits had undectectable levels of N (Table 1). Their C and Ca2+ contents and density were generally intermediate between those of calcium InsP6 treated in parallel and those of the same (untreated) compound. This probably reflected losses of crystalliza- tion water, taking place during sample treatment and affecting the biological samples to a lesser extent than the synthetic compound. InsP6 solids are known to lose crystallization water readily, even under mild condi- tions (N. Veiga & C. Kremer, unpublished results).
The solubility of InsP6 salts increases markedly from Ca2+ to Mg2+ and from Mg2+ to the monovalent cati- ons [13a]. Hence, assessing the presence of Mg2+ and monovalent cations in the deposits required precautions against solubilization of InsP6 solids, especially during the final washing steps. Therefore, we included, in our analysis, samples that had not been subjected to wash- ing (Table 2). The K+ contents of the Echinococcus solids were not significantly different from those of the control calcium InsP6 samples; neither did Na+ con- tents appear to be significant. In contrast, the biological solids contained significant amounts of Mg2+, which was present at a 6–9% molar ratio with respect to Ca2+. Mg2+ levels were similar between samples sub- jected and not subjected to washes (observed to cause some solubilization of solid), suggesting that the metal is found within the solid lattice rather than externally adsorbed. Taken together, the data are consistent with the biological solid in its native context being formed by Ca5H2LÆ16H2O, with a minor contribution from magnesium InsP6 (probably Mg5H2LÆ22H2O) [15].
Fig. 1. Purification of calcium InsP6 deposits through alkaline diges- tion of the laminated layer (LL) mucins. Pulverized bovine cyst walls were subjected to alkaline digestion for different periods of time, the remaining conditions being those detailed in the Experimental procedures. The insoluble residues were washed and dried by pro- cedure 2, also as detailed in the Experimental procedures. (A) Kin- etics of solubilization in terms of mass in the insoluble residue, carbohydrates in residue and in supernatant, and the absorbance (A) at 240 nm (indicative of b-elimination of O-glycosylated serine and threonine residues). Carbohydrates were estimated as the sum of masses of galactose, N-acetylgalactosamine and N-acetylgluco- samine, which together make up over 90% of the total LL sugar ([12,42] and our unpublished data). Solubilized carbohydrates are degraded under the hydrolysis conditions (which do not include a reducing agent for the free carbonyls generated), and thus the data after 24 h were not plotted. Similarly, the unsaturated amino acids generated by b-elimination are hydrolysed, with loss of A240 nm, and the corresponding data were thus only plotted up to 48 h. Error bars on the insoluble mass and A240 nm plots represent standard deviations for independent hydrolyses carried out in triplicate. (B) InsP6 is retained in the insoluble residue throughout the hydrolysis period as assessed by TLC in [8].
Yields of solid were (cid:1) 22 g and 11 g per 100 g of initial dry mass for bovine materials and for mouse materials, respectively (as assessed by procedure 2; see the Experimental procedures). These values are consid- erably lower than our estimates of the calcium InsP6 content in the HCW, namely 37% and 24% of the total dry mass for bovine and mouse materials, respectively [9]. Mass losses can be expected to result from loss of crystallization water, solubilization during washing and incomplete recovery of finely divided par- ticles in the centrifugation steps.
The purified InsP6 deposits appear to be similar to microcrystalline solids under transmission electron microscopy
had previous evidence that the major counterion of InsP6 in the LL was Ca2+ [8], we chose to compare our purified material with synthetic calcium InsP6. Calcium InsP6 prepared under a variety of conditions has the stoichiometry Ca5H2LÆ16H2O (L being fully deprotonated InsP6) [13a].
By infrared spectroscopy (data not shown), elemental analysis for C, H and N, and additional determination of Ca2+ (Table 1), the purified materials were very
When laid on coated copper grids and observed unstained under the transmission electron microscope, the naturally electron-dense solid (Fig. 2) was strongly
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Table 1. C, H, N, S and Ca2+ contents, and density of purified InsP6 deposits. InsP6 deposits were purified by alkaline hydrolysis of the Echinococcus granulous cyst wall followed by washing and drying by two different procedures, as detailed in the Experimental procedures. Synthetic calcium InsP6 was subjected to alkaline treatment, washing and drying in parallel for comparison. C, H, N and S were determined by automated elemental analysis, and Ca2+ by atomic absorption. Density measurements are given as the mean ± SD of at least three inde- pendent determinations. ND, not determined.
