A membrane-bound trehalase from Chironomus riparius larvae: Purification and sensitivity to inhibition

Article abstract of DOI:10.1093/glycob/cwq087

A preparation of a membrane-bound trehalase from the larvae of the midge Chironomus riparius (Diptera: Chironomidae) was obtained by detergent solubilization, ion-exchange chromatography and concanavalin A affinity chromatography. Trehalase was purified 1080-fold to a specific activity of 75 U mg^-1. The initial rate of trehalase activity followed Henri-Michaelis-Menten kinetics with a Km of 0.48 ± 0.04 mM. Catalytic efficiency was maximal at pH 6.5. The activity was highly inhibited by mono-and bicyclic iminosugar alkaloids such as (in order of potency) casuarine (IC50 = 0.25 ± 0.03 μM), deoxynojirimycin (IC50 = 2.83 ± 0.34 μM) and castanospermine (IC50 = 12.7 ± 1.4 μM). Increasing substrate concentration reduced the inhibition. However, in the presence of deoxynojirimycin, Lineweaver-Burk plots were curvilinear upward. Linear plots were obtained with porcine trehalase. Here, we propose that deoxynojirimycin inhibits the activity of trehalase from C. riparius according to a ligand exclusion model. Inhibition was further characterized by measuring enzyme activity in the presence of a series of casuarine and deoxynojirimycin derivatives. For comparison, inhibition studies were also performed with porcine trehalase. Results indicate substantial differences between midge trehalase and mammalian trehalase suggesting that, in principle, inhibitors against insect pests having trehalase as biochemical targets can be developed.

Full text of DOI:10.1093/glycob/cwq087

Glycobiology vol. 20 no. 9 pp. 1186–1195, 2010
doi:10.1093/glycob/cwq087
Advance Access publication on June 11, 2010
A membrane-bound trehalase from Chironomus riparius
larvae: purification and sensitivity to inhibition
Matilde Forcella2, Francesca Cardona4, Andrea Goti4,
Introduction
Camilla Parmeggiani4, Laura Cipolla3, Maria Gregori3,
Raffaella Schirone3, Paola Fusi3, and Paolo Parenti1,2
2Department of Environmental Sciences, and 3Department of Biotechnology
Trehalose (α-D-glucopyranosyl-α-D-glucopyranoside) is the
main circulating sugar in the hemolymph of most insects (Elbein
and Biosciences, University of Milano-Bicocca, Piazza della Scienza 2, 20126
et al. 2003). Insect tissues possess the enzyme α,α-trehalase
(EC 3.2.1.28), which promotes the irreversible hydrolysis of
trehalose into two molecules of D-glucose (Thompson 2003).
The enzyme has been mainly found in brain and thoracic gan-
glia (Strang and Clement 1980), asynchronous (Reed and
Sacktor 1971) and synchronous flight muscle (Gussin and Wyatt
1965; Wegener et al. 2003), midgut cells (Azuma and Yamashita
1985; Silva et al. 2004), midgut surrounding visceral muscle
(Mitsumasu et al. 2005), follicle cells (Shimada and Yamashita
1979), spermatophore (Yaginuma et al. 1996) and hemolymph
(van Handel 1978). Trehalase has been proved to exist in two dis-
tinct forms, a membrane-bound form and a soluble form (Wyatt
1967). The membrane-bound form has been described in flight
muscle, ovary cells, spermatophore and midgut. In flight muscle
cells, the situation is further complicated by the fact that part of
the membrane-bound enzyme is inactive and present in a latent
form that can be activated by treatments that destroy membrane
integrity (Wegener et al. 2003). The soluble trehalase has been
purified from the midgut, where it is distributed in the cavity
of the goblet cells (Mitsumasu et al. 2005), and from the he-
molymph, even though the presence in the latter compartment
has been questioned in some insects (Vaandrager et al. 1989).
For its role in glucose metabolism, trehalase has been re-
garded as an interesting target for the development of new
agents against insect pests (Asano et al. 1990; Wegener et al.
2003). Inhibition studies revealed that several compounds are
able to specifically block trehalase activity in a micromolar or
submicromolar range. Among the most powerful compounds,
there are validoxylamine A (Asano et al. 1990), trehazolin
(Ando et al. 1995) and suidatrestin (Knuesel et al. 1998). In-
jection of these compounds into insect hemolymph is lethal
(Wegener et al. 2003) since it produces unsuccessful metamor-
phosis (Asano et al. 1990). In addition, trehalase is inhibited by
iminosugars that strongly inhibit other glycosidases (Asano
2003), such as the mono- and bicyclic alkaloids deoxynojiri-
mycin and castanospermine (Asano et al. 1996), as well as
by a variety of glycosides (Silva et al. 2004). Together with
the selection of new potential insecticides, inhibition studies
have been proved useful to elucidate structural features of
the enzyme active site (Asano et al. 1996; Gibson et al.
2007; Cardona et al. 2009).
Milano, Italy, and 4Department of Chemistry “U. Schiff”, Laboratory of
Design, Synthesis and Study of Biologically Active Heterocycles
(HeteroBioLab), University of Florence, Via della Lastruccia 13, 50019
Florence, Italy
Received on November 12, 2009; revised on June 1, 2010; accepted on
June 7, 2010
A preparation of a membrane-bound trehalase from the
larvae of the midge Chironomus riparius (Diptera: Chiro-
nomidae) was obtained by detergent solubilization, ion-
exchange chromatography and concanavalin A affinity
chromatography. Trehalase was purified 1080-fold to a
specific activity of 75 U mg−1. The initial rate of trehalase
activity followed Henri–Michaelis–Menten kinetics with a
Km of 0.48 0.04 mM. Catalytic efficiency was maximal
at pH 6.5. The activity was highly inhibited by mono- and
bicyclic iminosugar alkaloids such as (in order of poten-
cy) casuarine (IC50 = 0.25 0.03 μM), deoxynojirimycin
(IC50 = 2.83
0.34 μM) and castanospermine (IC50 =
12.7 1.4 μM). Increasing substrate concentration re-
duced the inhibition. However, in the presence of
deoxynojirimycin, Lineweaver–Burk plots were curvilin-
ear upward. Linear plots were obtained with porcine
trehalase. Here, we propose that deoxynojirimycin inhi-
bits the activity of trehalase from C. riparius according
to a ligand exclusion model. Inhibition was further char-
acterized by measuring enzyme activity in the presence of a
series of casuarine and deoxynojirimycin derivatives. For
comparison, inhibition studies were also performed with
porcine trehalase. Results indicate substantial differences
between midge trehalase and mammalian trehalase sug-
gesting that, in principle, inhibitors against insect pests
having trehalase as biochemical targets can be developed.
