P. C. Effio*, A. V. Folgueras-Flatschart, W. R. Montor, F. M. Pernasetti, M. T. Pueyo and M. C. Sogayar
Instituto de Quimica, Universidade de São Paulo, São Paulo, Brazil
Abstract
The
Anopheles merus
(Diptera, Nematocera, Culicoi-dea) αααα
-amylase gene (AmerAmy, GenBank AccessionNumber U01210) was amplified with its own or withthe
Zabrotes subfasciatus
αααα
-amylase signal peptide(ZsAmerAmy, GenBank Accession Number AY270183)by PCR, using designed primers. The AmerAmy genewas sequenced from its promotor to the TGA codon.As a positive control, the
Z. subfasciatus
αααα
-amylase genewith its own signal peptide (ZsAmy, GenBank Acces-sion Number AF255722) was also amplified by PCR.These three sequences were inserted into the baculo-virus genome using the Bac-to-Bac™ system. Recom-binant baculovirus preparations were used to infect
Sf
9
Spodoptera frugiperda
insect cells. The
A. merusαααα
-amylase was successfully expressed as an activeenzyme detected mainly in cell culture supernatants.
Keywords: alpha amylase,
Anopheles merus
,expression, Baculovirus/
Spodoptera frugiperda
.
Introduction
Different
α
-amylase (EC 3.2.1.1) genes from archaebacteria,eubacteria, fungi, animal and plant sources have beencloned and sequenced (Vihinen & Matsala, 1989). Theseenzymes hydrolyse alpha-1,4 glycosidic bonds present inoligosaccharides and starch (Steup, 1988), playing a keyrole in the overall metabolism of these organisms. Fromanimals to bacteria,
α
-amylases display a wide variety of
kinetic parameters, optimum pH and substrate specificity(Bertoft
et al
., 1984; Akazawa
et al
., 1988; Knutson, 1993).The
α
-amylases are widely used by insects in their larvalstage, as has previously been reported (Terra & Ferreira,1994). Closely linked amylase genes have been detected inthe housefly,
Musca domestica
(Ogita, 1968), silkworm,
Bombyx mori
(Kikkawa, 1953), in several species of theflour beetle,
Tribolium
(Pope
et al
., 1986), and in the yellowfever mosquito,
Aedes aegypti
(Grossman
et al
., 1997). Todate, the only functional recombinant
α
-amylases to bereported are those from
Zabrotes subfasciatus
(Grossi de Sá& Chrispeels, 1997) and
Diabrotica virgifera virgifera
(Titarenko & Chrispeels, 2000). In Culicoidea, the expres-sion of an
α
-amylase gene was determined at the mRNAlevel in the salivary glands of
Ae. aegypti
(Grossman &James, 1993). However, that protein was not detected as anactive enzyme as was accomplished for an
α
-glucosidase(Marinotti & James, 1990) in these organs. The resultson
Anopheles merus
presented here evinced for the firsttime the expression, using the baculovirus/
Spodopterafrugiperda
cell system, of a Culicoidea
α
-amylase recom-binant gene as a functional enzyme. These results stronglysuggest that this protein could also be an active enzyme ofthe digestive secretion in these Diptera.
Results
Generation of recombinant
α
-amylase constructs
The coding sequence for
A. merus
α
-amylase (AmerAmy)was obtained and subcloned into the baculovirus-basedpFastBac1 plasmid vector as described in Experimentalprocedures. Two different constructs were sought, namelypFastBac1-AmerAmy (contains the Ameramy gene plusthe sequence corresponding to its own signal peptide) andpFastBac1-ZsAmerAmy (contains the AmerAmy gene plusthe sequence corresponding to the signal peptide of theamylase gene of
Z. subfasciatus
). A construct made withthe
Z. subfasciatus
α
-amylase gene (pFastBac1-ZsAmy)was used as a control. Transformation of plasmid DNA into
E. coli
DH10Bac allowed the production of the followingbacmid recombinants: Bd-AmerAmy, Bd-ZsAmerAmy andBd-ZsAmy.
Received 16 December 2002; accepted after revision 25 April 2003. Corre-spondence: Drs Manuel Troyano Pueyo and Mari Cleide Sogayar, Institutode Quimica, Universidade de São Paulo, CP 26077, São Paulo 05513–970,SP, Brazil. E-mail: [email protected] and [email protected]
*On leave from the Universidad Nacional Pedro Ruiz Gallo, Lambayeque,Perú.
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 2. Chromatographic pattern of starch cleavage by A. merus α-amylase. Starch was dissolved to 0.5% in activity buffer (100 mM phosphate buffer, pH 6.0, 20 mM NaCl, 0.1 mM CaCl2). To determine the cleavage pattern, a series of four Eppendorf tubes containing 480 µl of starch were prepared. To each of those tubes, 20 µl of the vacuum-concentrated supernatant from Sf9 cultures infected with the recombinant baculoviruses, Bv-AmerAmy, Bv-ZsAmerAmy and Bv-ZsAmy, was added. Tubes were incubated at 37 °C for 24 h and centrifuged at 13 000 g for 10 min at 4 °C. The supernatant was dried at 60 °C and redissolved in 25 µl of Milli-Q water. A total of 1 µl (five applications of 0.2 µl) of each sample was applied. The supernatant from the Sf9 culture infected with baculovirus lacking insert was used as negative control. Lane 1: glucose plus maltose. Supernatant samples from Sf9 cultures infected with recombinant baculoviruses are in: Lane 2, Bv-ZsAmy; Lane 4, Bv-AmerAmy; Lane 5, Bv-ZsAmerAmy. Lane 3, negative control.
