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Insect Molecular Biology (2003) 12(5), 415–425

Expression of functional Anopheles merus α-amylase in


Blackwell Publishing Ltd.

the baculovirus/Spodoptera frugiperda system

P. C. Effio*, A. V. Folgueras-Flatschart, W. R. Montor, kinetic parameters, optimum pH and substrate specificity


F. M. Pernasetti, M. T. Pueyo and M. C. Sogayar (Bertoft et al., 1984; Akazawa et al., 1988; Knutson, 1993).
Instituto de Quimica, Universidade de São Paulo, The α-amylases are widely used by insects in their larval
São Paulo, Brazil stage, as has previously been reported (Terra & Ferreira,
1994). Closely linked amylase genes have been detected in
the housefly, Musca domestica (Ogita, 1968), silkworm,
Abstract Bombyx mori (Kikkawa, 1953), in several species of the
The Anopheles merus (Diptera, Nematocera, Culicoi- flour beetle, Tribolium (Pope et al., 1986), and in the yellow
dea) α-amylase gene (AmerAmy, GenBank Accession fever mosquito, Aedes aegypti (Grossman et al., 1997). To
Number U01210) was amplified with its own or with date, the only functional recombinant α-amylases to be
the Zabrotes subfasciatus α-amylase signal peptide reported are those from Zabrotes subfasciatus (Grossi de Sá
(ZsAmerAmy, GenBank Accession Number AY270183) & Chrispeels, 1997) and Diabrotica virgifera virgifera
by PCR, using designed primers. The AmerAmy gene (Titarenko & Chrispeels, 2000). In Culicoidea, the expres-
was sequenced from its promotor to the TGA codon. sion of an α-amylase gene was determined at the mRNA
As a positive control, the Z. subfasciatus α-amylase gene level in the salivary glands of Ae. aegypti (Grossman &
with its own signal peptide (ZsAmy, GenBank Acces- James, 1993). However, that protein was not detected as an
sion Number AF255722) was also amplified by PCR. active enzyme as was accomplished for an α-glucosidase
These three sequences were inserted into the baculo- (Marinotti & James, 1990) in these organs. The results
virus genome using the Bac-to-Bac™ system. Recom- on Anopheles merus presented here evinced for the first
binant baculovirus preparations were used to infect time the expression, using the baculovirus/Spodoptera
Sf 9 Spodoptera frugiperda insect cells. The A. merus frugiperda cell system, of a Culicoidea α-amylase recom-
α-amylase was successfully expressed as an active binant gene as a functional enzyme. These results strongly
enzyme detected mainly in cell culture supernatants. suggest that this protein could also be an active enzyme of
the digestive secretion in these Diptera.
Keywords: alpha amylase, Anopheles merus,
expression, Baculovirus/Spodoptera frugiperda.
Results

Generation of recombinant α-amylase constructs


Introduction
The coding sequence for A. merus α-amylase (AmerAmy)
Different α-amylase (EC 3.2.1.1) genes from archaebacteria,
was obtained and subcloned into the baculovirus-based
eubacteria, fungi, animal and plant sources have been
pFastBac1 plasmid vector as described in Experimental
cloned and sequenced (Vihinen & Matsala, 1989). These
procedures. Two different constructs were sought, namely
enzymes hydrolyse alpha-1,4 glycosidic bonds present in
pFastBac1-AmerAmy (contains the Ameramy gene plus
oligosaccharides and starch (Steup, 1988), playing a key
the sequence corresponding to its own signal peptide) and
role in the overall metabolism of these organisms. From
pFastBac1-ZsAmerAmy (contains the AmerAmy gene plus
animals to bacteria, α-amylases display a wide variety of
the sequence corresponding to the signal peptide of the
amylase gene of Z. subfasciatus). A construct made with
Received 16 December 2002; accepted after revision 25 April 2003. Corre- the Z. subfasciatus α-amylase gene (pFastBac1-ZsAmy)
spondence: Drs Manuel Troyano Pueyo and Mari Cleide Sogayar, Instituto was used as a control. Transformation of plasmid DNA into
de Quimica, Universidade de São Paulo, CP 26077, São Paulo 05513–970,
SP, Brazil. E-mail: matpueyo@ig.com.br and mcsoga@iq.usp.br E. coli DH10Bac allowed the production of the following
*On leave from the Universidad Nacional Pedro Ruiz Gallo, Lambayeque, bacmid recombinants: Bd-AmerAmy, Bd-ZsAmerAmy and
Perú. Bd-ZsAmy.

© 2003 The Royal Entomological Society 415


416 P. C. Effio et al.

Figure 1. Lugol test of starch hydrolysis by the supernatant samples of Sf9 cells infected with the recombinant baculoviruses Bv-AmerAmy, Bv-ZsAmerAmy
and Bv-ZsAmy. 1, uninfected Sf9 cells; 2, Sf9 cells infected with baculovirus lacking insert. The following holes correspond repectively to cells infected with
recombinant baculovirus: 3, Bv-AmerAmy; 4, Bv-ZsAmerAmy; 5, Bv-ZsAmy. The extreme right hole corresponds to a negative control: 6, lugol in Milli-Q water.
Starch substrate (480 µl) was assayed for hydrolysis with 20 µl of supernatant of Sf9 cell cultures infected with recombinant baculoviruses. After 12 h at 37 °C,
10 µl was removed and mixed with 200 µl of lugol. Nonhydrolysed starch yields a blue colour (Sf9, Bv and Milli-Q water). Hydrolysis of starch to glucose and
maltose yields a yellow colour (Bv-ZsAmy). Hydrolysis to oligosaccharides yields a purple colour (Bv-AmerAmy, Bv-ZsAmerAmy).