C (% mass) H (% mass) N (% mass) S (% mass) Ca (% mass) Density (gÆcm)3)
6.3 6.4a 7.5 3.5 3.3a 2.6 – 0.0 0.0 – 0.0 0.0 17.6 17.7a 21.3 – 1.80 ± 0.05 2.10 ± 0.01 Procedure 1
Procedure 2 7.0 6.8 7.0 2.7 3.0 3.4 0.0 0.0 0.0 0.0 0.0 0.0 16.2 16.6 21.0 1.97 ± 0.07 ND ND
a Data from [13a].
Ca5H2LÆ16H2O (theoretical) Synthetic calcium InsP6 Synthetic calcium InsP6, subjected to alkaline treatment Purified bovine material Purified mouse material Synthetic calcium InsP6, subjected to alkaline treatment Purified bovine material 7.0 3.7 0.0 0.0 19.0 2.07 ± 0.05
Table 2. K+, Na+ and Mg2+ contents of InsP6 deposits. InsP6 deposits were purified by three different procedures, as detailed in Experimental procedures, synthetic calcium InsP6 being treated in parallel in each case. Note that in procedure 3, designed to pre- clude all solubilization of InsP6 solids, washes were omitted and the samples were freeze-dried as suspensions in 0.1 M NaOH. Hence, absolute metal contents cannot be given, and all data are presented as relative values with respect to Ca2+ content. ND, not determined.
K+a Na+a Mg2+a
ND ND 0.2 Procedure 1 Synthetic calcium InsP6,
ND ND ND ND 1.2 0.2 7.2 6.0 0.4 subjected to alkaline treatment Purified bovine material Purified mouse material Procedure 2 Synthetic calcium InsP6,
0.2 0.2 2.3 2.1 1.0 – 8.7 6.2 0.4 subjected to alkaline treatment Purified bovine material Purified mouse material Procedure 3 Synthetic calcium InsP6,
a % molar ratios with respect to Ca2+.
subjected to alkaline treatment Purified bovine material 2.1 – 8.8
Fig. 2. Transmission electron microscopy of purified InsP6 deposits. Purified deposits were suspended in chloroform, laid on copper grids and observed at 80 kV; the materials shown were processed by procedure 1, and equivalent results were obtained using proce- dure 2 (see the Experimental procedures).
reminiscent of the granules observed in tissue sections [13]. However, it was apparent that the individuality of the granules was partially lost during purification: although far from having formed a compact solid, the corpuscles appeared to have partially fused. Volumes, or lumps, of solid envisaged to derive from individual granules were between 20 and 100 nm in size, as opposed to the defined 41-nm size reported for the granules [13]. Within each ‘lump’, a clear substructure was observed, consisting of several, relatively electron- luscent, smaller subspherical volumes ((cid:1) 7–9 nm in
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the purified E. granulosus
InsP6 deposits
Table 3. Numerical estimations in relation to InsP6 deposits. ‘Ideal’ granules and the crystals that compose them were taken to be per- fectly spherical for the purpose of estimating their volumes or sur- face areas. InsP6 deposits were taken to have the stoichiometry Ca5H2LÆ16H2O (formula weight 1138.6). The density of the lamin- ated layer (LL) was calculated on the basis of it comprising an aqueous gel and a (dispersed) solid phase consisting of InsP6 deposits, by the expression D ¼ 1 ⁄ [(mInsP6 ⁄ DInsP6) + (mgel ⁄ Dgel)], where mInsP6 and DInsP6 represent the mass fraction in the LL and densities of the InsP6 deposits, and mgel and Dgel represent the same two parameters for the aqueous gel. The mass fractions were calculated from our previous estimations of the calcium InsP6 mass to total dry mass ratio (37%) and total dry to total wet mass ratio (13%) for bovine cyst walls ([9] and our unpublished results). The density of the aqueous gel was taken to be the same as that of the extracellular fluid bathing it (assumed to be 1 gÆcm)3), and the density of InsP6 deposits was taken to be that of untreated cal- cium InsP6 (i.e. 1.80 gÆcm)3) (Table 1). The estimation of total InsP6 deposit surface does not take into account granule coalescence interactions in vivo in the LL [9], and is thus and granule–fibril slightly inflated.
from 1.80 ± 0.05 gÆcm)3 to values similar to those (2.0– of 2.1 gÆcm)3; Table 1). As this increase was ascribed to loss of crystallization water, we took the lower figure to be the best estimate of the density of the deposits found in vivo. This figure, together with the stoichiometry Ca5H2LÆ16H2O, allowed us to estimate that an ‘ideal’ granule (taken to be a sphere 41 nm in diameter [13]) would contain some 3 · 104 InsP6 molecules (Table 3). Similarly, an ‘ideal’ putative individual crystal (taken to be 7.3 nm in diameter) [13] would contain in the order of 2 · 102 molecules of InsP6. Further calculations that take into account the estimated density of the LL show that (cid:1) 3% of the LL volume is occupied by InsP6 depos- its (Table 3). Finally, a 1 cm2 portion of LL, of typical 1 mm thickness, comprises some 4000 cm2 of InsP6 deposit surface area. Even if, because of granule coales- cence and interaction between granules and LL fibrils, part of this area is not actually available, the deposits still offer an enormous surface available for the adsorp- tion of diffusible molecules, including host proteins.
