Báo cáo khoa học: Characterization of myo-inositol hexakisphosphate deposits from larval Echinococcus granulosus pdf

Characterization of myo-inositol hexakisphosphate
deposits from larval Echinococcus granulosus
Cecilia Casaravilla
1
, Charles Brearley
2
, Silvia Soule
´
3
, Carolina Fontana
3
, Nicola
´
s Veiga
4
,
Marı
´
a I. Bessio
3
, Fernando Ferreira
3
, Carlos Kremer
4
and Alvaro Dı
´
az
1
1Ca
´
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
4Ca
´
tedra de Quı
´
mica Inorga
´
nica, Departamento Estrella Campos, Facultad de Quı
´
mica, Universidad de la Repu
´
blica, Montevideo, Uruguay
myo-Inositol hexakisphosphate (InsP
6

) 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 Mg
2+
⁄ K
+
⁄ Ca
2+
,
Mn
2+
and Zn
2+
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. 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:
(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 (InsP
6
) 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 InsP
6
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 InsP
6
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 Ca
5
H
2

LÆ16H
2
O (L representing fully deprotonated InsP
6
), and
additionally contain Mg
2+
(6–9% molar ratio with respect to Ca
2+
), but
not K
+
. Calculations employing recently available chemical constants show
that the precipitation of Ca
5
H
2
LÆ16H
2
O 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 InsP
6
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 InsP
6
,

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 InsP
6
, rather than arising from extracellular hydrolysis of InsP
6
.
Abbreviations
al, attolitres; GL, germinal layer; HCW, hydatid cyst wall; InsP
6
, myo-inositol hexakisphosphate; InsP
5
, 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 InsP
6
deposits
are also formed in an animal system, namely the larva
of the parasitic cestode Echinococcus granulosus.In
this system, InsP
6
reaches a vesicular compartment
and precipitates mainly with Ca

2+
, the resulting solid
then being exocytosed and accumulating in major
amounts in the extracellular medium [8,9].
The extracellular structure in which InsP
6
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 InsP
6
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 Ins P
6

-rich granules
[13]. This fact, together with the massive scale in which
InsP
6
accumulates, makes working with the InsP
6
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 InsP
6
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 InsP
6
in the solid phase
The carbohydrate-rich fibril meshwork of the LL is
singularly difficult to solubilize. Therefore, attempts to
purify the InsP
6
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 InsP
6
. In addition, the available
information on the chemistry of InsP
6
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 InsP
6
in
amounts similar to those in the starting material
(Fig. 1B). Furthermore, its
1
H-NMR spectrum (after
solubilization by use of Dowex resin in proton form)
showed strong signals corresponding only to InsP
6
and to an accompanying, less-abundant myo-inositol
pentakisphosphate (InsP
5
) (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 InsP
6
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 InsP
6
as the only abundant organic
molecule.
The major component of the purified InsP
6
deposits has the stoichiometry Ca
5
H

2
LÆ16H
2
O
(L being deprotonated InsP
6
)
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 InsP
6
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
InsP
6
in the LL was Ca
2+
[8], we chose to compare
our purified material with synthetic calcium InsP
6
.
Calcium InsP
6
prepared under a variety of conditions
has the stoichiometry Ca
5
H
2

LÆ16H
2
O (L being fully
deprotonated InsP
6
) [13a].
By infrared spectroscopy (data not shown), elemental
analysis for C, H and N, and additional determination
of Ca
2+
(Table 1), the purified materials were very
similar to synthetic calcium InsP
6
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 Ca
2+
contents and density were
generally intermediate between those of calcium InsP
6
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. InsP
6
solids are known to lose
crystallization water readily, even under mild condi-

tions (N. Veiga & C. Kremer, unpublished results).
The solubility of InsP
6
salts increases markedly from
Ca
2+
to Mg
2+
and from Mg
2+
to the monovalent cati-
ons [13a]. Hence, assessing the presence of Mg
2+
and
monovalent cations in the deposits required precautions
against solubilization of InsP
6
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 InsP
6
samples; neither did Na
+
con-
tents appear to be significant. In contrast, the biological