Figure 3. Activity assay of AmerAmy in blue starch gel. Supernatants from Sf9 cell cultures infected
Figure 2. Chromatographic pattern of starch with the recombinant baculoviruses Bv-AmerAmy, Bv-ZsAmerAmy and Bv-Zs were precipitated with
cleavage by A. merus α-amylase. Starch was cold acetone and redissolved in 10% of the initial volume with Milli-Q water. After electrophoretic
dissolved to 0.5% in activity buffer (100 mM separation in PAGE-SDS, the gel was washed three times with Milli-Q water for 30 min. Another
phosphate buffer, pH 6.0, 20 mM NaCl, 0.1 mM wash with activity buffer was made in order to reactivate the enzymatic activity as described in
CaCl2). To determine the cleavage pattern, a Experimental procedures. An agar gel containing blue starch was layered over the polyacrylamide gel.
series of four Eppendorf tubes containing 480 µl The set was wrapped in a PVC film and incubated at 37 °C for 12 h. Active amylases separated in the
of starch were prepared. To each of those tubes, polyacrylamide gel below are detected in samples from culture supernatants as clear bands in the agar/
20 µl of the vacuum-concentrated supernatant blue starch gel. Electrophoretic conditions were 100 V/20 mA. Samples of 5 and 0.5 µg were applied in
from Sf9 cultures infected with the recombinant lanes containing A. merus and Zabrotes proteins, respectively. Both pellet and supernatant samples
baculoviruses, Bv-AmerAmy, Bv-ZsAmerAmy and were analysed in lanes labelled as follows: 1 and 5, Sf9 from noninfected culture; 2 and 6, Bv from culture
Bv-ZsAmy, was added. Tubes were incubated at infected with baculovirus lacking insert; 3, Bv-AmerAmy, supernatant from culture infected with
37 °C for 24 h and centrifuged at 13 000 g for baculovirus carrying sequences corresponding to A. merus α-amylase and to its own signal peptide;
10 min at 4 °C. The supernatant was dried at 4, Bv-ZsAmerAmy, supernatant from culture infected with baculovirus carrying sequences
60 °C and redissolved in 25 µl of Milli-Q water. corresponding to the Zabrotes signal peptide and A. merus α-amylase; 7, Bv-AmerAmy, pellet from
A total of 1 µl (five applications of 0.2 µl) of each culture infected with baculovirus carrying sequences corresponding to its own signal peptide and
sample was applied. The supernatant from the A. merus α-amylase; 8, Bv-ZsAmerAmy, pellet from culture infected with baculovirus carrying
Sf9 culture infected with baculovirus lacking insert sequences corresponding to the Zabrotes signal peptide and A. merus α-amylase; 9, Bv-ZsAmy,
was used as negative control. Lane 1: glucose supernatant from culture infected with baculovirus carrying sequences corresponding to Zabrotes’s own
plus maltose. Supernatant samples from Sf9 signal peptide and α-amylase.
cultures infected with recombinant baculoviruses
are in: Lane 2, Bv-ZsAmy; Lane 4, Bv-AmerAmy;
Lane 5, Bv-ZsAmerAmy. Lane 3, negative control.

© 2003 The Royal Entomological Society, Insect Molecular Biology, 12, 415–425
Expression of the Anopheles merus α-amylase gene 417