Keywords: Chironomus riparius/iminosugar alkaloids/
inhibition/trehalase
1To whom correspondence should be addressed: Tel: +39 2 6448 2752; E-mail:
paolo.parenti@unimib.it
Most of what we know on insect trehalase derives from stud-
ies performed on terrestrial species such as Lepidoptera,
1186 © The Author 2010. Published by Oxford University Press. All rights reserved. For permissions, please e-mail: journals.permissions@oxfordjournals.org

Trehalase from Chironomus riparius larvae
Table I. Purification of the membrane-bound trehalase from C. riparius larvae
Diptera, Blattaria and Orthoptera (Thompson 2003; Wegener et
al. 2003; Mitsumasu et al. 2008). Little is known on trehalase
from aquatic insects. Chironomidae, commonly known as
midges, are widely distributed and often represent the most
abundant invertebrate taxon in freshwater ecosystems. In view
of their importance as potential sentinel organisms (Callaghan
et al. 2001; Forcella et al. 2007), it is of concernment that our
knowledge of their physiology is as wide as possible. Chirono-
mus riparius represents a good model for biochemical studies,
and we have recently demonstrated that its resistance to stress
factors is highly dependent on trehalose catabolism. Larval ex-
posure to sublethal concentrations of the two insecticides
fenitrothion and carbofuran has effects on the catabolism of
trehalose and on the trehalase activity (Forcella et al. 2007).
This result has prompted us to assume that trehalase could
be considered as a target for new and specific molecules having
insecticide activity.
Total
proteins
(mg)
Total
activity
(U)
Specific
activity
(U/mg)
Yield
(%)
Purification
factor
Procedure
Crude extract
DEAE
Mono Q
1404
312
5.96
0.193
194
162
84
0.138
0.52
14.1
149.7
100
1
83.5
51.7
34.2
3.8
102
1080
Concanavalin A
28.7
measured as a function of trehalose concentration showed sat-
uration kinetics fitting with a simple Henri–Michaelis–Menten
equation (Figure 2). Kinetics parameters are reported in Table II.
Apparently, pH mainly affects the maximal velocity with no sig-
nificant modification of the affinity for trehalose. Catalytic
efficiency decreased 4.1-fold when pH increased from 6.5 to
7.5. To check whether this decrease was due to buffer effect, ini-
tial rate measurements at saturating concentration of trehalose
were repeated in 20 mM 4-(2-hydroxyethyl)-1-piperazine-
ethansulfonic acid (HEPES)–2-amino-2-hydroxymethyl-1,3-
propanediol (Tris), pH 6.5: the specific activity was slightly
lower but not significantly different from that measured in ac-
etate buffer (3.95 0.15 vs 4.15 0.09, respectively). These
data are compatible with the slightly acidic pH found in the he-
molymph of this insect species (unpublished observations).
Results
Trehalase purification
About 38% of total trehalase activity in the midge larva ho-
mogenate is from membrane-bound trehalase. Activity was
recovered upon solubilization of the membrane pellet obtained
after centrifugation of the crude homogenate at 100,000 × g for
1 h. Extraction from membranes was obtained incubating the
pellet with the zwitterionic detergent 3-[(3-cholamidopropyl)-
dimethyl-ammonio]-1-propanesulfonate (CHAPS). The purifi-
cation of trehalase from C. riparius larvae is summarized in
Table I. The solubilized crude extract was subjected to three
chromatographic steps. The first one was included in order to
remove greenish pigments present in the preparation. After
Mono Q, the trehalase was concentrated in three 1-mL fractions.
The final step was based on data from literature describing the
membrane form of the trehalase as glycosylated. The molecular
mass of the purified trehalase was estimated to be 67 kDa both
by sodium dodecyl sulfate-polyacrylamide gel electrophoresis
(SDS-PAGE) (not shown) and by high-performance liquid
chromatography IC50 50% inhibitor concentration (HPLC)
Superdex 200 HR 10/30 (Figure 1A). In order to verify if
the preparation might be a mixture of isoforms, native gel elec-
trophoresis with staining activity was performed. As shown in
Figure 1B, a single band was detected, confirming the presence
of a single trehalase isoform (the faint band above is repre-
sented by small quantities of unsolubilized material unable to
enter the running gel). The apparent molecular mass was high-
er than that estimated by SDS-PAGE, and the discrepancy
could be explained by the presence of glycosylated residues
which reduce the mobility of the protein within the gel under
non-denaturating conditions.
Inhibition studies with glycosidase inhibitors
In order to characterize the sensitivity of this enzyme, inhibition
by a panel of compounds was considered, and dose–response
curves were established for each of them. Considering that
we expected a competitive effect, experiments were performed
at two substrate concentrations, one close to the Km value
(0.5 mM) and the second one at a 10-fold higher concentration.
Inhibitor concentrations varied in the experiments according to
their degree of inhibition as assayed in a preliminary screening.
Figure 3 shows dose–response curves for deoxynojirimycin,
castanospermine and casuarine at fixed trehalose concentration
(0.5 mM). Initial rates as a function of inhibitor concentration
were fitted to the following equation:
vi
v
1
ꢀ
ꢁ
=
ð1Þ
n
½Iꢀ
IC50
1 +
where vi and v are the initial rate in the presence and in the ab-
sence of inhibitor, respectively; [I] is the inhibitor concentration;
IC50 is the inhibitor concentration producing half-maximal inhi-
bition; and n is the Hill coefficient.