Observed precipitation of calcium InsP6 is predicted from the background chemistry applied to vesicular system conditions
a Estimations for LL from bovine hydatid cysts.
3.6 · 10)17 6.5 · 10)17 3 · 104 5.3 · 10)11 2.0 · 10)19 3.7 · 10)19 2 · 102 1.02 2.7% 8 · 1014 4 · 104 Volume of an ideal 41 nm granule (cm3) Mass of an ideal 41 nm granule (g) InsP6 molecules per ideal 41 nm granule Surface area of an ideal 41 nm granule (cm2) Volume of an ideal 7 nm crystal (cm3) Mass of an ideal 7 nm crystal (g) InsP6 molecules per ideal 7 nm crystal Density of LL (gÆcm)3)a LL volume occupied by InsP6 depositsa Number of granules per LL volume (cm)3)a InsP6 deposit surface area per LL volume (cm)1)a
diameter each) separated by areas of higher electron density. This corresponds very well with the observa- tion made by Richards [13], who reported ‘in favour- able sections’, ‘individual electron-dense bodies seen to be composed of several small, electron-luscent spheres’, each 7–8 nm in diameter. Our interpretation is that each granule consists of several individual crystals fused together during the precipitation process. In other words, individual deposits would be microcrys- talline solids; in such solids, it is usual that individual crystals appear more electron-luscent, while grain borders, formed by disordered molecules, are more electron-dense [16].
InsP6 deposits represent a host-exposed surface three orders of magnitude larger than the external cyst surface
The density of synthetic calcium InsP6 increased upon alkaline hydrolysis and washing ⁄ drying of the solid,
We wished to know if the vesicular precipitation of InsP6, as found in E. granulosus, was predictable solely in terms of the chemical interactions between InsP6, and Ca2+ and Mg2+; we were particularly interested in mildly acidic, secretory vesicle-like conditions. We assumed that each vesicle gives rise to a single 41 nm deposit, as documented in a previous publication [13], and considered two extreme possible vesicle volumes. The low extreme was taken to correspond to a vesicle tightly binding an ideal 41 nm granule [i.e. having a luminal volume of (cid:1) 4 · 10)2 attolitres (al) (Table 3)]. The high extreme was estimated from the dimensions of the (presumably mature) InsP6 deposit-containing secretory vesicles observed in the syncitial tegument of the GL cells. These measure (cid:1) 250 nm in length by 100 nm in diameter ([13] and our own unpublished images); approximating them to cylinder yields of a volume of 2 al (as a comparison, endosomal volumes in animal cells range between 0.7 and 88 al [17]). From this value and the InsP6 content of a granule (Table 3), InsP6 ‘total concentrations’ (encompassing soluble and precipitated compound) of 1.6 m and 0.03 m were derived. The pH was taken to range from 7.2 to 5.5 (i.e. the values thought to prevail in the Golgi appar- atus and secretory vesicles, respectively) [18,19]. Free Mg2+ concentration was fixed at 0.5 mm. Then appro- the above conditions were priate combinations of
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lar hydrolysis of InsP6, as it was also the second most abundant inositol phosphate in an extract from the GL cells (Fig. 4D). Upon labeling of small cysts in cul- ture with [3H]inositol, the only detectable labeled InsP5 (Fig. 4E) migrated with the Ins(2,3,4,5,6) ⁄ (1,2,4,5,6)P5 [3H]inositol-labeled standard (Fig. 4F). In addition, parasites showed labeled peaks corresponding to InsP6 and to two different inositol monophosphate species (Fig. 4E).