chromatographic pattern shown in Fig. 2 indicates that the


Transfection of Sf9 insect cells; production of baculoviruses amylases present in culture supernatants from cells infected
carrying the AmerAmy, ZsAmerAmy and ZsAmy genes with Bv-AmerAmy and Bv-ZsAmerAmy yield a cleavage
Sf 9 insect cells were transfected with the Bd-AmerAmy, Bd- pattern that is typical of α-amylase, with relatively large
ZsAmerAmy and Bd-ZsAmy bacmid constructs for primary amounts of oligosaccharides and small amounts of glucose
baculovirus particle production. After transfection, baculo- and maltose, whereas Bv-ZsAmy culture supernatants
virus production was confirmed by two different methods, degraded starch mainly to glucose and maltose and small
based on the work we previously reported (Folgueras- amounts of oligosaccharides, as shown in Fig. 1.
Flatschart et al., 2000), namely the observation of the The α-amylase activity present in Sf9 culture super-
cytopathic effect that occurs upon subculturing of trans- natants was also analysed by the blue starch overlay method
fected cells and amplification of specific DNA fragments, and by Western blotting. The results shown in Figs 3 and
by PCR, using culture supernatants potentially containing 4 indicate that proteins present in supernatants obtained
baculovirus particles, as a template and specific primers for from Sf9 cells infected with Bv-AmerAmy and Bv-ZsAmerAmy
AmerAmy-F, ZsAmerAmy-F and AmerAmy-R. Baculovirus display amylase activity. The Coomassie-blue-stained
master stocks were amplified for Sf9 infection and protein SDS-PAGE of proteins pelleted from culture medium of
production. After 96 h of massive infection [multiplicity of infected cells revealed a single protein band of approxim-
infectivity (MOI) = 10; numbers are relationships between ately 62 kDa, in both ZsAmy and ZsAmerAmy prepara-
viral particles and cells], Sf9 cultures displayed typical tions (Fig. 4A). However, the band corresponding to ZsAmy
cytophathic effects, i.e. low cell density and poor adherence is much more intense than that of ZsAmerAmy. When the
to the substrate, indicating that virus production was taking gel was silver stained, a second (faint) band, of approxim-
place. The nontransfected Sf9 cultures as well as those ately 52 kDa, was observed in ZsAmy supernatants (data
infected with empty baculoviruses displayed a normal cell not shown). Western blotting showed that two α-amylases
monolayer. were secreted by cells infected with the ZsAmy baculovirus.
The protein(s) present in the 62 kDa band reacted weakly
Protein determination in cell supernatants with the Z. subfaciatus anti-(α-amylase) antibody, whereas
The total protein content of cell supernatants was deter- the second band (≈ 52 kDa) reacted strongly with this
mined as described in Material and methods. Cells trans- antiserum. In the case of A. merus, only one band, cor-
fected with Bv lacking insert as a control gave a mean value responding to the secreted protein that exhibits an apparent
(three determinations) of 670 µg/ml. This figure corres- MW = 62 kDa, was detected in culture supernatants ob-
ponds in general to proteins of the bovine serum used in tained from cells infected with the ZsAmerAmy baculovirus
the culture medium. Thus, all determinations accomplished (Fig. 4B).
in the supernatants of cells transfected with baculoviruses
carrying inserted sequences were corrected by subtracting Sequence analysis
670 µg/ml from the actual data. Bv-ZsAmy transfected cells The promotor of the amylase gene is likely to be CCCAT-
were included as an internal control. Typical protein content CAACT with 97% confidence (Fig. 5).
values obtained when the insect cells were transfected with The conceptual translation product of the α-amylase
Bv-AmerAmy, Bv-ZsAmerAmy and Bv-ZsAmy are 7.5, 8.5 genomic sequence is also presented in Fig. 5. Features of
and 19.0 µg/ml, respectively. this product are: (a) MKLLVRLAPILLLALTGRPVAA, a plau-
sible signal peptide with 90% confidence, suggesting that
Detection of α-amylase activity the peptide is secreted; (b) a mature peptide consisting of
Amylase activity was detected both in culture supernatants 492 residues starting with the blocking N-terminal glutamine,
and in cell extracts using the lugol method, as described in an indication that the peptide may be in contact with digest-
Material and methods. The results, shown in Fig. 1, indicate ive secretion (Strobl et al., 1997); (c) 54.8 kDa, the mole-
that only amylases from culture supernatants obtained cular weight calculated for the unmodified mature peptide.
from cells infected with recombinant baculovirus Bv- Comparison with other amino acid sequences reveals that
AmerAmy and Bv-ZsAmerAmy were able to degrade the a glutamic acid (E) and two aspartic acid (D) residues that
starch substrate to dextrins displaying a number of glucose are conserved in the α-amylase family (Janecek, 1997) are
residues sufficient to give a purple-coloured lugol solution. known to be involved in catalysis at the active sites of por-
The Z. subfasciatus α-amylase, produced by Bv-ZsAmy- cine pancreatic (Qian et al., 1994) and Tenebrio molitor
infected Sf9 cells, degrades starch to products that have a (Strobl et al., 1998) α-amylases. They are found in Amer-
number of glucose residues not enough to give a colour Amy as D215, D318 and E252. Three conserved histidine
complex with l 3− (Bailey & Whelan, 1961). The products residues, which are thought to be involved in substrate
obtained upon starch hydrolysis by recombinant α- binding (Qian et al., 1994; Strobl et al., 1998), are also
amylases were analysed by paper chromatography. The present in AmerAmy as H125, H219, H317. By comparison

© 2003 The Royal Entomological Society, Insect Molecular Biology, 12, 415–425
418 P. C. Effio et al.

Figure 4. SDS-PAGE and Western blot of proteins present in culture supernatants from Sf9 cells infected with recombinant Bv-AmerAmy and Bv-ZsAmy
baculoviruses. A: Coomasie-blue staining of proteins fractionated by SDS-PAGE. Proteins recovered from supernatants of cell cultures transfected with
Bv-ZsAmy and Bv-ZsAmerAmy are in lanes 1 and 3, respectively. The 62 kDa band corresponding to ZsAmy is more intense than that of ZsAmerAmy. In
lane 2 are the molecular weight markers (Gibco). B: Western blot of proteins recovered from supernatants of cell cultures transfected with Bv-ZsAmy and
Bv-ZsAmerAmy are in lanes 1 and 3, respectively. Two bands of 62 and 52 kDa of different intensities are separated in lane 1. A unique band of 62 kDa is
observed in lane 3. A sketch of the molecular weight markers is shown in lane 2. Arrows point to α-amylase.