Figure 3. Activity assay of AmerAmy in blue starch gel. Supernatants from Sf9 cell cultures infected with the recombinant baculoviruses Bv-AmerAmy, Bv-ZsAmerAmy and Bv-Zs were precipitated with cold acetone and redissolved in 10% of the initial volume with Milli-Q water. After electrophoretic separation in PAGE-SDS, the gel was washed three times with Milli-Q water for 30 min. Another wash with activity buffer was made in order to reactivate the enzymatic activity as described in Experimental procedures. An agar gel containing blue starch was layered over the polyacrylamide gel. The set was wrapped in a PVC film and incubated at 37 °C for 12 h. Active amylases separated in the polyacrylamide gel below are detected in samples from culture supernatants as clear bands in the agar/blue starch gel. Electrophoretic conditions were 100 V/20 mA. Samples of 5 and 0.5 µg were applied in lanes containing A. merus and Zabrotes proteins, respectively. Both pellet and supernatant samples were analysed in lanes labelled as follows: 1 and 5, Sf9 from noninfected culture; 2 and 6, Bv from culture infected with baculovirus lacking insert; 3, Bv-AmerAmy, supernatant from culture infected with baculovirus carrying sequences corresponding to A. merus α-amylase and to its own signal peptide; 4, Bv-ZsAmerAmy, supernatant from culture infected with baculovirus carrying sequences corresponding to the Zabrotes signal peptide and A. merus α-amylase; 7, Bv-AmerAmy, pellet from culture infected with baculovirus carrying sequences corresponding to its own signal peptide and A. merus α-amylase; 8, Bv-ZsAmerAmy, pellet from culture infected with baculovirus carrying sequences corresponding to the Zabrotes signal peptide and A. merus α-amylase; 9, Bv-ZsAmy, supernatant from culture infected with baculovirus carrying sequences corresponding to Zabrotes’s own signal peptide and α-amylase.
9 insect cells; production of baculoviruses carrying the AmerAmy, ZsAmerAmy and ZsAmy genes
Sf
9 insect cells were transfected with the Bd-AmerAmy, Bd-ZsAmerAmy and Bd-ZsAmy bacmid constructs for primarybaculovirus particle production. After transfection, baculo-virus production was confirmed by two different methods,based on the work we previously reported (Folgueras-Flatschart
et al
., 2000), namely the observation of thecytopathic effect that occurs upon subculturing of trans-fected cells and amplification of specific DNA fragments,by PCR, using culture supernatants potentially containingbaculovirus particles, as a template and specific primers forAmerAmy-F, ZsAmerAmy-F and AmerAmy-R. Baculovirusmaster stocks were amplified for
Sf
9 infection and proteinproduction. After 96 h of massive infection [multiplicity ofinfectivity (MOI) = 10; numbers are relationships betweenviral particles and cells],
Sf
9 cultures displayed typicalcytophathic effects, i.e. low cell density and poor adherenceto the substrate, indicating that virus production was takingplace. The nontransfected
Sf
9 cultures as well as thoseinfected with empty baculoviruses displayed a normal cellmonolayer.
Protein determination in cell supernatants
The total protein content of cell supernatants was deter-mined as described in Material and methods. Cells trans-fected with Bv lacking insert as a control gave a mean value(three determinations) of 670
µ
g/ml. This figure corres-ponds in general to proteins of the bovine serum used inthe culture medium. Thus, all determinations accomplishedin the supernatants of cells transfected with baculovirusescarrying inserted sequences were corrected by subtracting670
µ
g/ml from the actual data. Bv-ZsAmy transfected cellswere included as an internal control. Typical protein contentvalues obtained when the insect cells were transfected withBv-AmerAmy, Bv-ZsAmerAmy and Bv-ZsAmy are 7.5, 8.5and 19.0
µ
g/ml, respectively.
Detection of
α
-amylase activity
Amylase activity was detected both in culture supernatantsand in cell extracts using the lugol method, as described inMaterial and methods. The results, shown in Fig. 1, indicatethat only amylases from culture supernatants obtainedfrom cells infected with recombinant baculovirus Bv-AmerAmy and Bv-ZsAmerAmy were able to degrade thestarch substrate to dextrins displaying a number of glucoseresidues sufficient to give a purple-coloured lugol solution.The
Z. subfasciatus
α
-amylase, produced by Bv-ZsAmy-infected
Sf
9 cells, degrades starch to products that have anumber of glucose residues not enough to give a colourcomplex with (Bailey & Whelan, 1961). The productsobtained upon starch hydrolysis by recombinant
α
-amylases were analysed by paper chromatography. The
chromatographic pattern shown in Fig. 2 indicates that theamylases present in culture supernatants from cells infectedwith Bv-AmerAmy and Bv-ZsAmerAmy yield a cleavagepattern that is typical of
α
-amylase, with relatively largeamounts of oligosaccharides and small amounts of glucoseand maltose, whereas Bv-ZsAmy culture supernatantsdegraded starch mainly to glucose and maltose and smallamounts of oligosaccharides, as shown in Fig. 1.