Discussion
plugged into the hyss program [20] loaded with all 22 relevant equilibrium contant values [13a]. When com- bined with the additional condition that free Ca2+ (in the mm range) should be present after precipitation, the single prediction obtained (throughout the range of pH and vesicle volumes) was the precipitation of 100% of the InsP6 present as Ca5H2LÆ16H2O. This means that the observed presence of Mg2+ in the solid must result from a nonequilibrium phenomenon. This phenomenon could be the occlusion of magnesium InsP6 during the (fast) precipitation of the calcium salt. Alternatively, it may concern the access of Ca2+ to the precipitation compartment: the mass of Ca2+ counterions required might outstretch the cellular capacity to deliver the cat- ion into that compartment. In this respect, coprecipita- tion of Mg2+ was predicted by hyss under the whole range of conditions detailed above, provided that equi- libration of free Ca2+ with the vesicular system at large was not allowed. Thus, using total Ca2+ values slightly below the 5 : 1 ratio with respect to InsP6, ratios of Mg2+ ⁄ Ca2+ : InsP6 encompassing the observed values (Table 2) were predicted, depending on the precise size of the Ca2+ deficit introduced.
Formation of solids is an important aspect of InsP6 biology, the understanding of which is still incomplete. In this article, we addressed this issue for InsP6 depos- its from larval E. granulosus. The deposits were puri- fied in native form and deduced to be composed largely of the Ca5H2LÆ16H2O salt. Mg2+ was also present, at a 6–9% molar ratio, with respect to Ca2+. Mg2+ is likely to have also InsP6 as counterion: the possibility that it is the specific counterion of the InsP5 also found in the solid is remote, as it is unlikely that the salt formed between these two ions is substantially less soluble than the salts formed by Mg2+ and InsP6, and by Ca2+ and InsP5.
[1 ⁄ 3-OH]Inositol pentakisphosphate accompanies InsP6 in the purified deposits and in intact parasite tissue
During our initial detection of InsP6 in the HCW, the compound was observed to be accompanied by mole- cule(s) present at a few percentage relative abundance, and migrating in TLC similarly to InsP5 [8]. In the purified deposits, InsP6 was also accompanied by an InsP5, which was determined by 1H-NMR spectrosco- py to correspond to the Ins(2,3,4,5,6) ⁄ (1,2,4,5,6)P5 enantiomeric pair (Fig. 3). Abundance of this com- pound relative to InsP6 was estimated on the basis of the NMR signals at 10% and 8% for material from bovine and mouse cysts, respectively.
Quantitative InsP6 chemistry clearly predicted the precipitation of Ca5H2LÆ16H2O in the conditions of the likely vesicular compartments for deposit forma- tion. However, the cell biology of the precipitation reaction remains obscure. The defined shape and size of the deposits suggests that precipitation may take place in secretory-like vesicles, as opposed to larger, (i.e. endoplasmic reticulum cisternal compartments the purified solids and Golgi). The structure of (Fig. 2) strongly suggests many nucleation centres, which are quite evenly distributed in the volume of each deposit. It is difficult to envisage this structure as arising from the transmembrane delivery of InsP6 into a compartment in which precipitation conditions already prevailed. On the other hand, it is difficult ‘free’ InsP6 could be delivered by to imagine that the conventional biosynthetic–exocytic pathway, in which high Ca2+ concentrations prevail, only to pre- cipitate in secretory vesicles. A more likely possibility is that InsP6 traffics along this route as a protein- containing complex, which dissociates at the acidic pH of secretory vesicles, thus allowing precipitation of the Ca2+ salt.
Anion-exchange HPLC with suppressed-ion conduc- tivity detection of the InsP6 deposits confirmed that the second most abundant inositol phosphate comi- grated with an Ins(2,3,4,5,6) ⁄ (1,2,4,5,6)P5 standard. In addition, minor amounts of what are likely to be other InsP5s and ⁄ or InsP4s were detected. Profiles for bovine and murine materials were identical (Fig. 4A,B). The inositol phosphates in the deposits could be expected to be protected from hydrolysis during the alkaline treatment as a result of being present within a solid phase. In agreement, the inositol phosphate profile of the LL of intact cysts was identical to that of the puri- fied materials (Fig. 4C). The abundant Ins(2,3,4,5,6) ⁄ (1,2,4,5,6)P5 was probably not a product of extracellu-
Monovalent cations were not found to be signifi- cant components of the deposits. This stands in con- trast to the situation in plant phytates, in which K+ is a major cation (together with Mg2+) [1,5–7]. As mixed monovalent–divalent cation salts of InsP6 do
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Fig. 3. 1H-NMR spectra of purified InsP6 deposits. (A) The 1D spectrum, with proton assignments and integral peak intensities given; asterisks denote interchangeable assignments (owing to the existence of an enantiomeric pair). The inset shows that across a wide range of chemical shifts, the only strong signals correspond to InsP6 (arrows). (B) The 1H-1H spectrum is shown in detail, with proton assignments and COSY correlations given; cross-peaks are not observable because of signal superimpo- sition (given in parentheses), while asterisks denote interchangeable assignments. The spin system revealing that the second major component in the material is Ins(1,2,4,5,6) ⁄ (2,3,4,5,6)P5 is indicated by arrows; the signal at 3.80 p.p.m. is diagnos- tic of a CHOH group and therefore of a non- phosphorylated position in the inositol ring. Spectra are shown for material from bovine cysts, processed by procedure 1 (see the Experimental procedures); similar data were obtained for material from mouse as well as for procedure 2.