with previous data (Strobl et al., 1998), other conserved structures. In addition, the insect cytoplasmic environment
residues can be related to the chloride (R213, N316, R351) is less reducing than that of the E. coli cells, thus enabling
and calcium (N124, R176, D185, H219) binding sites in the proper disulphide bridge (–S–S–) assembly. This feature is
A. merus enzyme (Fig. 5). important in the case of the A. merus α-amylase because
The similarity and identity of AmerAmy with other α-amylases it displays 11 cystein residues, seven of which are concen-
is shown in Table 1. AmerAmy has 51–59, 49–51, 47, 11, 24 trated between amino acids 384 and 506 (Fig. 5). The number
and 8–23% identity with α-amylases from, respectively, of –S–S– bonds in this protein is not known but the high
insects, mammals, birds, plants, fungi and bacteria. frequency of cystein residues probably enhances the com-
AmerAmy does not have any putative N-glycosylation plexity of folding. Note that the α-amylase nucleotide
site (consensus sequence Asn-X-Ser/Thr). Instead, Amer- sequence used throughout this work is of genomic origin.
Amy has the sites T131, S141, T142 and T359, which could The lack of introns (Pernasetti, 1991) is a valuable feature
be O-glycosylated, with probabilities of 86.32, 96.33, 94.14 for the expression of this gene as an active α-amylase that
and 74.57%, respectively (Table 2). can now be used to study structural, kinetic and inhibitory
behaviour in the presence of insect plant amylase inhibitors.
This could reveal clues for future mosquito control strategies.
Discussion
We previously used the baculovirus/insect cell expres-
We chose to express the AmerAmy sequence in the baculo- sion system to produce active mouse Fos and Jun proteins
virus/insect cell system not only because it is a versatile (Corvello et al., 1995), bovine Herpesvirus glycoproteins
and reliable system for the production of recombinant (Folgueras-Flatschart et al., 2000), the alpha7 subunit of
proteins (Possee, 1997; Grossi de Sá & Chrispeels, 1997; acetylcholine nicotinic receptor (Aztiria et al., 2000) and
Titarenko & Chrispeels, 2000) but also because it allows human prolactin (Pereira et al., 2001). Here we describe
expression of eukaryotic genes in a eukaryotic cell system, the results showing that, for the first time, it was possible to
taking advantage of their protein synthesis machinery, express the A. merus α-amylase in the active form.
thereby facilitating folding and post-translational modifications The attempt to express the A. merus α-amylase gene
(O’Reilly et al., 1992). These include N- and O-glycosilation, attached to the sequence that codes for the signal peptide
acylation, proper proteolysis and oligomerization of the of α-amylase from Z. sufasciatus stems from the fact that
protein product via processes that are often identical to that this enzyme was previously successfully expressed in the
occurring into the original cells. Therefore, proteins may be baculovirus/insect cell system (Grossi de Sá & Chrispeels,
secreted from the cell or be directed to other subcellular 1997). Signal peptide sequences derived from insect protein

© 2003 The Royal Entomological Society, Insect Molecular Biology, 12, 415–425
Expression of the Anopheles merus α-amylase gene 419

Figure 5. Nucleotide sequence of the AmerAmy gene and its conceptual translation. The vertical arrow indicates the probable cleavage site of the signal
peptide. Open boxes indicate conserved residues of the active site, and shaded boxes are conserved residues involved in substrate binding. Conserved residues
of the chloride and calcium binding sites are, respectively, outlined letters and letters into bold outlined boxes. The shaded bordered sequence corresponds to
that conserved in the C terminal region of several amylases. The wavy bordered sequence corresponds to the typical sequence of the AmerAmy C terminal
region. See Discussion for further details.

have been used with variable results in the baculovirus/ reported here, ZsAmy was two-fold (19.0 µg/ml) higher
insect cell system. Previous studies on the expression of (see Fig. 4 for comparative purposes). Our data as well as
a plant protein, papain (Tessier et al., 1991), have shown those referred to above suggest that the recognition and
that the use of an insect-derived signal peptide enhanced the processing of signal peptides from heterologous pro-
the secretion five-fold over that detected when using the teins are not the only factors involved in the secretion level
native signal peptide. However, other studies have found that when the baculovirus/insect cell system is used. It is likely
insect-derived signal peptide sequences were not optimal that, among other factorss, disulphide bond formation and
(Jarvis et al., 1993; Dupuis et al., 1997; Dahl et al., 2000) or folding could be rate-limiting steps.
were not linked to the secretion of heterologous proteins in The identity of AmerAmy with its mammalian, insect
the baculovirus/insect cell system (Tessier et al., 1991; and bird counterparts is relatively high (59, 51 and 47%,
Dupuis et al., 1997). The level of secretion of A. merus α- respectively), as shown in Table 1.
amylase and Z. subfasciatus reported here (7.5, 8.5 and We identified three amino acids (D215, E252, D318) that
19.0 µg/ml) are lower than those reported for other hetero- are known to be important for catalysis and three histidine
logous proteins: GRP78/BJP (50–75 µg/ml), hepatitis B residues (H125, H219, H317) that are known to be involved
surface antigen (90 µg/ml; Lanford et al., 1989) and plant in substrate binding (Qian et al., 1994). Residues R213,
phaseolin (90 µg/ml; Bustos et al., 1988). Although a large N316 and R351 were identified as belonging to the chloride
difference between secretion of AmerAmy (7.5 µg/ml) and binding site, whereas residues N124, R176, D185, H219
ZsAmerAmy (8.5 µg/ml) was not detected in the results were related to the calcium binding site (Strobl et al., 1998).

© 2003 The Royal Entomological Society, Insect Molecular Biology, 12, 415–425
420 P. C. Effio et al.

Table 1. Identity and similarity of A. merus


Amino acid Amino acid α-amylase residues when compared with other
Organism Accession no. identity (%) similarity (%) known α-amylases

Insects
Anopheles gambiae L04753 98 100
Tenebrio molitor S75702 59 71
Drosophila melanogaster AB043051 55 68
Drosophila simulans D11734 55 70
Culex tarsalis U01211 55 67
Aedes aegyptis O02652 56 67
Tribolium castaneum U04271 56 69
Diabrotica virgifera virgifera AF208002 53 65
Phaedon cochleriae Y17902 52 64
Lutzomyia longipalpis AF132512 51 66

Mammals
Mus musculus J00360 51 66
Homo sapiens M24895 49 65
Rattus norvegicus J00703 49 64
Sus scrofa AF06472 49 64

Birds
Gallus gallus U63411 47 63

Plants
Hordeum vulgare X15226 11 not determined
Oriza sativa X52240 11 not determined
Vigna mungo X73301 11 not determined