solids contained significant amounts of Mg
2+
, which
was present at a 6–9% molar ratio with respect to
Ca
2+
.Mg
2+
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 Ca
5
H
2
LÆ16H
2
O, with a minor contribution from
magnesium InsP
6
(probably Mg
5
H
2
LÆ22H
2
O) [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 InsP
6
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 InsP
6
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 InsP
6
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 A
240 nm
,and
the corresponding data were thus only plotted up to 48 h. Error
bars on the insoluble mass and A
240 nm
plots represent standard
deviations for independent hydrolyses carried out in triplicate. (B)
InsP
6
is retained in the insoluble residue throughout the hydrolysis
period as assessed by TLC in [8].
Echinococcus granulosus InsP
6
deposits C. Casaravilla et al.
3194 FEBS Journal 273 (2006) 3192–3203 ª 2006 The Authors Journal compilation ª 2006 FEBS
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 Ca
2+
contents, and density of purified InsP
6
deposits. InsP
6
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 InsP
6
was subjected to alkaline treatment, washing and drying in parallel for comparison. C, H, N and S were determined
by automated elemental analysis, and Ca
2+
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
)
Ca
5
H
2
LÆ16H
2
O (theoretical) 6.3 3.5 – – 17.6 –
Synthetic calcium InsP
6
6.4
a
3.3
a
0.0 0.0 17.7
a
1.80 ± 0.05
Procedure 1 Synthetic calcium InsP
6
,
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
Procedure 2 Synthetic calcium InsP
6
,

subjected to alkaline treatment
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 Mg
2+
contents of InsP
6
deposits. InsP
6
deposits were purified by three different procedures, as detailed in
Experimental procedures, synthetic calcium InsP
6
being treated in
parallel in each case. Note that in procedure 3, designed to pre-
clude all solubilization of InsP
6
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 Ca
2+
content. ND, not
determined.

K
+a
Na
+a
Mg
2+a
Procedure 1 Synthetic calcium InsP
6
,
subjected to alkaline treatment
ND ND 0.2
Purified bovine material ND ND 7.2
Purified mouse material ND ND 6.0
Procedure 2 Synthetic calcium InsP
6
,
subjected to alkaline treatment
0.2 1.2 0.4
Purified bovine material 0.2 2.1 8.7
Purified mouse material 0.2 1.0 6.2
Procedure 3 Synthetic calcium InsP
6
,
subjected to alkaline treatment
2.3 – 0.4
Purified bovine material 2.1 – 8.8
a
% molar ratios with respect to Ca
2+
.

Fig. 2. Transmission electron microscopy of purified InsP
6
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).
C. Casaravilla et al. Echinococcus granulosus InsP
6
deposits
FEBS Journal 273 (2006) 3192–3203 ª 2006 The Authors Journal compilation ª 2006 FEBS 3195
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].
InsP
6
deposits represent a host-exposed surface
three orders of magnitude larger than the
external cyst surface
The density of synthetic calcium InsP

6
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 InsP
6
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
Ca
5
H
2
LÆ16H
2
O, allowed us to estimate that an ‘ideal’
granule (taken to be a sphere 41 nm in diameter [13])
would contain some 3 · 10
4
InsP
6
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 · 10
2
molecules of InsP
6
. Further calculations that
take into account the estimated density of the LL show
that  3% of the LL volume is occupied by InsP
6
depos-
its (Table 3). Finally, a 1 cm
2
portion of LL, of typical
1 mm thickness, comprises some 4000 cm
2
of InsP
6
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 InsP
6
is
predicted from the background chemistry
applied to vesicular system conditions
We wished to know if the vesicular precipitation of
InsP
6
, as found in E. granulosus, was predictable solely

in terms of the chemical interactions between InsP
6
,
and Ca
2+
and Mg
2+
; 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) InsP
6
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 InsP
6
content of a granule (Table 3),