Among the compounds tested, casuarine has been proved to
be the most potent inhibitor with a calculated IC50 = 0.248
0.003 μM, whereas the dose–response curves for deoxynojir-
imycin and castanospermine fitted to Eq. (1) returned IC50
values of 2.83 0.34 and 12.4 1.14 µM, respectively. In
the presence of 5 mM trehalose, all dose–response curves
shifted to higher IC50 values as expected for competitive inhi-
bition: the calculated IC50 values were 2.93 0.84, 39.6 2.84
and 132 15 µM for casuarine, deoxynojirimycin, and casta-
nospermine, respectively.
Kinetic studies
Trehalase showed high specificity for trehalose and displayed
barely detectable activity or no activity at all against some dis-
accharides and trisaccharides such as lactose (0.1%), sucrose
(0.08%), cellobiose (0.05%), maltose (0%) and raffinose (0.9%).
Preliminary experiments showed that maximal activity was re-
corded at slightly acidic pH values. Therefore, kinetic
experiments were performed at three different pH values (5.5,
6.5 and 7.5). In all instances, the initial rate of trehalase activity
1187

M Forcella et al.
Fig. 1. Molecular mass of C. riparius trehalase. (A) Gel filtration co-elution profile of Mono Q active fractions from C. riparius homogenate and two standard proteins,
bovine serum albumin (BSA) and alcohol dehydrogenase (ADH), with a known molecular weight of 67 and 150 kDa, respectively. The trehalase activity of each
fraction was determined (– –). In the box, the calibration curve of Superdex 200 HR 10/30 with thyroglobulin (669 kDa), apoferritin (443 kDa), alcohol dehydrogenase
•
(150 kDa), bovine serum albumin (67 kDa) and deoxyribonuclease (31 kDa) as standard is reported. (B) Non-denaturating PAGE of pooled fractions eluted in
correspondence with BSA (see A) stained for trehalase activity.
ynojirimycin. Results are illustrated in Figure 4. The family of
Usingꢂthe foꢃllowing Cheng–Prusoff relationship,
½Sꢀ
curves of the double reciprocal plot converged to the same Vmax
value, suggesting a competitive mechanism of inhibition. How-
ever, in the presence of deoxynojirimycin, the plot was
curvilinear upward when C. riparius trehalase was used. The
plot was linear for porcine trehalase, and for simplicity, only
the results obtained in the presence of the highest concentration
of deoxynojirimycin (5 μM) are illustrated (Figure 4, inset). The
non-linearity of the Lineweaver–Burk plot could be explained
by three possible scenarios: (i) the enzyme is a dimer, (ii) the
IC50 = Ki 1 +
, the inhibition constant was calculated,
Km
and these results are reported in Table III together with data
obtained using porcine trehalase, a mammalian counterpart.
Fitting to Eq. (1) returned n values higher than 1 for all three
inhibitors, suggesting that more than one molecule of inhibitor
could bind to the enzyme (in inhibition study, the n coefficient
is not an index of cooperativity). To further explore this aspect,
we performed kinetics of trehalase activity as a function of
trehalose in the presence of increasing concentrations of deox-
1188

Trehalase from Chironomus riparius larvae
Fig. 3. Dose–response curves of glycosidase inhibitors on trehalase activity
from C. riparius larvae. The enzyme was assayed in the presence of the
indicated inhibitor concentrations and in the presence of 0.5 mM trehalose at
Fig. 2. Kinetics of C. riparius trehalase. Activity was measured as a function
of medium pH: initial rates were at pH 7.5 (▾), 6.5 (○) and 5.5 ( ).
•
pH 6.5. Symbols refer to casuarine ( ), deoxynojirimycin (○) and
•
castanospermine (▾). The assay was started by the addition of trehalose after
inhibitor is a slow-binding inhibitor or (iii) a more complex non-
linear inhibition pattern is present. From SDS-PAGE and gel fil-
tration, we have no evidences that trehalase is a dimer.
Therefore, to discriminate between the other two alternatives,
the progress curve of product formation in the presence of an
inhibitor was followed for a prolonged period of time in assay
mixtures in which the reaction was started either by the addition
of enzyme or substrate. According to literature, in the presence
of a slow-binding inhibitor, in the first case the relatively rapid
initial rate decreases to a slower steady-state rate, while in the
second case there is a slow release of inhibitor, and ultimately
a steady state is reached (Sculley et al. 1996). The reaction
was followed over 30 min, and none of such slow-binding be-
haviors were observed: the time course of the reaction was in all
instances linear. Deoxynojirimycin was chosen because its
slow-binding inhibition is well documented toward sucrase-
isomaltase, where the aforementioned effects are evident with-
in 5–10 min of incubation (Hanozet et al. 1981). We conclude
that the explanation of the observed results should be sought
among complex non-linear inhibition patterns: this aspect will
be discussed below.
10 min of pre-incubation. Data are plotted according to Eq. (1).
of inhibiting compounds were synthesized, introducing a sub-
stituent of the iminoglucitol ring of deoxynojirimycin. Their
structure is reported in Figure 5. Eight compounds were assayed
on trehalase activity, and their effect is reported in Table IV. The
most active molecule was compound (4), and its inhibition was
partially displaced by the enzyme substrate. Compound (1) was
the second most active, but its effect seemed to be non-compet-
itive as well as that of inhibitors (3) and (2). The remaining four
compounds did not inhibit trehalase activity. For comparison,
the same assay was carried out using porcine kidney trehalase,
and no inhibition was observed for all but compound (4). More-
over, the degree of inhibition of compound (4) on porcine
trehalase was significantly lower than that on midge trehalase.
Inhibition kinetics was performed on midge trehalase for com-
pound (4) (Figure 6). Fitting the data to the Henri–Michaelis–
Menten equation returned the following kinetic parameters: Km
(mM) = 1.67 0.12 and 0.34 0.05, Vmax (U/mL) = 0.25 0.01
and 0.24 0.02, in the presence and in the absence of an
inhibitor, respectively. Competitive inhibition was confirmed
by double reciprocal plot (Figure 6, inset). Using the Cheng–
Prusoff relationship for competitive inhibition, the value Ki =
25 μM was calculated.