Fungi
Aspergillus orizae X12726 24 43

Bacteria
Xanthomonas campestris M85252 23 35
Pirococcus woesei AF240464 11 not determined
Geobacillus stearothermophilus AF032864 08 not determined

These findings indicate that, as with other amylases, AmerAmy of certain α-amylases. The significance and the extent of
has a canonical composition and distribution of residues its distribution in living organisms remains to be ascert-
in order to attain its function. The amino acid C-terminal ained. Interestingly, the sequence determined for the C-
sequences of α-amylases from organisms from taxonomic terminal region in A. merus amylase displays DGVIAIH as
classes Mammalia, Insecta (orders Diptera, Hymenoptera, expected, but does not follow the rule of similarity or length
Lepidoptera, Coleoptera), Arachnida, Nematoda, Osteich- after it. Instead, a stretched SEVSVCVCCRMRSDGRV
thyes (order Teleostea), Bivalvia and Crustaceae were sequence was detected. This seems to be restricted to the
compiled (not shown) in order to contrast them with the amylase of A. merus and in part to the same protein of
corresponding sequence of A. merus α-amylase deter- A. gambiae (accession number L04753). A part of this
mined in this work. As a general rule, the C terminal tail sequence, VSVCVC, is also found in proteins that are not
starting at D comprises 12 amino acids. An example of related to amylases of several organisms and viruses,
this terminal C sequence is D G V L A I H V N A K L from including an inner region of agCP8155 (accession number
T. molitor (Strobl et al., 1997). According to our analysis, it EAA13780.1), an unidentified protein from A. gambiae.
can be noted as the widely conserved DGX1X2AIH sequence. Thus, concerning its C-terminal portion, the A. merus pro-
In addition, the similarity of residues between DG and A tein seems to be unique within the amylases from several
and after H was noted. In most of the sequences collected, higher organisms. Nevertheless, it is possible that the odd
the two amino acids preceding AIH are hydrophobic: VL, IL, C-terminal portion described may be present only in the
VI, VV, FV or FI. The amino acids immediately following A I conceptual translation of the genomic sequence, but not in
H are hydrophobic (A, V, I, G) and hydrophilic (KXK, NXK, the functional protein itself.
NXR, SXK, EXK, DXK, DXR) with lysine being highly con- O-glycosylation is expected to occur in AmerAmy at the
served, or in a few examples substituted by arginine or T138, T142, and S141 residues and in ZsAmy at the T425
glutamine. However, this tail is not observed in sequences residue (Table 2). AmerAmy does not display classical
that are currently available from bacteria, fungi, arachnida, N-glycosylation sites (Asn-X-Ser/Thr) whereas ZsAmy has
nematoda or plant amylases. It seems likely that the C- one starting at N442, close to the C-terminus (Grossi de Sá
terminal region, as described before, is a well-defined feature & Chrispeels, 1997).

© 2003 The Royal Entomological Society, Insect Molecular Biology, 12, 415–425
Expression of the Anopheles merus α-amylase gene 421

Table 2. Prediction of O-glycosylation sites on α-amylases from different of the relationship between the intensity of the ZsAmy and
insects based on protein and DNA sequence data
ZsAmerAmy 62 kDa bands reveals that it is inverted in
O-glycosylation Fig. 4A and Fig. 4B. To a smaller mass of ZsAmerAmy cor-
Insect Residue probability (%) responds a stronger antibody reaction. This may suggest
that the hypothetical modification at the mosquito protein
Aedes aegypti, Amy 1 T90 84.45
S23 75.63 does not interfere with the antibody cross-reaction and that
S93 73.06 it is not probably placed at the amylase epitope. Together,
S149 69.06 these two observations suggest that the epitope of these
S703 71.20
Aedes aegypti, Amy 2 T64 71.30 amylases is located near the C terminus, the domain C
Anopheles merus T138 86.32 (Strobl et al., 1998) of these proteins.
T142 94.14 The very faint band below the main band of Anopheles
T359 74.57
S141 96.33 amylase in Fig. 4(B), lane 3, (Western blot) is not distin-
Apis melifera None guishable from the sharp band of Fig. 4(A), lane 3
Culex tarsalis T63 97.96 (stained). The faint band is probably due to the lesser
T138 77.00
S460 73.06 O-glycosylation of T359 (see Table 2) and seems to be
Dermatophagoides pteronyssimus None the product of a residual event.
Diabrotica virgifera virgifera T427 93.66 The unique band of Anopheles amylase in Fig. 4(A,B),
Drosophila melanogaster, Amy distal T130 98.09
T358 59.83 lane 3, has a unique band of activity in Fig. 3, lanes 3 and 4.
S129 97.53 This suggests that the putative O-glycosylation of this protein
S135 99.85 is compatible with enzymatic activity. The two bands detected
S457 79.19
Drosophila melanogaster, Amy prox. T131 97.65 for the amylase of Zabrotes in Fig. 4A,B, lane 1, also have
T359 59.83 a unique activity band in Fig. 3, lane 9. Clearly, only one of
S130 81.06 the bands in Fig. 4A,B, lane 1, retains activity. This is con-
S136 99.90
S458 79.19 sistent with previous results (Grossi de Sá & Chrispeels,
Euroglyphus maynei None 1997). As judged by the slightly faster migration of the band
Lutzomyia longipalpis S357 94.95 in Fig. 3, lane 9, the activity is probably on the glycosylated
Phaedon cochleriae S136 78.50
T355 95.83 band (62 kDa) of Zabrotes α-amylase. If this interpretation
Tenebrio molitor None is correct, in Z. subfasciatus only the α-amylase modified
Tribolium castaneum None by a putative N-glycosylation retains enzymatic activity.
Zabrotes subfasciatus T425 83.24
The exact role played by glycosylation in protein function
is as yet not completely understood, but the data available
point towards variable roles for different proteins. Questions
on how N- or O-linked glycosylation might affect protein
In AmerAmy, the S141 T142 site has a high (94–96%) function are still to be adressed. A systematic compilation
probability of being O-glycosylated (Table 2). It occurs near of data on glycosylation regarding protein nature and func-
the consensus region I (DIIINH), into the B domain of tion, cell type, subcellular localization and secretion may
the enzyme. The results in Fig. 4 showed a single 62 kDa prove to be useful in establishing the importance of this
band in AmerAmy supernatant that cross-reacts with the post-translational modification.
ZsubAmy anti-amylase antibody. Considering that the The colour of the iodine–iodide complex with starch is
expected molecular weight for AmerAmy is 54.8 kDa, this dependent on the number of glucose residues in the chain
indicates that the enzyme was post-translationally modified, (Bailey & Whelan, 1961). A chain of less than 10 glucose
probably by glycosylation, increasing its molecular weight residues does not yield any colour. However, as the number
up to an apparent value of 62 kDa as occurs with ZsAmy of residues increases, a change from red to red–purple,
(Grossi de Sá & Chrispeels, 1997; Fig. 4B). purple and blue is observed. Thus, Fig. 1 indicates that the
The Western blot exihibited in Fig. 4(B) suggests that the Zabrotes enzyme produces mainly oligosaccharides with
putative glycosylation is a different event for these enzymes few glucose residues. Instead, the sugars released by the
in Spodoptera cells. The bruchide amylase may or may not mosquito amylase may have a higher number of glucose
be modified whereas that of the mosquito was entirely mod- residues. In accordance with these observations are the
ified. The strong cross-reaction observed for the 52 kDa results presented in Fig. 2. This shows a quite different
band of Zabrotes contrasts with the relatively faint 62 kDa pattern of extensive (12 h) starch hydrolysis for the two
band (Fig. 4B). This may be an indication that the modifica- enzymes. That of Zabrotes releases mainly glucose and
tion is a disturbance factor in the antibody attachment to maltose and relatively smaller amounts of oligosaccha-
the amylase epitope, which is probably located at the C- rides. The mosquito enzyme produces low levels of glucose
terminus and contains the N442 residue. Visual inspection and maltose and relatively higher levels of oligosaccharides.