not form readily [13a], the major Mg2+ ⁄ K+ ⁄ Ca2+ phytate in plant seeds may be a physical mixture of different compounds. It can be further reasoned that while in E. granulosus the availability of Ca2+ in the precipitation compartment is enough to produce over 90% pure calcium InsP6, this would not be the case in the analogous compartment in plant seeds; here the sequential exhaustion of available Ca2+ and Mg2+ in the presence of excess InsP6 would lead to the precipitation of relatively more soluble K+-con- taining salt(s).
InsP6 in the purified deposits, in the intact LL and (GL) was in the tissue synthesizing the deposits accompanied by Ins(2,3,4,5,6)P5 and ⁄ or (1,2,4,5,6)P5 (Figs 3 and 4A–D). The similarity in InsP profiles between GL and LL is expected, as GL extracts will include important amounts of InsPs from deposits not yet exocytosed. However, it does mean that the [1 ⁄ 3- OH]InsP5 detected does not arise from extracellular
hydrolysis; consistently, this was also the only InsP5 species detected after short-term metabolic labeling (Fig. 4E,F). The dominant InsP5 in mammalian sys- tems [21–28], as well as in yeasts [29] and a parti- is [2-OH]InsP5. In contrast, cular plant system [30], [1 ⁄ 3-OH]InsP5 is abundant, as in E. granulosus, in sev- eral plant systems [21,23,31–33]. It is plausible that the [1 ⁄ 3-OH]InsP5 reflects low-level dep- abundance of hosphorylation of InsP6 present in vesicular compart- [1 ⁄ 3-OH]InsP5 has ments. In animals in particular, been suggested to be (together with its [4 ⁄ 6-OH]iso- mer) a signature of the activity on InsP6 of the vesicu- lar system-cloistered multiple inositol polyphosphate phosphatase [34,35]; multiple inositol polyphosphate phosphatase is present in platyhelminths, as evidenced by a Schistosoma expressed sequence tag (accession number: CD080141 [36]). Alternatively, it is also poss- ible that [1 ⁄ 3-OH]InsP5 is a by-product of the syn- thetic pathways leading to InsP6.
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Fig. 4. Inositol phosphate profiles of purified myo-inositol hexakisphosphate (InsP6) deposits and parasite tissues, and metabolic labeling of parasites in culture. Suppressed- anion conductivity traces of the inositol phosphate profiles are shown for purified deposits from bovine (A) and mouse (B) materials (obtained by procedure 2; identical results were obtained for procedure 1, see the Experimental procedures). Similar pro- files were obtained for extracts obtained from the intact laminated layer (C) and the germinal layer (D) from mouse cysts. The two major peaks in these samples, InsP6 and D- and ⁄ or L-Ins(1,2,4,5,6)P5, were identi- fied on the basis of elution times in compar- ison with standards. The column used was an IonPac AS11, 2-mm bore, anion- exchange column. The profile of radiola- belled inositol phosphates from cysts cul- tured for 20 h with myo-[3H]inositol, separated on a Partisphere SAX HPLC col- umn, is shown in (E). The same [3H] sample was run on an IonPac AS11, 4-mm bore, column (F) alongside [14C]InsP5 standards. The [3H]radioactivity and suppressed-ion conductivity traces are shown, together with the elution positions of [14C]Ins(1,3,4,5,6)P5 (radioactivity trace shown) and D-and ⁄ or L-[14C]Ins(1,2,4,5,6)P5 (trace not shown). The [3H]InsP5 peak co-elutes with the D- and ⁄ or L-[14C]Ins(1,2,4,5,6)P5 standard and with the major InsP5 in samples detected on mass basis (by suppressed-ion conductivity).
From the point of view of the biology of E. granulo- sus, the observation that InsP6 deposits from bovine- and mouse-derived cysts are essentially indistinguish- able (Fig. 2 and Tables 1 and 2; IR and NMR data not shown), suggests that biosynthesis of InsP6 by the para- site deposits is robust with respect to host responses.