The
α
-amylase activity present in
Sf
9 culture super-natants was also analysed by the blue starch overlay methodand by Western blotting. The results shown in Figs 3 and4 indicate that proteins present in supernatants obtainedfrom
Sf
9 cells infected with Bv-AmerAmy and Bv-ZsAmerAmydisplay amylase activity. The Coomassie-blue-stainedSDS-PAGE of proteins pelleted from culture medium ofinfected cells revealed a single protein band of approxim-ately 62 kDa, in both ZsAmy and ZsAmerAmy prepara-tions (Fig. 4A). However, the band corresponding to ZsAmyis much more intense than that of ZsAmerAmy. When thegel was silver stained, a second (faint) band, of approxim-ately 52 kDa, was observed in ZsAmy supernatants (datanot shown). Western blotting showed that two
α
-amylaseswere secreted by cells infected with the ZsAmy baculovirus.The protein(s) present in the 62 kDa band reacted weaklywith the
Z. subfaciatus
anti-(
α
-amylase) antibody, whereasthe second band (
≈
52 kDa) reacted strongly with thisantiserum. In the case of A. merus, only one band, cor-responding to the secreted protein that exhibits an apparentMW = 62 kDa, was detected in culture supernatants ob-tained from cells infected with the ZsAmerAmy baculovirus(Fig. 4B).
Sequence analysis
The promotor of the amylase gene is likely to be CCCAT-CAACT with 97% confidence (Fig. 5).
The conceptual translation product of the α-amylasegenomic sequence is also presented in Fig. 5. Features ofthis product are: (a) MKLLVRLAPILLLALTGRPVAA, a plau-sible signal peptide with 90% confidence, suggesting thatthe peptide is secreted; (b) a mature peptide consisting of492 residues starting with the blocking N-terminal glutamine,an indication that the peptide may be in contact with digest-ive secretion (Strobl et al., 1997); (c) 54.8 kDa, the mole-cular weight calculated for the unmodified mature peptide.Comparison with other amino acid sequences reveals thata glutamic acid (E) and two aspartic acid (D) residues thatare conserved in the α-amylase family (Janecek, 1997) areknown to be involved in catalysis at the active sites of por-cine pancreatic (Qian et al., 1994) and Tenebrio molitor(Strobl et al., 1998) α-amylases. They are found in Amer-Amy as D215, D318 and E252. Three conserved histidineresidues, which are thought to be involved in substratebinding (Qian et al., 1994; Strobl et al., 1998), are alsopresent in AmerAmy as H125, H219, H317. By comparison
with previous data (Strobl et al., 1998), other conservedresidues can be related to the chloride (R213, N316, R351)and calcium (N124, R176, D185, H219) binding sites in theA. merus enzyme (Fig. 5).
The similarity and identity of AmerAmy with other α-amylasesis shown in Table 1. AmerAmy has 51–59, 49–51, 47, 11, 24and 8–23% identity with α-amylases from, respectively,insects, mammals, birds, plants, fungi and bacteria.
AmerAmy does not have any putative N-glycosylationsite (consensus sequence Asn-X-Ser/Thr). Instead, Amer-Amy has the sites T131, S141, T142 and T359, which couldbe O-glycosylated, with probabilities of 86.32, 96.33, 94.14and 74.57%, respectively (Table 2).
Discussion
We chose to express the AmerAmy sequence in the baculo-virus/insect cell system not only because it is a versatileand reliable system for the production of recombinantproteins (Possee, 1997; Grossi de Sá & Chrispeels, 1997;Titarenko & Chrispeels, 2000) but also because it allowsexpression of eukaryotic genes in a eukaryotic cell system,taking advantage of their protein synthesis machinery,thereby facilitating folding and post-translational modifications(O’Reilly et al., 1992). These include N- and O-glycosilation,acylation, proper proteolysis and oligomerization of theprotein product via processes that are often identical to thatoccurring into the original cells. Therefore, proteins may besecreted from the cell or be directed to other subcellular
structures. In addition, the insect cytoplasmic environmentis less reducing than that of the E. coli cells, thus enablingproper disulphide bridge (–S–S–) assembly. This feature isimportant in the case of the A. merus α-amylase becauseit displays 11 cystein residues, seven of which are concen-trated between amino acids 384 and 506 (Fig. 5). The numberof –S–S– bonds in this protein is not known but the highfrequency of cystein residues probably enhances the com-plexity of folding. Note that the α-amylase nucleotidesequence used throughout this work is of genomic origin.The lack of introns (Pernasetti, 1991) is a valuable featurefor the expression of this gene as an active α-amylase thatcan now be used to study structural, kinetic and inhibitorybehaviour in the presence of insect plant amylase inhibitors.This could reveal clues for future mosquito control strategies.