InsP
6
‘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
Mg
2+
concentration was fixed at 0.5 mm. Then appro-
priate combinations of the above conditions were
Table 3. Numerical estimations in relation to InsP
6
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. InsP
6
deposits were taken to have the stoichiometry
Ca
5
H
2
LÆ16H
2
O (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 InsP
6
deposits, by the expression D ¼ 1 ⁄ [(m

InsP6
⁄ D
InsP6
)+(m
gel
⁄ D
gel
)],
where m
InsP6
and D
InsP6
represent the mass fraction in the LL and
densities of the InsP
6
deposits, and m
gel
and D
gel
represent the
same two parameters for the aqueous gel. The mass fractions
were calculated from our previous estimations of the calcium InsP
6
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 InsP

6
deposits was taken to be that of untreated cal-
cium InsP
6
(i.e. 1.80 gÆcm
)3
) (Table 1). The estimation of total InsP
6
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 (cm
3
)3.6· 10
)17
Mass of an ideal 41 nm granule (g) 6.5 · 10
)17
InsP
6
molecules per ideal 41 nm granule 3 · 10
4
Surface area of an ideal 41 nm granule (cm
2
)5.3· 10
)11
Volume of an ideal 7 nm crystal (cm
3
)2.0· 10
)19
Mass of an ideal 7 nm crystal (g) 3.7 · 10

)19
InsP
6
molecules per ideal 7 nm crystal 2 · 10
2
Density of LL (gÆcm
)3
)
a
1.02
LL volume occupied by InsP
6
deposits
a
2.7%
Number of granules per LL volume (cm
)3
)
a
8 · 10
14
InsP
6
deposit surface area per LL volume (cm
)1
)
a
4 · 10
4
a

Estimations for LL from bovine hydatid cysts.
Echinococcus granulosus InsP
6
deposits C. Casaravilla et al.
3196 FEBS Journal 273 (2006) 3192–3203 ª 2006 The Authors Journal compilation ª 2006 FEBS
plugged into the hyss program [20] loaded with all 22
relevant equilibrium contant values [13a]. When com-
bined with the additional condition that free Ca
2+
(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 InsP
6
present as Ca
5
H
2
LÆ16H
2
O. This means that
the observed presence of Mg
2+
in the solid must result
from a nonequilibrium phenomenon. This phenomenon
could be the occlusion of magnesium InsP
6
during the
(fast) precipitation of the calcium salt. Alternatively, it

may concern the access of Ca
2+
to the precipitation
compartment: the mass of Ca
2+
counterions required
might outstretch the cellular capacity to deliver the cat-
ion into that compartment. In this respect, coprecipita-
tion of Mg
2+
was predicted by hyss under the whole
range of conditions detailed above, provided that equi-
libration of free Ca
2+
with the vesicular system at large
was not allowed. Thus, using total Ca
2+
values slightly
below the 5 : 1 ratio with respect to InsP
6
, ratios of
Mg
2+
⁄ Ca
2+
: InsP
6
encompassing the observed values
(Table 2) were predicted, depending on the precise size
of the Ca

2+
deficit introduced.
[1 ⁄ 3-OH]Inositol pentakisphosphate accompanies
InsP
6
in the purified deposits and in intact
parasite tissue
During our initial detection of InsP
6
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 InsP
5
[8]. In the
purified deposits, InsP
6
was also accompanied by an
InsP
5
, which was determined by
1
H-NMR spectrosco-
py to correspond to the Ins(2,3,4,5,6) ⁄ (1,2,4,5,6)P
5
enantiomeric pair (Fig. 3). Abundance of this com-
pound relative to InsP
6
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 InsP
6
deposits confirmed that
the second most abundant inositol phosphate comi-
grated with an Ins(2,3,4,5,6) ⁄ (1,2,4,5,6)P
5
standard. In
addition, minor amounts of what are likely to be other
InsP
5
s and ⁄ or InsP
4
s 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)P
5
was probably not a product of extracellu-
lar hydrolysis of InsP
6
, 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 [
3
H]inositol, the only detectable labeled InsP
5
(Fig. 4E) migrated with the Ins(2,3,4,5,6) ⁄ (1,2,4,5,6)P
5
standard (Fig. 4F). In addition, [
3
H]inositol-labeled
parasites showed labeled peaks corresponding to InsP
6
and to two different inositol monophosphate species
(Fig. 4E).
Discussion
Formation of solids is an important aspect of InsP
6
biology, the understanding of which is still incomplete.
In this article, we addressed this issue for InsP
6
depos-
its from larval E. granulosus. The deposits were puri-
fied in native form and deduced to be composed
largely of the Ca
5
H
2
LÆ16H
2
O salt. Mg
2+