Inhibition studies with deoxynojirimycin derivatives
The difference in the affinity for trehalose and in the inhibition
pattern between midge trehalase and porcine trehalase suggests
the presence of a different structure for the substrate/inhibitor
binding site. If this were to be confirmed, it may be helpful in
developing specific inhibitors for the insect enzyme. A series
Inhibition studies with casuarine derivatives
To further explore the potentiality of the pyrrolizidine ring of
casuarine, a number of derivatives were assayed as inhibitors
of trehalase activity from C. riparius. The structure of mole-
cules included in this study is illustrated in Figure 7, and their
effect is reported in Table V. None of the compounds tested
was proven to be more effective than casuarine itself in inhi-
biting trehalase activity; thus, the hydroxyl group in position
7 of casuarine seems to play a role in enhancing inhibitory
activity. In particular, substitution of the hydroxyl group with
a hydroxy methyl group dramatically increased the Ki value
(273-fold in C. riparius), whereas removing the hydroxyl
group (7-deoxycasuarine) had a relatively slight effect with
Table II. Kinetic parameters of trehalase activity from C. riparius larvae. Fixed
amounts of trehalase were incubated in the presence of increasing concentrations
of trehalose as illustrated in Figure 1 and described in Materials and methods.
Data are means SE of three independent experiments
pH of the medium
Km (mM)
Vmax (U/mg)
Vmax/Km
5.5
6.5
7.5
0.44 0.02
0.48 0.04
0.52 0.04
2.73 0.04
4.57 0.09
1.22 0.02
6.2
9.5
2.3
1189

M Forcella et al.
Table III. Inhibition of trehalase activity. Fixed amounts of trehalase from C. riparius larvae and from porcine kidney were incubated in the presence of fixed
trehalose concentrations at Km value and increasing concentrations of the indicated inhibitors. Parameters were calculated as described in the text. Data are means SE
of three independent experiments
C. riparius
Porcine kidney
Inhibitor
IC50 (μM)
n
Ki (μM)
IC50 (μM)
n
Ki (μM)
Casuarine
Deoxynojirimycin
Castanospermine
0.25 0.03
2.83 0.34
9.93 1.80
1.59
1.25
1.19
0.12 0.01
1.39 0.18
4.86 0.91
12a
–
1.08
–
–
5.96 0.62 4.3b
2.5b
2.98 0.32
–
aData from Kato et al. 2003.
bData from Asano et al. 2003.
a 4.9-fold increase of the Ki value. Also, this latter compound
had a Ki value 19.8-fold lower for trehalase from C. riparius
larvae than that for trehalase from porcine kidney. Reducing
the size of the heterocyclic ring from pyrrolizidine to pyrro-
lidine (as in 6-deoxy-DMDP and DAB-1) drastically
decreased the inhibitory activity. In this case, the value of
the inhibition constants for C. riparius larvae and porcine
kidney was similar.
oxic environments (Rosenberg and Resh 1993). C. riparius
larvae, as other insects, contain both soluble and membrane-
bound isoforms of trehalase, known as trehalase-1 and treha-
lase-2, respectively. It is believed that the membrane isoform
represents a latent or inactive form that can be activated in vitro
by detergents (Wegener et al. 2003). In Bombyx mori, the pre-
dominant trehalase switches from trehalase-2 to trehalase-1 at
the onset of spinning, marking the beginning of the prepupal pe-
riod (Yamashita et al. 1974). Previous studies performed in our
laboratory suggested that anoxia might trigger activation pro-
cesses on trehalase-2 in C. riparius larvae (Forcella et al. 2007).
Trehalase-2 purified from midge larvae is a glycoprotein as
demonstrated by its retention on concanavalin A affinity col-
umn. Its apparent molecular mass is 67 kDa as determined
with SDS-PAGE and gel filtration. Also, the enzyme preparation
is apparently homogeneous and made of a single isoform as de-
termined by native gel electrophoresis. This latter aspect is
particularly important for the interpretation of the kinetic results.
First of all, as expected, the larval enzyme is highly specific for
trehalose with an affinity about 5-fold higher when compared to
the mammalian trehalase. Secondly, the enzyme is competitively
inhibited by casuarine, deoxynojirimycin and castanospermine.
However, we observed that, in the presence of glycosidase inhi-
bitors, in particular deoxynojirimycin, the double reciprocal plot
of the initial rate as a function of trehalose concentration was cur-
vilinear upward. This phenomenon has neither been described for
other trehalases from insect tissues nor from other organisms. In
fact, it is not evident in inhibitory studies performed with porcine
kidney trehalase. In principle, this pattern suggests the presence of
a multisite system. For example, it could be compatible with a sit-
uation in which the enzyme is a dimer and the inhibitor I binds at
the interface between the two subunities, inducing a conforma-
tional change that excludes the substrate S from both catalytic
sites. If the binding of S to one catalytic site prevents I from bind-
ing, then the v versus [S] is sigmoidal, and the Lineweaver–Burk
plot is non-linear (Segel 1975, p.385). This scenario is ruled out
because there is no evidence that trehalase from C. riparius is a
dimer or oligomer in its active form: after gel filtration, the en-
zyme eluted as a single peak in correspondence of the expected
molecular weight as determined with standard proteins.