We previously used the baculovirus/insect cell expres-sion system to produce active mouse Fos and Jun proteins(Corvello et al., 1995), bovine Herpesvirus glycoproteins(Folgueras-Flatschart et al., 2000), the alpha7 subunit ofacetylcholine nicotinic receptor (Aztiria et al., 2000) andhuman prolactin (Pereira et al., 2001). Here we describethe results showing that, for the first time, it was possible toexpress the A. merus α-amylase in the active form.
The attempt to express the A. merus α-amylase geneattached to the sequence that codes for the signal peptideof α-amylase from Z. sufasciatus stems from the fact thatthis enzyme was previously successfully expressed in thebaculovirus/insect cell system (Grossi de Sá & Chrispeels,1997). Signal peptide sequences derived from insect protein
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.
have been used with variable results in the baculovirus/insect cell system. Previous studies on the expression ofa plant protein, papain (Tessier et al., 1991), have shownthat the use of an insect-derived signal peptide enhancedthe secretion five-fold over that detected when using thenative signal peptide. However, other studies have found thatinsect-derived signal peptide sequences were not optimal(Jarvis et al., 1993; Dupuis et al., 1997; Dahl et al., 2000) orwere not linked to the secretion of heterologous proteins inthe baculovirus/insect cell system (Tessier et al., 1991;Dupuis et al., 1997). The level of secretion of A. merus α-amylase and Z. subfasciatus reported here (7.5, 8.5 and19.0 µg/ml) are lower than those reported for other hetero-logous proteins: GRP78/BJP (50–75 µg/ml), hepatitis Bsurface antigen (90 µg/ml; Lanford et al., 1989) and plantphaseolin (90 µg/ml; Bustos et al., 1988). Although a largedifference between secretion of AmerAmy (7.5 µg/ml) andZsAmerAmy (8.5 µg/ml) was not detected in the results
reported here, ZsAmy was two-fold (19.0 µg/ml) higher(see Fig. 4 for comparative purposes). Our data as well asthose referred to above suggest that the recognition andthe processing of signal peptides from heterologous pro-teins are not the only factors involved in the secretion levelwhen the baculovirus/insect cell system is used. It is likelythat, among other factorss, disulphide bond formation andfolding could be rate-limiting steps.
The identity of AmerAmy with its mammalian, insectand bird counterparts is relatively high (59, 51 and 47%,respectively), as shown in Table 1.
We identified three amino acids (D215, E252, D318) thatare known to be important for catalysis and three histidineresidues (H125, H219, H317) that are known to be involvedin substrate binding (Qian et al., 1994). Residues R213,N316 and R351 were identified as belonging to the chloridebinding site, whereas residues N124, R176, D185, H219were related to the calcium binding site (Strobl et al., 1998).
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.
These findings indicate that, as with other amylases, AmerAmyhas a canonical composition and distribution of residuesin order to attain its function. The amino acid C-terminalsequences of α-amylases from organisms from taxonomicclasses Mammalia, Insecta (orders Diptera, Hymenoptera,Lepidoptera, Coleoptera), Arachnida, Nematoda, Osteich-thyes (order Teleostea), Bivalvia and Crustaceae werecompiled (not shown) in order to contrast them with thecorresponding sequence of A. merus α-amylase deter-mined in this work. As a general rule, the C terminal tailstarting at D comprises 12 amino acids. An example ofthis terminal C sequence is D G V L A I H V N A K L fromT. molitor (Strobl et al., 1997). According to our analysis, itcan be noted as the widely conserved DGX1X2AIH sequence.In addition, the similarity of residues between DG and Aand after H was noted. In most of the sequences collected,the two amino acids preceding AIH are hydrophobic: VL, IL,VI, VV, FV or FI. The amino acids immediately following A IH are hydrophobic (A, V, I, G) and hydrophilic (KXK, NXK,NXR, SXK, EXK, DXK, DXR) with lysine being highly con-served, or in a few examples substituted by arginine orglutamine. However, this tail is not observed in sequencesthat are currently available from bacteria, fungi, arachnida,nematoda or plant amylases. It seems likely that the C-terminal region, as described before, is a well-defined feature
of certain α-amylases. The significance and the extent ofits distribution in living organisms remains to be ascert-ained. Interestingly, the sequence determined for the C-terminal region in A. merus amylase displays DGVIAIH asexpected, but does not follow the rule of similarity or lengthafter it. Instead, a stretched SEVSVCVCCRMRSDGRVsequence was detected. This seems to be restricted to theamylase of A. merus and in part to the same protein ofA. gambiae (accession number L04753). A part of thissequence, VSVCVC, is also found in proteins that are notrelated to amylases of several organisms and viruses,including an inner region of agCP8155 (accession numberEAA13780.1), an unidentified protein from A. gambiae.Thus, concerning its C-terminal portion, the A. merus pro-tein seems to be unique within the amylases from severalhigher organisms. Nevertheless, it is possible that the oddC-terminal portion described may be present only in theconceptual translation of the genomic sequence, but not inthe functional protein itself.