© 2003 The Royal Entomological Society, Insect Molecular Biology, 12, 415–425
422 P. C. Effio et al.

Figures 1 and 2 strongly suggest that the bruchide and other α-amylase substrates, were included in the diet, even
the mosquito amylases act on starch by different if at a smaller ratio.
mechanisms. The former enzyme resembles a glucosi-
dase, which uses oligosaccharides formed by a few
glucose units as a substrate, yielding glucose and maltose Experimental procedures
(Gray et al., 1979). This raises the question of how these
Cells and bacterial strains
oligosaccharides are produced because glucosidases
Spodoptera frugiperda Sf 9 cells were obtained from the American
do not attack starch (Gray et al., 1979). The Anopheles
Type Culture Collection (ATCC, CRL-1711, Manassas, VA, USA).
enzyme displays the normal α-amylase pattern of starch E. coli DH5alpha and E. coli JM103 were purchased from New
degradation. England Biolabs Inc. (Beverly, MA, USA). E. coli DH 10B was
Mosquitoes transmit more human parasitic diseases purchased from Gibco-BRL (Rockville, MD, USA).
than any other arthropod group. The Anophelinae, mem-
bers of the gambiae complex that comprises several mor-
Isolation of the α-amylase gene from A. merus
phologically indistinguishable species (White, 1985), were
The α-amylase gene from A. merus (Diptera, Nematocera, Culi-
responsible for nearly 500 million clinical cases of malaria
coidea; GenBank accession number U01210) was isolated from a
each year in the 1980s (Sturchler, 1989). Knowledge of the genomic library prepared by Frank H. Collins (Center for Infectious
feeding habits of these diptera is of utmost importance in Diseases, Atlanta, Georgia, USA) in lambda phage EMBL 3. A
order to design control strategies. Studies on the physiolo- recombinant phage was separated by probing the library with a
gical role of the yellow fever mosquito (Ae. aegypti) salivary 0.6 kbp Pvu II fragment of a Drosophila melanogaster α-amylase
glands showed that these organs are sexually dimorphic. In gene (Boer & Hickey, 1986). The cloned fragment was isolated,
restricted with HindIII and ligated to the plasmid pIBI24 (Interna-
females, they express different gene products from unique
tional Biotechnologies Inc.). The ligation mixture was used to
regions of the gland enabling the female to adopt two dif-
transform E. coli JM 103 competent cells. By employing the same
ferent feeding modes based on blood or sugar meals. By 0.6 kbp PvuII probe, a recombinant plasmid containing a 1.7 kbp,
contrast, the male is only able to use the sugar feeding mode. HindIII fragment was isolated, characterized by standard restric-
The results presented here suggest that the genomic tion analysis and partially sequenced (Pernasetti, 1991). The
sequence of A. merus α-amylase may be expressed 1.7 kbp fragment contains a complete α-amylase gene from
as a fully active enzyme during the mosquito’s life cycle, A. merus (AmerAmy) that was studied throughout this work.
in accordance with previous data demonstrating the expres-
sion of α-amylase mRNA in salivary glands of Ae. aegypti Cloning the AmerAmy gene DNA into the pGEM-T plasmid
(Grossman & James, 1993). Together, these point towards The AmerAmy gene was amplified by PCR from the pIBI24-
a sugar feeding mode in both Culicinae and Anophelinae AmerAmy construct, using two distinct forward primers (F) in order
mosquitoes, which probably use starch as the primary to obtain AmerAmy with its proper signal peptide or, alternatively, the
target of α-amylases. However, a starch diet requires AmerAmy coding sequence plus the Z. subfasciatus α-amylase
both α-amylase and glucosidase acting as part of digest- signal peptide (ZsAmerAmy). The same reverse primer (R) was
used in both amplifications.
ive secretion in order to achieve the ultimate goal of the
The primers used are shown below:
polysaccharide metabolism: D-glucose. In addition to α-
amylase (Grossman & James, 1993), Ae. aegypti also AmerAmy-F. 5′-TCGGGATCCATGAAACTTTTAGTGCGCCTAG-
expresses an active glucosidase (Marinotti & James, 1990) CACCG-3′
in compliance with the above requirement. This suggests
that a glucosidase may also be present in Anophelinae, ZsAmerAmy-F. 5′-TCGGGATCCATGAAGTTAGGAGTAGTGCA-
thus rendering starch an efficient and widespread substrate GTTGATCCTCGGTTTGGCCGTGGGGTTCACCCAGCACGAT-
CCCCACTTTGTCCGC-3′
for a sugar feeding mode that is decisive for both male and
species survival. The observation that salivary glands are AmerAmy-R. 5′-CCGCTCGAGAAGCTTGAATTCTCACACCCG-
not able to hydrolyse starch (Marinotti & James, 1990) is GCCATCAGACCTCATTCG-3′
not consonant with these assumptions but it suggests an
alternative hypothesis. The α-amylase could be partially PCR was performed under the following conditions: 200 m M
secreted, remaining insoluble and attached to the external Tris-HCl, pH 8.0; 50 mM KCl; 0.2 mM of each dNTP; 1.5 mM
surface of the cell membrane. It would be interesting to MgCl2; 0.5 µM Forward primer; 0.5 µM Reverse primer; 0.1 µg DNA
assess the role of the atypical C terminus discussed above template (pIBI24-AmerAmy construct); 1 µl (5 U) Taq DNA-
Polymerase; Milli-Q water to a final volume of 200 µl. Amplification
in a putative anchoring of α-amylase. Nevertheless, the
was achieved upon twenty-eight cycles of 45 s at 95 °C, 30 s at
data presented here and previously (Grossman & James, 55 °C, 2 min at 72 °C and an extension step, at 72 °C, for 10 min
1993) may indicate that the number of mosquitoes feeding The PCR products were extracted from the agarose gel using a
on flowers and nectar (Van Handel et al., 1972) would be recovering DNA kit (Amersham Biosciences, Uppsala, Sweden)
more accurate if other carbohydrates, such as starch and and cloned into the T/A cloning plasmid pGEM-T (Promega, Madison,