Discussion
Insect trehalase has been purified from several species belong-
ing to main insect orders, and new trehalases continue to be
added to this list (Jin and Zheng 2009). The enzyme is of in-
creasing interest in insect physiology since the soluble isoform
of the enzyme is under hormonal regulation (Tatun et al. 2008;
Gu et al. 2009). For its central role in glucose catabolism, treha-
lase has been considered (Asano et al. 1990) and continues to be
regarded as a possible target for insect pest control (Jin and
Zheng 2009). The larvae of the midge C. riparius play a funda-
mental role in the freshwater food web and are included as
sentinel organisms for their ability to cope with polluted and an-
Among complex inhibitory patterns which can be compatible
with our experimental data, the “hit-and-run” substrate-inhibi-
tor displacement system proposed by Reiner (1969) and further
developed by Fisher et al. (1970) as a ligand exclusion theory
seems to give a plausible explanation of our observations. The
model assumes the presence of subsites for the binding of sub-
Fig. 4. Lineweaver–Burk plot in the presence of deoxynojirimycin. Main
panel: trehalase kinetics was measured as a function of deoxynojirimycin:
0 μM ( ), 2.5 μM (▾) and 5 μM (○). Inset: kinetics of porcine kidney
•
trehalase in the presence of 5 μM deoxynojirimycin.
1190

Trehalase from Chironomus riparius larvae
Fig. 5. Structure of deoxynojirimycin derivatives used as inhibitors of trehalase from C. riparius larvae and from porcine kidney.
1191

M Forcella et al.
Table IV. Inhibition by deoxynojirimycin derivatives of membrane-bound tre-
halase from C. riparius larvae and trehalase from porcine kidney. Initial rate of
trehalase was measured in the presence of 100 μM inhibitor (#) at two fixed
substrate concentrations, namely at the corresponding Km value and in the
presence of an excess of trehalose. Data represent relative activities (vi,v) and are
expressed as a percentage of a control without inhibitors
Trehalose concentration
C. riparius
Porcine kidney
2.5 mM
Inhibitor (#)
0.5 mM
5 mM
20 mM
(1)
(2)
(3)
(4)
(5)
(6)
(7)
(8)
50
68
59
46
101
91
100
95
51
67
63
63
102
83
95
96
103
97
106
75
94
102
98
103
100
102
91
98
103
100
104
103
strate and inhibitor, and there are two routes to the complex
enzyme substrate according to the following equilibria (Segel
1975, p.836):
Fig. 7. Structure of casuarine and analogs used as inhibitors of trehalase from
C. riparius.
binding to C. riparius trehalase and with binding of the inhib-
itor that modifies the kinetics as a function of trehalose
concentration. A cartoon illustrating the interaction of deoxy-
nojirimycin with trehalase from midge larvae is shown in
Figure 8.
There is significant dissimilarity in the kinetic behavior be-
tween trehalase from C. riparius and trehalase from porcine
kidney. This is clearly evidenced by several aspects: first of
all, there is a significant difference in substrate affinity; second,
the degree of inhibition caused by iminosugar alkaloids casuar-
ine, castanospermine, deoxynojirimycin and their derivatives is
markedly different between the two enzymes; third, the effect
of deoxynojirimycin is different since it modifies the v versus
[S] relationship from hyperbolic to sigmoidal in C. riparius
trehalase. Taken together, these data support the idea that, al-
though trehalase is an evolutionary old and well-conserved
protein, the midge trehalase bears molecular features that could
be exploited to develop compounds having insecticidal activity
against dipterans. Comparative studies with trehalases from
species belonging to other insect orders are needed to explore
whether these features are shared also by major agricultural
insect pests.
It is now well accepted that trehalase catalytic site contains
two subsites (Asano et al. 1996; Silva et al. 2004; Gibson et al.
2007; Cardona et al. 2009), a catalytic site and a recognition
site. It was demonstrated that deoxynojirimycin can bind to
each subsite of the porcine trehalase (Asano et al. 1996). Our
results can be interpreted with more than one deoxynojirimycin
Table V. Inhibition by casuarine derivatives of membrane-bound trehalase from
C. riparius larvae and trehalase from porcine kidney. Initial rate of trehalase
activity was measured in the presence of 0.5 mM trehalose (C. riparius) and
5 mM trehalose (porcine kidney) and increasing concentrations of the indi-
cated inhibitors ranging from 0.1 to 200 μM. Data are means SE of three
independent experiments
C. riparius
Porcine kidney
IC50(µM)
IC50(µM)
Ki (µM)
Ki (µM)
7-Deoxycasuarine 1.2 0.04
7-Homocasuarine 65.5 1.5
6-Deoxy-DMDP 52.4 2.2
0.59 0.02 21.5 2.8
33.0 0.8 214.0 28.8
25.6 1.1 110.8 16.4
11.7 1.4
107.0 14.4
55.4 8.2
5.3 1.2
Fig. 6. Inhibition kinetics by compound 4 on C. riparius trehalase. Closed
symbols represent a control in the absence of inhibitor, open symbols initial rates
in the presence of 100 μM inhibitor. Inset: Lineweaver–Burk plot of the data.
DAB-1
19.0 0.9
9.3 0.4
10.6 2.4
1192

Trehalase from Chironomus riparius larvae
Fig. 8. Cartoon of the inhibition pattern by deoxynojirimycin on C. riparius trehalase. The schematic structure of trehalose (substrate), glucose (products), and
deoxynojirimycin (inhibitor) are shown.
Materials and methods
Animals
synthesized as described by Cardona et al. (2003), Merino et
al. (2008) and Cardona et al. (2009). Deoxynojirimycin deriva-
tives (5R,6S,7S,8S)-5,6,7-trihydroxy-8-(hydroxymethyl)-3-
methylhexahydropyrido[1,2-c][1,3]oxazin-1(3H)-one (6),
(3R,4S,5S,6S)-N-benzyl-2-ethyl-3,4,5-trihydroxy-6-(hydroxy-
methyl)piperidine-1-carboxamide (4), (5S,6S,7R,8R)-6,7,8-
trihydroxy-5-(hydroxymethyl)-1-methyltetrahydro-1H-oxazolo
[3,4-a]pyridin-3(5H)-one (7), (6R,7S,8S)-6,7,8-trihydroxy-5-
(2-oxopropyl)tetrahydro-1H-oxazolo[3,4-a]pyridin-3(5H)-one
(8), (5S,6S,7S,8R)-1-(aminomethyl)-6,7,8-trihydroxy-5-
(hydroxymethyl) hexahydroimidazo [1,5-a]pyridin-3(5H)-
one (5), (5S,6S,7R,8R)-1-(aminomethyl)-6,7,8-trihydroxy-5-
(hydroxymethyl)tetrahydro-1H-oxazolo[3,4-a]pyridin-3(5H)-one
(1), (5R,6S,7S,8R)-3-(aminomethyl)-5,6,7-trihydroxy-1-oxoocta-
hydropyrido[1,2-c][1,3]oxazine-8-carboxylic acid (3) and
(5R,6S,7S,8R)-1-(aminomethyl)-6,7,8-trihydroxy-3-oxo-
hexahydro-1H-oxazolo[3,4-a]pyridine-5-carboxylic acid (2)
were synthesized as previously described (Cipolla et al. 2007).