O-glycosylation is expected to occur in AmerAmy at theT138, T142, and S141 residues and in ZsAmy at the T425residue (Table 2). AmerAmy does not display classicalN-glycosylation sites (Asn-X-Ser/Thr) whereas ZsAmy hasone starting at N442, close to the C-terminus (Grossi de Sá& Chrispeels, 1997).
In AmerAmy, the S141 T142 site has a high (94–96%)probability of being O-glycosylated (Table 2). It occurs nearthe consensus region I (DIIINH), into the B domain ofthe enzyme. The results in Fig. 4 showed a single 62 kDaband in AmerAmy supernatant that cross-reacts with theZsubAmy anti-amylase antibody. Considering that theexpected molecular weight for AmerAmy is 54.8 kDa, thisindicates that the enzyme was post-translationally modified,probably by glycosylation, increasing its molecular weightup to an apparent value of 62 kDa as occurs with ZsAmy(Grossi de Sá & Chrispeels, 1997; Fig. 4B).
The Western blot exihibited in Fig. 4(B) suggests that theputative glycosylation is a different event for these enzymesin Spodoptera cells. The bruchide amylase may or may notbe modified whereas that of the mosquito was entirely mod-ified. The strong cross-reaction observed for the 52 kDaband of Zabrotes contrasts with the relatively faint 62 kDaband (Fig. 4B). This may be an indication that the modifica-tion is a disturbance factor in the antibody attachment tothe amylase epitope, which is probably located at the C-terminus and contains the N442 residue. Visual inspection
of the relationship between the intensity of the ZsAmy andZsAmerAmy 62 kDa bands reveals that it is inverted inFig. 4A and Fig. 4B. To a smaller mass of ZsAmerAmy cor-responds a stronger antibody reaction. This may suggestthat the hypothetical modification at the mosquito proteindoes not interfere with the antibody cross-reaction and thatit is not probably placed at the amylase epitope. Together,these two observations suggest that the epitope of theseamylases is located near the C terminus, the domain C(Strobl et al., 1998) of these proteins.
The very faint band below the main band of Anophelesamylase in Fig. 4(B), lane 3, (Western blot) is not distin-guishable from the sharp band of Fig. 4(A), lane 3(stained). The faint band is probably due to the lesserO-glycosylation of T359 (see Table 2) and seems to bethe product of a residual event.
The unique band of Anopheles amylase in Fig. 4(A,B),lane 3, has a unique band of activity in Fig. 3, lanes 3 and 4.This suggests that the putative O-glycosylation of this proteinis compatible with enzymatic activity. The two bands detectedfor the amylase of Zabrotes in Fig. 4A,B, lane 1, also havea unique activity band in Fig. 3, lane 9. Clearly, only one ofthe bands in Fig. 4A,B, lane 1, retains activity. This is con-sistent with previous results (Grossi de Sá & Chrispeels,1997). As judged by the slightly faster migration of the bandin Fig. 3, lane 9, the activity is probably on the glycosylatedband (62 kDa) of Zabrotes α-amylase. If this interpretationis correct, in Z. subfasciatus only the α-amylase modifiedby a putative N-glycosylation retains enzymatic activity.
The exact role played by glycosylation in protein functionis as yet not completely understood, but the data availablepoint towards variable roles for different proteins. Questionson how N- or O-linked glycosylation might affect proteinfunction are still to be adressed. A systematic compilationof data on glycosylation regarding protein nature and func-tion, cell type, subcellular localization and secretion mayprove to be useful in establishing the importance of thispost-translational modification.
The colour of the iodine–iodide complex with starch isdependent on the number of glucose residues in the chain(Bailey & Whelan, 1961). A chain of less than 10 glucoseresidues does not yield any colour. However, as the numberof residues increases, a change from red to red–purple,purple and blue is observed. Thus, Fig. 1 indicates that theZabrotes enzyme produces mainly oligosaccharides withfew glucose residues. Instead, the sugars released by themosquito amylase may have a higher number of glucoseresidues. In accordance with these observations are theresults presented in Fig. 2. This shows a quite differentpattern of extensive (12 h) starch hydrolysis for the twoenzymes. That of Zabrotes releases mainly glucose andmaltose and relatively smaller amounts of oligosaccha-rides. The mosquito enzyme produces low levels of glucoseand maltose and relatively higher levels of oligosaccharides.
Table 2. Prediction of O-glycosylation sites on α-amylases from different insects based on protein and DNA sequence data
Figures 1 and 2 strongly suggest that the bruchide andthe mosquito amylases act on starch by differentmechanisms. The former enzyme resembles a glucosi-dase, which uses oligosaccharides formed by a fewglucose units as a substrate, yielding glucose and maltose(Gray et al., 1979). This raises the question of how theseoligosaccharides are produced because glucosidasesdo not attack starch (Gray et al., 1979). The Anophelesenzyme displays the normal α-amylase pattern of starchdegradation.