was also
present, at a 6–9% molar ratio, with respect to Ca
2+
.
Mg
2+
is likely to have also InsP
6
as counterion: the
possibility that it is the specific counterion of the InsP
5
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 Mg
2+
and InsP
6
,
and by Ca
2+
and InsP
5
.
Quantitative InsP
6
chemistry clearly predicted the
precipitation of Ca
5
H
2

LÆ16H
2
O 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 InsP
6
into a compartment in which precipitation conditions
already prevailed. On the other hand, it is difficult
to imagine that ‘free’ InsP
6
could be delivered by
the conventional biosynthetic–exocytic pathway, in
which high Ca
2+
concentrations prevail, only to pre-
cipitate in secretory vesicles. A more likely possibility
is that InsP
6
traffics along this route as a protein-
containing complex, which dissociates at the acidic
pH of secretory vesicles, thus allowing precipitation

of the Ca
2+
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 Mg
2+
) [1,5–7]. As
mixed monovalent–divalent cation salts of InsP
6
do
C. Casaravilla et al. Echinococcus granulosus InsP
6
deposits
FEBS Journal 273 (2006) 3192–3203 ª 2006 The Authors Journal compilation ª 2006 FEBS 3197
not form readily [13a], the major Mg
2+
⁄ K
+
⁄ Ca
2+
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 Ca
2+
in the
precipitation compartment is enough to produce over
90% pure calcium InsP

6
, this would not be the case
in the analogous compartment in plant seeds; here
the sequential exhaustion of available Ca
2+
and
Mg
2+
in the presence of excess InsP
6
would lead to
the precipitation of relatively more soluble K
+
-con-
taining salt(s).
InsP
6
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)P
5
and ⁄ or (1,2,4,5,6)P
5
(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]InsP
5
detected does not arise from extracellular

hydrolysis; consistently, this was also the only InsP
5
species detected after short-term metabolic labeling
(Fig. 4E,F). The dominant InsP
5
in mammalian sys-
tems [21–28], as well as in yeasts [29] and a parti-
cular plant system [30], is [2-OH]InsP
5.
In contrast,
[1 ⁄ 3-OH]InsP
5
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]InsP
5
reflects low-level dep-
hosphorylation of InsP
6
present in vesicular compart-
ments. In animals in particular, [1 ⁄ 3-OH]InsP
5
has
been suggested to be (together with its [4 ⁄ 6-OH]iso-
mer) a signature of the activity on InsP
6
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]InsP
5
is a by-product of the syn-
thetic pathways leading to InsP
6
.
Fig. 3.
1
H-NMR spectra of purified InsP
6
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 InsP
6
(arrows). (B) The
1
H-
1
H 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)P
5
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.
Echinococcus granulosus InsP
6
deposits C. Casaravilla et al.
3198 FEBS Journal 273 (2006) 3192–3203 ª 2006 The Authors Journal compilation ª 2006 FEBS
From the point of view of the biology of E. granulo-
sus, the observation that InsP
6
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 InsP
6
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 (InsP
6
)
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, InsP
6
and D- and ⁄ or L-Ins(1,2,4,5,6)P
5
, 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-[
3