C. riparius larvae were purchased alive from a wholesale bait
and fish stocking farm (Eschematteo, Parma, Italy). Larvae
were maintained at constant photoperiod (16 h light/8 h dark)
and temperature (4°C) in large vessels containing 2 cm of de-
chlorinated tap water, continuously aerated, and reared with
Tetramin or yeast powder. The water was changed as soon it
became turbid. The larvae used for the experiments were in
the fourth instar.
Chemicals
Tris, 4-morpholine-ethansulfonic acid (MES), HEPES, CHAPS,
phenylmethylsulfonyl fluoride (PMSF), sodium azide, nitro blue
tetrazolium (NBT), 5-bromo-4-chloro-3-indolyl phosphate
(BCIP) trehalase from porcine kidney, α-methyl-D-mannoside,
deoxynojirimycin, castanospermine, salts, substrates and coen-
zymes were purchased from Sigma-Aldrich (Italy). The
following compounds, (1R,2R,3R,6S,7S,7aR)-3-(hydroxymethyl)
hexahydro-1H-pyrrolizine-1,2,6,7-tetrol (casuarine),
(1R,2R,3R,6R,7aR)-3-(hydroxymethyl) hexahydro-1H-pyrrolizine-
1,2,6-triol (7-deoxycasuarine), (1R,2R,3R,6R,7R,7aR)-3,7-bis
(hydroxymethyl)hexahydro-1H-pyrrolizine-1,2,6-triol
(7-homocasuarine), (2R,3R,4R,5R)-3,4-dihydroxy-2-(hydroxy-
methyl)-5-methylpyrrolidine (6-deoxy-DMDP) and (2R,3R,4R)-
3,4-dihydroxy-2-(hydroxymethyl)pyrrolidine (DAB-1) were
Tissue homogenization and protein extraction
Midge larvae were suspended in 9 mL/g fresh weight of a hy-
potonic buffer (50 mM MES, pH 6.5) containing 0.1 mM
PMSF and 0.01% sodium azide and homogenized at 4°C in
a Warring mixer, set at 11,400 rpm, for two 2-min periods sep-
arated by a 2-min cooling interval. The suspension was then
filtered through two layers of surgical gauze, and one small al-
1193

M Forcella et al.
iquot was withdrawn for enzyme assays. The crude extract was
centrifuged at 100,000 × g for 1 h at 4°C (XL-90 Ultracentri-
fuge, Beckman), and the resulting supernatant was discharged.
The sediment was resuspended in four volumes of homogeni-
zation buffer in a glass and teflon Potter-Helvehjem, two 6-
strokes at 2000 rpm, separated by 1 min in ice and incubated
with 30 mM CHAPS, stirring gently at room temperature for
30 min. The preparation was centrifuged at 100,000 × g for 1 h
at 4°C, and the supernatant containing the solubilized mem-
brane-bound trehalase was used for further processing.
pH 5.5, sodium acetate 20 mM, pH 6.5 and HEPES–Tris
20 mM, pH 7.5. The effect of inhibitors was evaluated as a
function of trehalose concentrations from 0.1 to 5 mM in the
presence of fixed inhibitor concentrations (see figure legends)
and at fixed substrate concentration in the presence of increas-
ing inhibitor concentrations as specified in the figure legends.
Kinetic parameters were calculated using a multiparameter, it-
erative, non-linear regression program based on the
Marquardt–Levenberg algorithm (Sigma Plot, Jandel, CA).
Data are given SD of three independent experiments.
Determination of molecular mass
Purification of membrane-bound trehalase
The molecular weight of membrane-bound trehalase was deter-
mined by SDS gel electrophoresis according to a standard
protocol and by gel filtration on the Superdex 200 HR 10/30
(Amersham) column in 50 mM sodium phosphate buffer (pH 7)
containing 150 mM NaCl into FPLC system, with the following
proteins (from Sigma Aldrich) as standards: thyroglobulin (669
kDa), apoferritin (443 kDa), alcohol dehydrogenase (150 kDa),
bovine serum albumin (67 kDa) and deoxyribonuclease (31 kDa).
The supernatant containing membrane-bound trehalase activ-
ity was applied on a weak anion exchanger (DEAE
Sephacel, Pharmacia Biotech). The column was equilibrated
and washed with 20 mM Tris-HCl, pH 6.8, and trehalase
was eluted by 1 M NaCl in the same buffer. Fractions con-
taining trehalase activity were combined, dialyzed (cutoff
13 kDa) against 20 mM Tris-HCl, pH 6.8 and concentrated
by ultrafiltration under N2 at 200–300 KPa of pressure
(Amicon unit, Millipore cellulose filter, 30 kDa). Fractions
were then chromatographed on Mono Q anion exchange col-
umn in a fast protein liquid chromatography (FPLC) system
(Pharmacia): the column was equilibrated and washed with
20 mM Tris-HCl, pH 6.8, and fractions were eluted,
performing a linear gradient with increasing concentrations
of sodium chloride (from 0 to 1 mol L−1) at a flow rate
of 1 mL min−1. Fractions with trehalase activity were com-
bined, dialyzed, concentrated on Centricon YM-30 (Millipore)
and subjected to affinity chromatography using a concanavalin
A column (Con A-Sepharose 4B, Amersham) according to Ja-
hagirdar et al. (1990). The column was washed with 20 mM
Tris-HCl, pH 6.8 containing 0.5 M NaCl, 1 mM MnCl2,
1 mM CaCl2, and elution was achieved with 0.2 M α-methyl-
D-mannoside in the same buffer. As before, trehalase fractions
were combined, dialyzed against 20 mM Tris-HCl, pH 6.8 and
concentrated on Centricon YM-30 (Millipore). Protein concen-
tration was determined with the method of Bradford (1976)
using bovine serum albumin as standard. The enzymatic activ-
ity was expressed in international units and referred to protein
concentration.