Mosquitoes transmit more human parasitic diseasesthan any other arthropod group. The Anophelinae, mem-bers of the gambiae complex that comprises several mor-phologically indistinguishable species (White, 1985), wereresponsible for nearly 500 million clinical cases of malariaeach year in the 1980s (Sturchler, 1989). Knowledge of thefeeding habits of these diptera is of utmost importance inorder to design control strategies. Studies on the physiolo-gical role of the yellow fever mosquito (Ae. aegypti) salivaryglands showed that these organs are sexually dimorphic. Infemales, they express different gene products from uniqueregions of the gland enabling the female to adopt two dif-ferent feeding modes based on blood or sugar meals. Bycontrast, the male is only able to use the sugar feeding mode.The results presented here suggest that the genomicsequence of A. merus α-amylase may be expressedas a fully active enzyme during the mosquito’s life cycle,in accordance with previous data demonstrating the expres-sion of α-amylase mRNA in salivary glands of Ae. aegypti(Grossman & James, 1993). Together, these point towardsa sugar feeding mode in both Culicinae and Anophelinaemosquitoes, which probably use starch as the primarytarget of α-amylases. However, a starch diet requiresboth α-amylase and glucosidase acting as part of digest-ive secretion in order to achieve the ultimate goal of thepolysaccharide metabolism: D-glucose. In addition to α-amylase (Grossman & James, 1993), Ae. aegypti alsoexpresses an active glucosidase (Marinotti & James, 1990)in compliance with the above requirement. This suggeststhat a glucosidase may also be present in Anophelinae,thus rendering starch an efficient and widespread substratefor a sugar feeding mode that is decisive for both male andspecies survival. The observation that salivary glands arenot able to hydrolyse starch (Marinotti & James, 1990) isnot consonant with these assumptions but it suggests analternative hypothesis. The α-amylase could be partiallysecreted, remaining insoluble and attached to the externalsurface of the cell membrane. It would be interesting toassess the role of the atypical C terminus discussed abovein a putative anchoring of α-amylase. Nevertheless, thedata presented here and previously (Grossman & James,1993) may indicate that the number of mosquitoes feedingon flowers and nectar (Van Handel et al., 1972) would bemore accurate if other carbohydrates, such as starch and
other α-amylase substrates, were included in the diet, evenif at a smaller ratio.
Experimental procedures
Cells and bacterial strains
Spodoptera frugiperda Sf9 cells were obtained from the AmericanType Culture Collection (ATCC, CRL-1711, Manassas, VA, USA).E. coli DH5alpha and E. coli JM103 were purchased from NewEngland Biolabs Inc. (Beverly, MA, USA). E. coli DH 10B waspurchased from Gibco-BRL (Rockville, MD, USA).
Isolation of the α-amylase gene from A. merus
The α-amylase gene from A. merus (Diptera, Nematocera, Culi-coidea; GenBank accession number U01210) was isolated from agenomic library prepared by Frank H. Collins (Center for InfectiousDiseases, Atlanta, Georgia, USA) in lambda phage EMBL 3. Arecombinant phage was separated by probing the library with a0.6 kbp Pvu II fragment of a Drosophila melanogaster α-amylasegene (Boer & Hickey, 1986). The cloned fragment was isolated,restricted with HindIII and ligated to the plasmid pIBI24 (Interna-tional Biotechnologies Inc.). The ligation mixture was used totransform E. coli JM 103 competent cells. By employing the same0.6 kbp PvuII probe, a recombinant plasmid containing a 1.7 kbp,HindIII fragment was isolated, characterized by standard restric-tion analysis and partially sequenced (Pernasetti, 1991). The1.7 kbp fragment contains a complete α-amylase gene fromA. merus (AmerAmy) that was studied throughout this work.
Cloning the AmerAmy gene DNA into the pGEM-T plasmid
The AmerAmy gene was amplified by PCR from the pIBI24-AmerAmy construct, using two distinct forward primers (F) in orderto obtain AmerAmy with its proper signal peptide or, alternatively, theAmerAmy coding sequence plus the Z. subfasciatus α-amylasesignal peptide (ZsAmerAmy). The same reverse primer (R) wasused in both amplifications.
PCR was performed under the following conditions: 200 mM
Tris-HCl, pH 8.0; 50 mM KCl; 0.2 mM of each dNTP; 1.5 mM
MgCl2; 0.5 µM Forward primer; 0.5 µM Reverse primer; 0.1 µg DNAtemplate (pIBI24-AmerAmy construct); 1 µl (5 U) Taq DNA-Polymerase; Milli-Q water to a final volume of 200 µl. Amplificationwas achieved upon twenty-eight cycles of 45 s at 95 °C, 30 s at55 °C, 2 min at 72 °C and an extension step, at 72 °C, for 10 minThe PCR products were extracted from the agarose gel using arecovering DNA kit (Amersham Biosciences, Uppsala, Sweden)and cloned into the T/A cloning plasmid pGEM-T (Promega, Madison,
WI, USA). As a control, the amylase gene from Z. subfasciatus(ZsAmy) was also amplified by PCR and cloned into the pGEM-Tplasmid. To amplify ZsAmy, a recombinant plasmid containingthe sequence that codes for Z. subfasciatus α-amylase wasused as template in a PCR reaction with specific primers ZsAmy-Fand ZsAmy-R (see below). The template DNA and primers weregently provided by Dr Fatima Grossi de Sá (EMBRAPA, Brazilia,Brazil).