Cattle is a nonpermissive host species for common E. granulosus strains, because it maintains a granuloma- tous reaction around the cyst [37]. In contrast, inflam- matory resolution is the predominant outcome in other natural hosts, such as sheep [38], and curiously, in experimental murine infections [39]. We believe that the
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primary function of the deposits is structural. Notwith- standing this, InsP6 salts must comply with being non- inflammatory to the (appropriate) hosts, or else they would not have been evolutionarily incorporated to the parasite’s outermost structure. The thick, macromole- cule-permeable, LL is noted for its large capacity to adsorb host proteins [40,41], and data in this work sug- gest that the InsP6 deposits may contribute importantly to this capacity. Therefore, the immune system might not encounter the deposits as such, but rather as scaf- folds for a mosaic of adsorbed proteins. Such functional issues are made amenable to detailed study by the possi- bility of purifying the deposits, as well as synthetically imitating them. A precipitate with the appropriate Mg2+ : Ca2+ ratio can be prepared by mixing MgCl2 (5 mm final), CaCl2 (46 mm final) and sodium InsP6 (previously adjusted to pH 11 with NaOH; 10 mm final, and added last). The presence of [1 ⁄ 3-OH]InsP5 could be imitated by substituting 10% of the InsP6 by this compound, which is, however, not readily available.
dilute (6 mm) NH4OH solution and freeze-dried. Procedure 2 was similar, except that the final solids were washed with 165 mm NH4OH followed by ethanol and ether, and air dried at 37 (cid:1)C. In these two procedures, the use of alkaline solutions for washing was designed to minimize the loss of InsP6 solids through solubilization [13a]; ammonia had the advantage of being removable through freeze-drying (proce- dure 1) or by washing the solid with organic solvents (proce- dure 2). In procedure 3, CaCl2 was not included in the hydrolysis medium, and the 2 h incubation step in fresh med- ium, as well as washing of the solid, were omitted; hence samples were freeze-dried directly after removal of the bulk of the 0.1 m NaOH supernatant. This procedure did not allow absolute compositional data (or obviously Na+ con- tents) to be obtained, but permitted assessment of relative K+, Mg2+ and Ca2+ contents, without any bias due to par- tial solubilization. In all cases, samples of synthetic calcium InsP6, corresponding to the approximate amounts of InsP6 expected to be present in the E. granulosus samples, were subjected to alkaline digestion, washing and drying in paral- lel for comparison purposes; calcium InsP6 was prepared from sodium InsP6 (Sigma, St Louis, MO, USA), as detailed in [13a].
Experimental procedures
Parasite materials
Sugar analysis
Dried samples were spiked with rhamnose as an internal standard and subjected to total hydrolysis of oligosaccha- rides (2 m trifluoroacetic acid, 2 h, 120 (cid:1)C), reduction of monosaccharides to alditols (10 mgÆmL)1 NaBH4 in 1 m NH4OH, 20 min at room temperature) and peracetylation (acetic anhydride ⁄ pyridine; 1 : 1, v ⁄ v; 20 min at 100 (cid:1)C). The peracetylated alditols were identified on the basis of retention times upon gas chromatography on an HP-5 fused-silica capillary column in an HP6890 series instru- ment (Hewlett-Packard Co., Palo Alto, CA, USA) using a temperature ramp from 180 (cid:1)C (2 min) to 260 (cid:1)C (5 min). Quantification was carried out by comparison of peak areas with the internal standard, assuming equivalent sensitivity for all alditol acetates.
NMR spectra
E. granulosus HCW were obtained from natural cattle infections and experimental mouse infections, as described previously [9]. For the purification of InsP6 deposits, cyst walls were dehydrated and pulverized as described previ- ously [9]; the much bulkier LL can be expected to contrib- ute most of the mass to this starting material, from which the GL was nonetheless not removed. For the solubilization and extraction of extracellular InsPs (i.e. those present in the LL), whole cysts from mice were extracted in 50 mm Tris ⁄ HCl pH 7.4, 10 mm EDTA, 5 mm NaF, for 30 min on ice; this procedure has been previously shown to extract InsP6 from the LL without affecting the integrity of the underlying GL cells [8]. Inositol phosphates from the GL cells were obtained by depleting cysts of extracellular inosi- tol phosphates, as described above (except for NaF being omitted), then cutting them open and extraction with 10% (w ⁄ v) trichloroacetic acid for 30 min on ice.
