Characterization of myo-inositol hexakisphosphate deposits from larval Echinococcus granulosus

Characterization of myo-inositol hexakisphosphatedeposits from larval Echinococcus granulosusCecilia Casaravilla1, Charles Brearley2, Silvia Soule3, Carolina Fontana3, Nicolas Veiga4,Marıa I. Bessio3, Fernando Ferreira3, Carlos Kremer4 and Alvaro Dıaz1

1 Catedra de Inmunologıa, Facultad de Quımica ⁄Ciencias, Universidad de la Republica, Montevideo, Uruguay

2 School of Biological Sciences, University of East Anglia, Norwich, UK

3 Laboratorio de Carbohidratos y Glicoconjugados, Departamento de Quımica Organica, Facultad de Quımica ⁄ Facultad de Ciencias ⁄ Facultad

de Medicina, Universidad de la Republica, Montevideo, Uruguay

4 Catedra de Quımica Inorganica, Departamento Estrella Campos, Facultad de Quımica, Universidad de la Republica, Montevideo, Uruguay

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-

uble deposits with inorganic cations (often called

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

Keywords

calcium; inositol hexakisphosphate; inositol

pentakisphosphate; magnesium; phytic acid

Correspondence

A. Dıaz, Catedra de Inmunologıa, 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: [email protected]

(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.

Abbreviations

al, attolitres; GL, germinal layer; HCW, hydatid cyst wall; InsP6, myo-inositol hexakisphosphate; InsP5, myo-inositol pentakisphosphate;

LL, laminated layer.

3192 FEBS Journal 273 (2006) 3192–3203 ª 2006 The Authors Journal compilation ª 2006 FEBS

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

of the parasitic cestode Echinococcus granulosus. In

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].

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.

Results

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

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 � 80% solubilization

towards 48 h of treatment (Fig. 1A). The remaining

solid, but not the supernatant, contained InsP6 in

amounts similar to those in the starting material

(Fig. 1B). Furthermore, its 1H-NMR spectrum (after

solubilization by use of Dowex resin in proton form)

showed strong signals corresponding only to InsP6

and to an accompanying, less-abundant myo-inositol

pentakisphosphate (InsP5) (discussed below, and

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.

The major component of the purified InsP6

deposits has the stoichiometry Ca5H2LÆ16H2O

(L being deprotonated InsP6)

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

C. Casaravilla et al. Echinococcus granulosus InsP6 deposits

FEBS Journal 273 (2006) 3192–3203 ª 2006 The Authors Journal compilation ª 2006 FEBS 3193

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

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].

Yields of solid were � 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

When laid on coated copper grids and observed

unstained under the transmission electron microscope,

the naturally electron-dense solid (Fig. 2) was strongly

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].

Echinococcus granulosus InsP6 deposits C. Casaravilla et al.


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 (� 7–9 nm in

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)

Ca5H2LÆ16H2O (theoretical) 6.3 3.5 – – 17.6 –

Synthetic calcium InsP6 6.4a 3.3a 0.0 0.0 17.7a 1.80 ± 0.05

Procedure 1 Synthetic calcium InsP6,

subjected to alkaline treatment

7.5 2.6 0.0 0.0 21.3 2.10 ± 0.01

Purified bovine material 7.0 2.7 0.0 0.0 16.2 1.97 ± 0.07

Purified mouse material 6.8 3.0 0.0 0.0 16.6 ND



7.0 3.4 0.0 0.0 21.0 ND

Purified bovine material 7.0 3.7 0.0 0.0 19.0 2.07 ± 0.05

a Data from [13a].

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

Purified bovine material ND ND 7.2

Purified mouse material ND ND 6.0



0.2 1.2 0.4

Purified bovine material 0.2 2.1 8.7

Purified mouse material 0.2 1.0 6.2



2.3 – 0.4

Purified bovine material 2.1 – 8.8

a % molar ratios with respect to Ca2+.

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).



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,

from 1.80 ± 0.05 gÆcm)3 to values similar to those

of the purified E. granulosus InsP6 deposits (2.0–

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 � 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

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 � 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 � 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-

priate combinations of the above conditions were

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

and granule–fibril interactions in vivo in the LL [9], and is thus

slightly inflated.

Volume of an ideal 41 nm granule (cm3) 3.6 · 10)17

Mass of an ideal 41 nm granule (g) 6.5 · 10)17

InsP6 molecules per ideal 41 nm granule 3 · 104

Surface area of an ideal 41 nm granule (cm2) 5.3 · 10)11

Volume of an ideal 7 nm crystal (cm3) 2.0 · 10)19

Mass of an ideal 7 nm crystal (g) 3.7 · 10)19

InsP6 molecules per ideal 7 nm crystal 2 · 102

Density of LL (gÆcm)3)a 1.02

LL volume occupied by InsP6 depositsa 2.7%

Number of granules per LL volume (cm)3)a 8 · 1014

InsP6 deposit surface area per LL volume (cm)1)a 4 · 104

a Estimations for LL from bovine hydatid cysts.



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.

[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.

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-

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

standard (Fig. 4F). In addition, [3H]inositol-labeled

parasites showed labeled peaks corresponding to InsP6

and to two different inositol monophosphate species

(Fig. 4E).

Discussion

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.

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,

cisternal compartments (i.e. endoplasmic reticulum

and Golgi). The structure of the purified solids

(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

to imagine that ‘free’ InsP6 could be delivered by

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.

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



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

in the tissue synthesizing the deposits (GL) was

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-

cular plant system [30], is [2-OH]InsP5. In contrast,

[1 ⁄ 3-OH]InsP5 is abundant, as in E. granulosus, in sev-

eral plant systems [21,23,31–33]. It is plausible that the

abundance of [1 ⁄ 3-OH]InsP5 reflects low-level dep-

hosphorylation of InsP6 present in vesicular compart-

ments. In animals in particular, [1 ⁄ 3-OH]InsP5 has

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.

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.



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

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).



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.

Experimental procedures

Parasite materials

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 �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

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 �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].

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 �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 �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 �C (2 min) to 260 �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

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.



Infrared spectroscopy and elemental analysis

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

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

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

balanced salt solution with 5 lCiÆmL)1 of [3H]inositol

(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.

HPLC for inositol phosphates

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

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.

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.

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.

[14C]Ins(1,3,4,5,6)P5 standards were obtained from

[U-14C]inositol-labelled Spirodela polyrhiza [30]. d-and ⁄orl-[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.

Density measurements of solids

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

This work was funded by CSIC (University of the

Republic, Uruguay), and DINACYT (Ministry of

Education, Uruguay) through ‘I+D’ and ‘Fondo

Clemente Estable’ grants to AD, respectively. CK is

funded by a PDT-DINACYT grant (Ministry of



Education, Uruguay). We are grateful to Rosario

Duran (Laboratorio de Bioquımica Analıtica, IIBCE,

Montevideo, Uruguay) for MALDI-TOF peptide

fingerprinting, and to Cecilia Fernandez (Catedra de

Inmunologıa, University of Uruguay) for help with

searching for multiple inositol polyphosphate phospha-

tase analogs in platyhelminths. We are further

indebted to Ana Marıa Ferreira (Catedra de

Inmunologıa, 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 Piston and Moises Knochen (Catedra

de Analisis Instrumental, DEC, Facultad de Quımica,

Montevideo, Uruguay). Electron microscopy images

were obtained by Gabriela Casanova and Alvaro

Olivera, from the Transmission Electron Microscopy

facility of the Faculty of Sciences, Montevideo,

Uruguay.

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Characterization of myo-inositol hexakisphosphate deposits from larval Echinococcus granulosus

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