Subcloning of the α-amylase genes into the Bac-to-Bac™ system
The resulting pGEM-T, constructs containing the amylase DNAs,were digested with BamHI and HindIII restriction enzymes in orderto insert the DNA, in frame, into the pFastBacl transfer vector ofthe Bac-To-Bac™ Baculovirus Expression System (Life Techno-logies, Rockville, MD, USA). The recombinant plasmids obtained(pFastBac1-AmerAmy, pFastBac1-ZsAmerAmy and pFastBac1-ZsAmy) were used to transform competent E. coli DH10 Bac (con-taining the wild-type baculovirus genome in the bacmid form) togenerate the corresponding recombinant bacmids upon trans-position, namely Bd-AmerAmy, Bd-ZsAmerAmy and Bd-ZsAmy.
Production of recombinant baculovirus
Spodoptera frugiperda Sf9 cells were transfected with recom-binant bacmid DNA for production of the baculovirus particles.Cells were cultured at 28 °C in TNM-FH medium (Life Technolo-gies) supplemented with 50 U/ml ampicilin and 50 µg/ml strepto-mycin. For transfection, 9 × 105 cells were plated in 35-mm tissueculture dishes and incubated for 1 h in 2 ml Sf900-II SFM (LifeTechnologies) without antibiotics to allow adhesion of the cells tothe dish. The medium was then changed to 1 ml serum-freeSf900-II without antibiotics, containing recombinant bacmid DNA(5 µl of a standard mini-preparation of plasmid DNA) that hadbeen pre-incubated for 30 min at room temperature with CellFectin(6 µl) (Life Technologies). Cells were incubated with the liposome–DNA complex at 27 °C for 5 h. The transfection medium wasremoved and 2 ml of TNF-FH medium, containing antibiotics,was added. Bd-AmerAmy and Bd-ZsAmerAmy DNA were trans-fected into Sf9 cells and nonrecombinant bacmid (Bd) DNA andBd-ZsAmy were used as, respectively, negative and positive con-trols. Transfected cells were incubated at 27 °C for 72 h allowingbaculovirus production and release into the culture medium. Theculture medium from each transfection was collected, clarified(500 g for 5 min) and stored at 4 °C as a master virus stock. Trans-fection efficiency, recombinant baculovirus (Bv-AmerAmy, Bv-ZsAmerAmy and Bv-ZsAmy) and nonrecombinant baculovirus(Bv) production were monitored by visualization of the cytopathiceffect displayed by transfected cells within 48 h after subculturing,under a phase contrast microscope and assaying the presence ofbaculovirus DNA through PCR analysis (Folgueras-Flatschartet al., 2000). To this end, baculovirus present in 50 µl of infectedculture supernatant was sedimented at 12 000 g for 10 min in amicrocentrifuge tube, and a volume (25 µl) of proteinase K buffer(10 mM Tris-HCl, pH 7.8; 5 mM EDTA; 0.5% SDS) containing50 µg/ml of proteinase K (Sambrook et al., 1989) was added to the
pellet to digest viral proteins during 1 h at 56 °C. An additionalheating at 95 °C for 20 min was done in order to inactivate theenzyme before proceeding to the PCR step. Viral DNA amplifica-tion was carried out using 2 µl of this DNA preparation as the tem-plate at the same conditions and primers described above. Thisprocedure was used to obtain the AmerAmy, ZsAmerAmy andZsAmy DNAs.
Recombinant baculovirus stocks
Samples of Bv-AmerAmy and Bv-ZsAmerAmy have been properlydeposited in the Cell and Molecular Biology Laboratory, Depart-ment of Biochemistry, Institute of Chemistry, University of São Paulo,under the care of M.C.S. They are available upon request.
Amplification of baculovirus stocks
For amplification of baculovirus master stocks, 1 × 106 Sf9 cellswere plated in a 25-cm2 flask and incubated for 1 h with 10 µl ofbaculovirus master stock in 1 ml of TNM-FH medium containingantibiotics (corresponding to an MOI of 0.01–0.1). After thisperiod, the medium was completed to 4.5 ml and the infected cellswere incubated for 48 h at 27 °C. The culture medium was col-lected, clarified (500 g for 5 min) and stored at 4 °C as viral stocksfor recombinant protein production.
Recombinant protein production
For massive infection and recombinant protein production, 25-cm2
culture flasks containing 1 × 106 Sf9 cells, plated and grown over-night in TNM-FH medium, at 27 °C, were infected with 500 µl ofamplified baculovirus stock (corresponding to MOI of 10) andincubated at 27 °C for 96 h or until detection of cytopathic effect in90–100% under an inverted phase contrast Nikon microscope.Clarified culture medium (supernatant) and cells (pellet) were col-lected after centrifugation (500 g for 5 min).