Purification of E. granulosus InsP6 deposits
Purification of E. granulosus InsP6 deposits was carried out by three similar procedures involving prolonged alkaline hydrolysis of the LL mucins. In procedure 1, pulverized HCW were suspended at 10 mgÆmL)1 in 0.1 m NaOH, 0.5 mm CaCl2, and incubated at 45 (cid:1)C for 3 days. Then the supernatants were separated by centrifugation, the insoluble residues were incubated for 2 h in fresh hydrolysis medium and the supernatants were separated again and pooled with the previous ones. The solid residues were then washed in
Samples were treated with a Dowex-50 W resin (Dow Chemical Comp., Pevely, MI, USA) in H+ form and then subjected to three cycles of freeze-drying and redissolution in D2O (99.9% atom D). Spectra were obtained in a Bruker Advance DPX-400 spectrometer, using the standard Bruker software throughout (Bruker BioSpin GmbH, Rheinstetten, Germany). For 1D 1H-NMR spectra, 8–72 free induction decays were typically acquired, each with 8000 data points, 1.34 s acquisition time and 5995 Hz spectral width. Proton shift-correlated 2D spectra (COSY) were acquired in one scan for each of 256 free induction decays, which contained 1024 data points in F2, and at 4006 Hz spectral width.
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Infrared spectroscopy and elemental analysis
1 mLÆmin)1 and the buffers were mixed as follows: time (min), % B; 0, 0; 5, 0; 20, 66.6. Samples were diluted to 0.1 or 1 mm with respect to total inositol polyphosphates and 10 or 50 lL samples were injected via a 200 lL sample loop. In this system, InsP5 and some InsP4 species elute counterintuitively after InsP6.
Infrared spectroscopy was carried out on a Bomen FT-IR spectrophotometer (ABB Ltd, Zurich, Swizterzland), with samples present as 1% KBr pellets. Elemental analysis (C, H, N, S) was performed on a Carlo Erba EA 1108 instru- ment (Limito, Italy).
Ca2+, Mg2+, Na+ and K+ analyses
For some experiments, fractions (0.1 min) were collected after anion-suppression. Radioactivity in these fractions was estimated by dual-label scintillation counting in a Wal- lac (Turku, Finland) 1409 DSA Liquid Scintillation Coun- ter after the addition of 2 mL of EcoScintTM A (National Diagnostics, Atlanta, GA, USA) scintillation fluid.
Ca2+ and Mg2+ were determined by flame atomic absorp- tion on a Perkin Elmer 380 spectrometer (Boston, MA, USA), using a multi-element hollow cathode lamp for Ca ⁄ Mg ⁄ Al (Perkin Elmer) at 20 mA and wavelengths of 285.2 and 422.7 nm, respectively. Samples for Ca2+ deter- mination were diluted in 0.5% (w ⁄ v) lanthanum in order to avoid interference from phosphates. Ca2+ was, alternatively, quantified gravimetrically by calcium oxalate precipitation, as described in [13a]. Na+ and K+ were measured by flame atomic emission, with atomization of the samples and refer- ence solutions directly into the flame, using wavelengths of 589.0 and 766.5 nm, respectively.
[3H]Inositol labeling of cysts in culture
Samples for online radioactivity ([3H]) detection were run on a Whatman (Maidstone, UK) Partisphere 5 l SAX (4.6 mm · 20 cm) column on a Jasco (Great Dunmow, Essex, UK) HPLC system. Separations were performed with gradients derived from buffer reservoirs containing water (A) and 2.5 m NaH2PO4 (B), mixed as follows: time (min), % B; 0, 0; 60, 100; at a flow rate of 1 mLÆmin)1. Samples were injected in a 2 mL volume. Radioactivity in column eluates was estimated in a Canberra Packard A515TR flow scintillation analyser, fitted with a 0.5 mL flow cell, by admixture of Optima Flo AP (Canberra-Pack- ard Co., Pangbourne, UK) scintillation fluid at 2 mLÆmin)1 to column eluate.
obtained
standards were
[14C]Ins(1,3,4,5,6)P5
from [U-14C]inositol-labelled Spirodela polyrhiza [30]. d-and ⁄ or l-[14C]Ins(1,2,4,5,6)P5 was obtained by limited acid-cata- lyzed phosphate migration of [14C]Ins(1,3,4,5,6)P5. Unla- belled standards of inositol phosphates were obtained from Sigma (Poole, Dorset, UK).
Transmission electron microscopy
Purified InsP6 deposits were suspended in chloroform, laid on copper grids and observed unstained under a JEM-1010 transmission electron microscope (JEOL, Tokyo, Japan), at 80 kV.