H]inositol,
separated on a Partisphere SAX HPLC col-
umn, is shown in (E). The same [
3
H] sample
was run on an IonPac AS11, 4-mm bore,
column (F) alongside [
14
C]InsP
5
standards.
The [
3
H]radioactivity and suppressed-ion
conductivity traces are shown, together
with the elution positions of
[
14
C]Ins(1,3,4,5,6)P
5
(radioactivity trace
shown) and
D-and ⁄ or L-[
14
C]Ins(1,2,4,5,6)P
5
(trace not shown). The [
3
H]InsP
5

peak
co-elutes with the
D-and⁄ or
L-[
14
C]Ins(1,2,4,5,6)P
5
standard and with the
major InsP
5
in samples detected on mass
basis (by suppressed-ion conductivity).
C. Casaravilla et al. Echinococcus granulosus InsP
6
deposits
FEBS Journal 273 (2006) 3192–3203 ª 2006 The Authors Journal compilation ª 2006 FEBS 3199
primary function of the deposits is structural. Notwith-
standing this, InsP
6
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 InsP
6
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
Mg
2+
:Ca
2+
ratio can be prepared by mixing MgCl
2
(5 mm final), CaCl
2
(46 mm final) and sodium InsP
6
(previously adjusted to pH 11 with NaOH; 10 mm final,
and added last). The presence of [1 ⁄ 3-OH]InsP
5
could
be imitated by substituting 10% of the InsP
6
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 InsP
6
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
InsP
6
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 InsP
6
deposits
Purification of E. granulosus InsP
6
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 CaCl
2
, 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)NH
4
OH solution and freeze-dried. Procedure
2 was similar, except that the final solids were washed with
165 mm NH
4
OH 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
InsP
6
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, CaCl
2
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
+
,Mg
2+
and Ca
2+
contents, without any bias due to par-
tial solubilization. In all cases, samples of synthetic calcium
InsP
6
, corresponding to the approximate amounts of InsP
6
expected to be present in the E. granulosus samples, were
subjected to alkaline digestion, washing and drying in paral-
lel for comparison purposes; calcium InsP
6
was prepared
from sodium InsP
6
(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
NaBH
4
in 1 m

NH
4
OH, 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 D
2
O (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
1
H-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.
Echinococcus granulosus InsP
6
deposits C. Casaravilla et al.
3200 FEBS Journal 273 (2006) 3192–3203 ª 2006 The Authors Journal compilation ª 2006 FEBS
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).
Ca
2+
,Mg
2+
,Na
+
and K
+
analyses
Ca
2+
and Mg
2+
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 Ca
2+

deter-
mination were diluted in 0.5% (w ⁄ v) lanthanum in order to
avoid interference from phosphates. Ca
2+
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.
[
3
H]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 [
3
H]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
NH
4
OH 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
1mLÆmin
)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
1mm with respect to total inositol polyphosphates and 10 or
50 lL samples were injected via a 200 lL sample loop. In this
system, InsP
5

and some InsP
4
species elute counterintuitively
after InsP
6
.
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 EcoScint
TM
A (National
Diagnostics, Atlanta, GA, USA) scintillation fluid.
Samples for online radioactivity ([
3
H]) 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 NaH
2
PO
4
(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.
[
14
C]Ins(1,3,4,5,6)P
5
standards were obtained from
[U-
14
C]inositol-labelled Spirodela polyrhiza [30]. d-and ⁄ or
l-[
14
C]Ins(1,2,4,5,6)P
5
was obtained by limited acid-cata-
lyzed phosphate migration of [
14
C]Ins(1,3,4,5,6)P
5
. Unla-
belled standards of inositol phosphates were obtained from
Sigma (Poole, Dorset, UK).
Transmission electron microscopy
Purified InsP
6
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 CCl
4
and CHBr
3
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
Repu´ blic, 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
C. Casaravilla et al. Echinococcus granulosus InsP
6
deposits
FEBS Journal 273 (2006) 3192–3203 ª 2006 The Authors Journal compilation ª 2006 FEBS 3201
Education, Uruguay). We are grateful to Rosario
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-
tase analogs in platyhelminths. We are further
indebted to Ana Marı
´
a Ferreira (Ca
´
tedra 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 Ca
2+
and Mg
2+
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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