Native electrophoresis
Non-denaturating electrophoresis was done on gradient from
20% to 6% polyacrylamide gel at constant voltage of 60 V
overnight at 4°C until equilibrium. No SDS nor β-mercap-
toethanol was added, and the samples were not heated before
being loaded onto the gel. After the run, trehalase activity was
detected by incubating the gel at 37°C overnight in 120 mM
sodium acetate, pH 6.5, 10 mM MgCl2, 1 mM ATP, 0.6 mM
NADP+, 20 mM trehalose, 1.34 U/mL hexokinase, 0.86 U/mL
glucose-6-phosphate dehydrogenase and 0.6 mM BCIP in 70%
dimethylformamide and 0.5 mM NBT in dimethylformamide.
Acknowledgments
We thank Carla Smeraldi for proofreading the manuscript in its
final form. This work was supported by a grant from the
University of Milano-Bicocca (FAR 2008). We also thank
Consorzio Interuniversitario Nazionale “Metodologie e
Processi Innovativi di Sintesi” (C.I.N.M.P.I.S.), MIUR (PRIN
2008) and Ente Cassa di Risparmio di Firenze, Italy, for
financial support.
Enzyme assay
Trehalase activity was measured through a coupled assay with
glucose-6-phosphate dehydrogenase and hexokinase according
to Wegener et al. (2003). All enzyme assays were performed in
triplicates at 30°C using sample volumes varying from 5 to 20
µL in a 1-mL test and using a Cary3 UV–Vis Spectrophotom-
eter. Enzyme activities were analyzed by Cary Win UV
application software for Windows XP. The specific activity
(U mg−1) was expressed as µmol min−1 mg protein−1. Values
were expressed as mean SE of replicated.
Abbreviations
BCIP, 5-bromo-4-chloro-3-indolyl phosphate; CHAPS, 3-[(3-
cholamidopropyl)-dimethyl-ammonio]-1-propanesulfonate;
FPLC, fast protein liquid chromatography; HEPES, 4-(2-
hydroxyethyl)-1-piperazine-ethansulfonic acid; HPLC,
high-performance liquid chromatography IC50 50% inhibitor
concentration; MES, 4-morpholine-ethansulfonic acid; NBT,
nitro blue tetrazolium; PMSF, phenylmethylsulfonyl fluoride;
SDS-PAGE, sodium dodecyl sulfate-polyacrylamide gel elec-
trophoresis; Tris, 2-amino-2-hydroxymethyl-1,3-propanediol.
Kinetic analyses
Kinetic experiments were performed using purified trehalase
from C. riparius and commercial porcine trehalase measuring
enzymatic activity at different trehalose concentrations from
0.1 to 20 mM. Kinetic experiments were carried out at different
pH values in the following buffer systems: MES–Tris 20 mM
References
Ando O, Nakajima M, Kifune M, Fang H, Tanazawa K. 1995. Trehazolin, a
slow, tight-binding inhibitor of silkworm trehalase. Biochim Biophys Acta.
1224:295–302.
1194

Trehalase from Chironomus riparius larvae
Asano N. 2003. Glycosidase inhibitors: update and perspectives on practical
use. Glycobiology. 13:93R–104R.
Asano N, Kato A, Matsui K. 1996. Two subsites on the active center of porcine
kidney trehalase. Eur J Biochem. 240:692–698.
Asano N, Takeuchi M, Kameda Y, Matsui K, Kono Y. 1990. Trehalase inhibi-
tors, validoxylamine A and related compounds as insecticides. J Antibiot.
43:722–726.
Azuma M, Yamashita O. 1985. Cellular localization and proposed function of
midgut trehalase in the silkworm larva, Bombyx mori Tiss. Cell. 17:539–551.
Bradford MM. 1976. A rapid and sensitive method for the quantification of
microgram quantities of proteins utilizing the principle of protein dye bind-
ing. Anal Biochem. 72:248–252.
Callaghan A, Hirthe G, Fisher T, Crane M. 2001. Effect of short-term exposure
to chlorpyriphos on developmental parameters and biochemical biomarkers
in Chironomus riparius Meigen. Ecotoxicol Environm Safety. 50:19–24.
Cardona F, Faggi E, Liguori F, Cacciarini M, Goti A. 2003. Total syntheses of
hyacinthacine A2 and 7-deoxycasuarine by cycloaddition to a carbohydrate
derived nitrone. Tetrahedron Lett. 44:2315–2318.
Cardona F, Parmeggiani C, Faggi E, Bonaccini C, Gratteri P, Sim L, Gloster
TM, Roberts S, Davies GJ, Rose DR, et al. 2009. Total syntheses of casuarine
and its 6-O-alpha-glucoside: complementary inhibition towards glycoside
hydrolases of the GH31 and GH37 families. Chem Eur J. 15:1627–1636.
Cipolla L, Reis Fernandes M, Gregori M, Airoldi C, Nicotra F. 2007. Synthesis
and biological evaluation of a small library of nojirimycin derived bicyclic
iminosugars. Carbohydr Res. 342:1813–1830.
Mitsumasu K, Azuma M, Niimi T, Yamashita O, Yaginuma T. 2008. Changes
in the expression of soluble and integral-membrane trehalases in the midgut
during metamorphosis in Bombyx mori Zool Sci. 25:693–698.
Jin L-Q, Zheng Y-G. 2009. Inhibitory effects of validamycin compounds on the
termites trehalase. Pest Biochem Physiol Sci. 95:28–32.