Cysts for this purpose were obtained after intraperitoneal infection of mice with protoscoleces. Mouse procedures (intraperitoneal inoculation and killing by cervical disloca- tion) were carried out by personnel licenced by the CHEA (Honorary Commission for Animal Experimentation, Uruguay) and in agreement with CHEA guidelines. Cysts were dissected, separated in size-matched batches of 8 cysts (between 3 and 6 cm in diameter), and cultured in Eagle’s solution with 5 lCiÆmL)1 of [3H]inositol balanced salt (Perkin Elmer). At various time-points, cysts were cut open and the cyst walls were extracted for 10 min in 0.5 mL per cyst batch of 0.5 m trichloroacetic acid. Samples were extracted with water-saturated ethyl ether, neutralized with NH4OH solution and freeze-dried until analysis.
Density measurements of solids
HPLC for inositol phosphates
Mixtures of CCl4 and CHBr3 were prepared such that the solids under study would neither float nor sink as deter- mined upon visual inspection. Three such independent mix- tures were prepared for each sample, and 100 lL aliquots of each were weighed.
Acknowledgements
Samples for suppressed-ion conductivity detection were run on 25 cm Dionex IonPac AS11 strong anion exchange columns (2- or 4 mm bore, as indicated in each case) with IonPac AG11 guard columns on a Dionex DX500 HPLC sys- tem with an ED50 electrochemical detector (Dionex, Cam- berley, UK). The ASRSII anion suppressor of this system was operated in the autosuppression mode. Elution was per- formed with linear gradients derived from buffer reservoirs containing water (A) and 150 mm NaOH (B). For the 2 mm bore column, the flow rate was 0.4 mLÆmin)1 and buffers were mixed as follows: time (min), % B; 0, 3.3; 5, 3.3; 40, 66.6. For the 4 mm bore column, the flow rate was
This work was funded by CSIC (University of the Repu´ blic, Uruguay), and DINACYT (Ministry of Education, Uruguay) through ‘I+D’ and ‘Fondo Clemente Estable’ grants to AD, respectively. CK is (Ministry of funded by a PDT-DINACYT grant
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9 Irigoı´ n F, Casaravilla C, Iborra F, Sim RB, Ferreira F
& Dı´ az A (2004) Unique precipitation and exocytosis of a calcium salt of myo-inositol hexakisphosphate in lar- val Echinococcus granulosus. J Cell Biochem 93, 1271– 1281.
10 Morseth DJ (1967) Fine structure of the hydatid cyst
C. Casaravilla et al. Echinococcus granulosus InsP6 deposits
in platyhelminths. We are
and protoscolex of Echinococcus granulosus. J Parasitol 53, 312–325.
(Ca´ tedra
to Ana Marı´ a Ferreira
11 Richards KS (1984) Echinococcus granulosus equinus:
the histochemistry of the laminated layer of the hydatid cyst. Folia Histochem Cytobiol 22, 21–31.
12 Kilejian A, Sauer K & Schwabe C (1962) Host–parasite relationship in Echinococcosis. VIII. Infrared spectra and chemical composition of the hydatid cyst. Exp Parasitol 12, 377–392.
13 Richards KS, Arme C & Bridges JF (1983) Echinococcus granulosus equinus: an ultrastructural study of the lami- nated layer, including changes on incubating cysts in various media. Parasitology 86, 399–405.
13a Veiga N, Torres J, Domı´ nguez S, Mederos S, Irvine
to Rosario Education, Uruguay). We are grateful Dura´ n (Laboratorio de Bioquı´ mica Analı´ tica, IIBCE, Montevideo, Uruguay) for MALDI-TOF peptide fingerprinting, and to Cecilia Ferna´ ndez (Ca´ tedra de Inmunologı´ a, University of Uruguay) for help with searching for multiple inositol polyphosphate phospha- further tase analogs indebted de Inmunologı´ a, University of Uruguay) and her collabo- rators for experimental data that helped us optimize metabolic labelling in the parasite, and to Julia Torres (Inorganic Chemistry Laboratory, University of Uruguay) for help with Ca2+ and Mg2+ quantitations. Atomic absorption and emission analyses were carried out by Mariela Pisto´ n and Moise´ s Knochen (Ca´ tedra de Ana´ lisis Instrumental, DEC, Facultad de Quı´ mica, Montevideo, Uruguay). Electron microscopy images were obtained by Gabriela Casanova and A´ lvaro Olivera, from the Transmission Electron Microscopy facility of the Faculty of Sciences, Montevideo, Uruguay.
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