© 2003 The Royal Entomological Society, Insect Molecular Biology, 12, 415–425
Expression of the Anopheles merus α-amylase gene 423

WI, USA). As a control, the amylase gene from Z. subfasciatus pellet to digest viral proteins during 1 h at 56 °C. An additional
(ZsAmy) was also amplified by PCR and cloned into the pGEM-T heating at 95 °C for 20 min was done in order to inactivate the
plasmid. To amplify ZsAmy, a recombinant plasmid containing enzyme before proceeding to the PCR step. Viral DNA amplifica-
the sequence that codes for Z. subfasciatus α-amylase was tion was carried out using 2 µl of this DNA preparation as the tem-
used as template in a PCR reaction with specific primers ZsAmy-F plate at the same conditions and primers described above. This
and ZsAmy-R (see below). The template DNA and primers were procedure was used to obtain the AmerAmy, ZsAmerAmy and
gently provided by Dr Fatima Grossi de Sá (EMBRAPA, Brazilia, ZsAmy DNAs.
Brazil).

Recombinant baculovirus stocks


ZsAmy-F. 5′-CGCGGAATCAAACGATGAAGTTAGGAGTAGTGC-3′
Samples of Bv-AmerAmy and Bv-ZsAmerAmy have been properly
ZsAmy-R. 5′-CCGCTCGAGAAGCTTGCGCCCGCTTACAGTT- deposited in the Cell and Molecular Biology Laboratory, Depart-
TGGAAGTGAG-3′ ment of Biochemistry, Institute of Chemistry, University of São Paulo,
under the care of M.C.S. They are available upon request.