Merino P, Delso I, Tejero T, Cardona F, Marradi M, Faggi E, Parmeggiani C,
Goti A. 2008. Nucleophilic additions to cyclic nitrones en route to imino-
cyclitols—total syntheses of DMDP, 6-deoxy DMDP, DAB-1, CYB-3,
nectrisine, and radicamine B. Eur J Org Chem. 17:2929–2947.
Mitsumasu K, Azuma M, Niimi T, Yamashita O, Yaginuma T. 2005. Mem-
brane-penetrating trehalase from silkworm Bombyx mori. Molecular
cloning and localization in larval midgut. Insect Mol Biol. 14:501–508.
Reed WD, Sacktor B. 1971. Localization of trehalase in flight muscle of the
blowfly Phormia regina. Arch Biochem Biophys. 145:392–401.
Reiner JM. 1969. Behavior of enzyme systems.. N.Y: Van Nostrand Reinhold.
Rosenberg DM, Resh VH. 1993. Freshwater biomonitoring and benthic
macroinvertebrates.. New York: Chapman and Hall. p. 488.
Sculley MJ, Morrison JF, Cleland WW. 1996. Slow-binding inhibition: the
general case. Biochim Biophys Acta. 1298:78–86.
Segel H. 1975. Enzyme kinetics. NY: John Wiley & sons.
Shimada S, Yamashita O. 1979. Trehalose absorption related with trehalase in
developing ovaries of the silkworm Bombyx mori. J Comp Physiol.
131:333–339.
Silva MCP, Terra WR, Ferreira C. 2004. The role of carboxyl, guanidine and
imidazole groups in catalysis by a midgut trehalase purified from an insect
larvae. Insect Biochem Mol Biol. 34:1089–1099.
Strang RHC, Clement EM. 1980. The relative importance of glucose and tre-
halose in the nutrition of the nervous system of the locust Schistocerca
americana gregaria. Insect Biochem.. 10:155–161.
Elbein AD, Pan YT, Pastuszak I, Carroll D. 2003. New insights on trehalose: a
multifunctional molecule. Glycobiology. 13:17R–27R.
Fisher HF, Gates RE, Cross DG. 1970. A ligand exclusion theory of allosteric
effects. Nature. 228:247–249.
Forcella M, Berra E, Giacchini R, Rossaro B, Parenti P. 2007. Increased alanine
concentration is associated with exposure to fenitrothion but not carbamates in
Chironomus riparius larvae. Ecotoxicol Environm Safety.. 66:326–334.
Gibson RP, Gloster TM, Roberts S, Warren RAJ, Storch de-Gracia I, García Á,
Chiara JL, Davies GJ. 2007. Molecular basis for trehalase inhibition re-
vealed by the structure of trehalase in complex with potent inhibitors.
Angew Chem Int Ed. 46:4115–4119.
Gu J, Shao Y, Zhang C, Liu Z, Zhang Y. 2009. Characterization of putative
soluble and membrane-bound trehalases in a hemipteran insect, Nilaparvata
lugens J Insect Physiol.. 55:997–1002.
Tatun N, Singtripop T, Tungjitwitayakul J, Sakurai S. 2008. Regulation of sol-
uble and membrane-bound trehalase activity and expression of the enzyme
in the larval midgut of the bamboo borer Omphisa fuscidentalis. Insect Bio-
chem Mol Biol. 38:788–795.
Thompson SN. 2003. Trehalose—the insect “blood” sugar. Adv Insect Physiol.
31:205–285.
Vaandrager SH, Haller TB, Van Marrewijk WJA, Beenakkers AMT. 1989.
Fractionation and kinetic properties of trehalase from flight muscle and he-
molymph of the locust Locusta migratoria. Insect Biochem. 19:89–94.
van Handel E. 1978. Trehalose turnover and the coexistance of trehalose and
active trehalase in cockroach haemolymph. J Insect Physiol. 24:151–153.
Wegener G, Tschiedel V, Schlöder P, Ando O. 2003. The toxic and lethal ef-
fects of the trehalase inhibitor trehazolin in locusts are caused by
hypoglycemia. J Exp Biol. 206:1233–1240.
Gussin AES, Wyatt GR. 1965. Membrane-bound trehalase from Cecropia silk-
worm muscle. Arch Biochem Biophys. 112:626–634.
Hanozet G, Pircher HP, Vanni P, Oesch B, Semenza G. 1981. An example of
enzyme hysteresis. J Biol Chem. 256:3703–3711.
Jahagindar AP, Downer RGH, Viswanatha T. 1990. Purification and character-
ization of trehalose-hydrolyzing enzymes from thoracic musculature of the
American cockroach Periplaneta americana. Insect Biochem. 20:511–516.
Kato A, Kano E, Adachi I, Molyneux RJ, Watson AA, Nash RJ, Fleet GWJ,
Wormald MW, Kizu H, Ikeda K, et al. 2003. Australine and related alka-
loids: easy structural confirmation by 13C NMR spectral data and
biological activities. Tetrahedron Asymmetr.. 14:325–331.
Wyatt GR. 1967. The biochemistry of sugars and polysaccharides in insects.
Adv Insect Physiol. 4:287–360.
Yaginuma T, Mizuno T, Mizuno C, Ikeda M, Wada T, Hattori K, Yamashita O,
Happ GM. 1996. Trehalase in the spermatophore from the bean-shaped acces-
sory gland of the male mealworm beetle, Tenebrio molitor: purification,
kinetic properties and localization of the enzyme. J Comp Physiol.
20:1079–1085.
Knuesel I, Murao S, Shin T, Amachi T, Kayser H. 1998. Comparative studies of
suidatrestin, a specific inhibitor of trehalases. Comp Biochem Physiol Part B.
120:639–646.
Yamashita O, Sumida M, Hasegawa K. 1974. Developmental changes in mid-
gut trehalase activity and its localization in the silkworm, Bombyx mori. J
Insect Physiol. 20:1079–1085.
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