Amylase purification from culture supernatants
Amylase secreted to the culture medium by baculovirus-infectedcells was concentrated to 10% of the initial volume by precipitatingproteins with three volumes of cold acetone (stored at −20 °C) for3 h and centrifugation at 12 000 g at 4 °C for 30 min The pelletwas redissolved in 100 µl of Milli-Q water and stored at −20 °C foranalysis of extracellular activity of the expressed α-amylase.
Protein determination
Quantification of proteins present in supernatants of pelleted cellswas accomplished by using the methods described previously(Bradford, 1976; Smith et al., 1985; Morton & Evans, 1992). Eggalbumine was used as the standard protein.
Preparation of baculovirus-infected cell extracts
Infected cells were lysed essentially as described (Grossi de Sá& Chrispeels, 1997), for 1 h with lysis buffer (10 mM Tris-HCl;130 mM NaCl; 0.1% SDS; 5 mM phenylmethylsulphonil fluoride,pH 7.5). To pellet cellular debris, the lysate was clarified bycentrifugation at 12 000 g at 4 °C for 10 min. The supernatantwas stored at −20 °C for analysis of intracellular activity of theexpressed amylase.
α-amylase activity was detected by the lugol (I2 + KI) procedure(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) ofsupernatants or infected cell extracts were added to 480 µl of thesubstrate solution. The reaction mixture was incubated for 12 h at37 °C. A sample (10 µl) of this reaction was then mixed with 200 µlof lugol in 0.05 N HCl. A nearly complete starch hydrolysis pro-duces a clear yellowish solution. The hydrolysed substrate (480 µl)was dried at 60 °C, redissolved with 25 µl of Milli-Q water andassayed by paper chromatography. To a 12 × 6-cm strip of Whatman1MM paper, 1 µl of each sample was applied. The mobile phasewas a mixture of isopropanol /acetic acid/water (7 : 1 : 2). Chroma-tography was ascendent up to 10 cm. Reducing sugars weredetected by precipitation of metalic silver (Trevelyan et al., 1950;Robyt & French, 1963).
SDS-PAGE and amylase activity
Proteins (5 µg) present in supernatants or extracts of infectedcell culture were fractionated by SDS-PAGE in 20 × 20-cm 15%polyacrylamide-SDS gels (Silva, 1989) at 100 V/20 mA. Owing tothe high activity observed in previous analysis, only 0.5 µg of ZsAmywas analysed. Upon fractionation, the gel was washed three timeswith Milli-Q water for 30 min and once with activity buffer (100 mM
phosphate buffer, pH 6.0; 20 mM NaCl; 0.1 mM CaCl2) in order toremove the SDS and allow the enzyme to renature. A layer of agarcontaining 1% blue starch in the activity buffer was cast at 40 °Con to the polyacrylamide gel to function as the indicator gel. Activeamylase was detected on the indicator gel as clear bands resultingfrom the higher diffusion rate of the hydrolysis products in a darkblue background.
SDS-PAGE and Western blotting
Protein samples from culture supernatants (10 µg) or extracts(20 µg) of cells infected with recombinant or wild-type baculoviruswere separated by SDS-PAGE in 10% gels (20 × 20 cm) accordingto Sambrook et al. (1989). Two gels were prepared: one to detectthe protein bands after Coomasie blue (Sambrook et al., 1989) orsilver staining (Silver Stain Plus kit, BIO-RAD, Hercules, CA, USA);the other was used for Western blotting. Proteins were transferedto nitrocellulose membrane by a Transblot-SD Semi-Dry TransferCell. Rabbit anti-Z. subfasciatus α-amylase antibody (kindly pro-vided by Dr Fatima Grossi de Sá), diluted 1 : 10 000, was used forimmunodetection. Western blotting was performed as describedpreviously (Sambrook et al., 1989).
Sequence analysis
The nucleotide sequence of A. merus α-amylase (accessionnumber U01210, GenBank) was processed using the Clone 4software to obtain the conceptual translation product. This wasthen processed in the http://www.up.univ-mrs.fr/∼wabim/d_abim/compo-p.html server to calculate its molecular weight (Mr). Thepromoter sequence was predicted from the http://www.fruitfly.org/seq-tools/promoter.html server (Waibel et al., 1989), the signalpeptide from http://www.cbs.dtu.dk/services/signalp/ (Nielsenet al., 1997) and glycosylation sites from http://www.cbs.dtu.dk/services/netoglyc/ (Hansen et al., 1998). Sequence alignments ofα-amylases were obtained by using the CLUSTAL W program(Thompson et al., 1994).
Acknowledgements
The expert technical assistance of Irenice Cairo da Silva,Sandra de Souza, Sirlei Mendes de Oliveira and Zizi deMendonça is greatly appreciated. This work was supportedby FAPESP, CNPq, FINEP, ICGEB and PRP-USP. P.C.E.was the recipient of a predoctoral Fellowship from CNPq.This work is a partial fulfilment of a Doctoral thesis require-ments (P.C.E.).
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