Subcloning of the α-amylase genes into the Bac-to-Bac™ system


Amplification of baculovirus stocks
The resulting pGEM-T, constructs containing the amylase DNAs,
For amplification of baculovirus master stocks, 1 × 106 Sf 9 cells
were digested with Bam HI and Hin dIII restriction enzymes in order
were plated in a 25-cm2 flask and incubated for 1 h with 10 µl of
to insert the DNA, in frame, into the pFastBacl transfer vector of
baculovirus master stock in 1 ml of TNM-FH medium containing
the Bac-To-Bac™ Baculovirus Expression System (Life Techno-
antibiotics (corresponding to an MOI of 0.01– 0.1). After this
logies, Rockville, MD, USA). The recombinant plasmids obtained
period, the medium was completed to 4.5 ml and the infected cells
(pFastBac1-AmerAmy, pFastBac1-ZsAmerAmy and pFastBac1-
were incubated for 48 h at 27 °C. The culture medium was col-
ZsAmy) were used to transform competent E. coli DH10 Bac (con-
lected, clarified (500 g for 5 min) and stored at 4 °C as viral stocks
taining the wild-type baculovirus genome in the bacmid form) to
for recombinant protein production.
generate the corresponding recombinant bacmids upon trans-
position, namely Bd-AmerAmy, Bd-ZsAmerAmy and Bd-ZsAmy.
Recombinant protein production
For massive infection and recombinant protein production, 25-cm2
Production of recombinant baculovirus
culture flasks containing 1 × 106 Sf 9 cells, plated and grown over-
Spodoptera frugiperda Sf 9 cells were transfected with recom- night in TNM-FH medium, at 27 °C, were infected with 500 µl of
binant bacmid DNA for production of the baculovirus particles. amplified baculovirus stock (corresponding to MOI of 10) and
Cells were cultured at 28 °C in TNM-FH medium (Life Technolo- incubated at 27 °C for 96 h or until detection of cytopathic effect in
gies) supplemented with 50 U/ml ampicilin and 50 µg/ml strepto- 90–100% under an inverted phase contrast Nikon microscope.
mycin. For transfection, 9 × 105 cells were plated in 35-mm tissue Clarified culture medium (supernatant) and cells (pellet) were col-
culture dishes and incubated for 1 h in 2 ml Sf900-II SFM (Life lected after centrifugation (500 g for 5 min).
Technologies) without antibiotics to allow adhesion of the cells to
the dish. The medium was then changed to 1 ml serum-free
Sf900-II without antibiotics, containing recombinant bacmid DNA Amylase purification from culture supernatants
(5 µl of a standard mini-preparation of plasmid DNA) that had Amylase secreted to the culture medium by baculovirus-infected
been pre-incubated for 30 min at room temperature with CellFectin cells was concentrated to 10% of the initial volume by precipitating
(6 µl) (Life Technologies). Cells were incubated with the liposome– proteins with three volumes of cold acetone (stored at −20 °C) for
DNA complex at 27 °C for 5 h. The transfection medium was 3 h and centrifugation at 12 000 g at 4 °C for 30 min The pellet
removed and 2 ml of TNF-FH medium, containing antibiotics, was redissolved in 100 µl of Milli-Q water and stored at −20 °C for
was added. Bd-AmerAmy and Bd-ZsAmerAmy DNA were trans- analysis of extracellular activity of the expressed α-amylase.
fected into Sf 9 cells and nonrecombinant bacmid (Bd) DNA and
Bd-ZsAmy were used as, respectively, negative and positive con-
trols. Transfected cells were incubated at 27 °C for 72 h allowing Protein determination
baculovirus production and release into the culture medium. The Quantification of proteins present in supernatants of pelleted cells
culture medium from each transfection was collected, clarified was accomplished by using the methods described previously
(500 g for 5 min) and stored at 4 °C as a master virus stock. Trans- (Bradford, 1976; Smith et al., 1985; Morton & Evans, 1992). Egg
fection efficiency, recombinant baculovirus (Bv-AmerAmy, Bv- albumine was used as the standard protein.
ZsAmerAmy and Bv-ZsAmy) and nonrecombinant baculovirus
(Bv) production were monitored by visualization of the cytopathic
effect displayed by transfected cells within 48 h after subculturing, Preparation of baculovirus-infected cell extracts
under a phase contrast microscope and assaying the presence of Infected cells were lysed essentially as described (Grossi de Sá
baculovirus DNA through PCR analysis (Folgueras-Flatschart & Chrispeels, 1997), for 1 h with lysis buffer (10 mM Tris-HCl;
et al., 2000). To this end, baculovirus present in 50 µl of infected 130 mM NaCl; 0.1% SDS; 5 mM phenylmethylsulphonil fluoride,
culture supernatant was sedimented at 12 000 g for 10 min in a pH 7.5). To pellet cellular debris, the lysate was clarified by
microcentrifuge tube, and a volume (25 µl) of proteinase K buffer centrifugation at 12 000 g at 4 °C for 10 min. The supernatant
(10 mM Tris-HCl, pH 7.8; 5 mM EDTA; 0.5% SDS) containing was stored at −20 °C for analysis of intracellular activity of the
50 µg/ml of proteinase K (Sambrook et al., 1989) was added to the expressed amylase.

© 2003 The Royal Entomological Society, Insect Molecular Biology, 12, 415–425
424 P. C. Effio et al.

α-amylase assays and paper chromatography Acknowledgements


α-amylase activity was detected by the lugol (I2 + KI) procedure
The expert technical assistance of Irenice Cairo da Silva,
(Silva, 1989) using soluble starch as substrate (in 100 mM phos-
phate, pH 6.0; 20 mM NaCl; 0.1 mM CaCl2). Samples (20 µl) of Sandra de Souza, Sirlei Mendes de Oliveira and Zizi de
supernatants or infected cell extracts were added to 480 µl of the Mendonça is greatly appreciated. This work was supported
substrate solution. The reaction mixture was incubated for 12 h at by FAPESP, CNPq, FINEP, ICGEB and PRP-USP. P.C.E.
37 °C. A sample (10 µl) of this reaction was then mixed with 200 µl was the recipient of a predoctoral Fellowship from CNPq.
of lugol in 0.05 N HCl. A nearly complete starch hydrolysis pro- This work is a partial fulfilment of a Doctoral thesis require-
duces a clear yellowish solution. The hydrolysed substrate (480 µl) ments (P.C.E.).
was dried at 60 °C, redissolved with 25 µl of Milli-Q water and
assayed by paper chromatography. To a 12 × 6-cm strip of Whatman
1MM paper, 1 µl of each sample was applied. The mobile phase
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