Nature of the anchors of membrane-bound aminopeptidase, amylase, and trypsin and secretory mechanisms in Spodoptera frugiperda (Lepidoptera) midgut cells

Journal of Insect Physiology 45 (1999) 29–37

Nature of the anchors of membrane-bound aminopeptidase, amylase, and trypsin and secretory mechanisms in Spodoptera frugiperda (Lepidoptera) midgut cells Beatriz P. Jorda˜o a, Adriana N. Capella c, Walter R. Terra c, Alberto F. Ribeiro b, Cle´lia Ferreira c,* a

c

b

Departamento de Biologia, Instituto de Biocieˆncias, Universidade de Sa˜o Paulo, C.P. 11461, 05422-970 Sa˜o Paulo, Brazil Departamento de Bioquı´mica, Instituto de Quı´mica, Universidade de Sa˜o Paulo, C.P. 26077, 05599-970 Sa˜o Paulo, Brazil

Departamento de Fisiologia,

Received 11 March 1998; received in revised form 14 May 1998; accepted 1 June 1998

Abstract Spodoptera frugiperda larvae have a microvillar aminopeptidase and both soluble and membrane-bound forms of amylase and trypsin. Membrane-bound aminopeptidase is solubilized by glycosyl phosphatidylinositol-specific phospholipase C (GPI-PLC) and detergents, suggesting it has a GPI anchor. Membrane-bound trypsin is not affected by GPI-PLC, although it is solubilized by papain and by different detergents. Membrane-bound amylase is similar to trypsin, although once solubilized in detergent it behaves as a hydrophilic protein. Musca domestica trypsin antiserum cross-reacts with only one polypeptide from S. frugiperda midgut. With this antiserum, trypsin was immunolocalized in the anterior midgut cells at the microvillar surface and on the membranes of secretory vesicles found in the apical cytoplasm and inside the microvilli. The data suggest that in this region trypsin is bound to the secretory vesicle membrane by a hydrophobic anchor. Vesicles migrate through the microvilli and are discharged into the lumen by a pinching-off process. Trypsin is then partly processed to a soluble form and partly, still bound to vesicle membranes, incorporated into the peritrophic membrane. In posterior midgut cells, trypsin immunolabelling is randomly distributed inside the secretory vesicles and at the microvilli surface, suggesting exocytosis. Amylase probably follows a route similar to that described for trypsin in anterior midgut, although membrane-bound forms (peptide anchor) solubilize apparently as a consequence of a pH increase inside the vesicles.  1998 Elsevier Science Ltd. All rights reserved. Keywords: Exocytosis; Microapocrine secretion; Peritrophic membrane; Trypsin immunolocalization

1. Introduction The processing of precursors and the secretory mechanism of digestive enzymes in insects seem to include aspects which are not found in other animals (Terra and Ferreira, 1994). Thus, the study of these processes may result in an important contribution to cell biology and may provide new targets for the development of methods of insect control. The first attempt to study midgut secretory mechanisms in lepidopteran larvae was performed with Bombyx mori using biochemical methods. The results sug-

* Corresponding author. [email protected]

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55-11-818-2186;

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gested that membrane-bound trypsin is somehow transported from the tissue to the lumen, where it is solubilized and converted to its main form. Part of the membrane-bound trypsin is incorporated into the peritrophic membrane (Eguchi et al., 1982; Kuriyama and Eguchi, 1985). Later on, using a combination of biochemical and cytological methods, it was suggested that in Erinnyis ello larvae glycosidases are secreted by exocytosis, but soluble amylase and soluble trypsin derive from membrane-bound precursors and are secreted by a microapocrine process. Membrane-bound amylase and trypsin which are found in E. ello ectoperitrophic luminal contents are presumed to be part of membrane vesicles which are incorporated into the peritrophic membrane (Santos and Terra, 1986; Santos et al., 1984, 1986). More recently, biochemical studies performed with

0022–1910/98/$ - see front matter  1998 Elsevier Science Ltd. All rights reserved. PII: S 0 0 2 2 - 1 9 1 0 ( 9 8 ) 0 0 0 9 8 - 5

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Spodoptera frugiperda larvae provided further evidence that amylase- and trypsin-carrying membranes occur in lepidopteran ectoperitrophic contents and that they are partly incorporated into the peritrophic membrane (Ferreira et al., 1994). This incorporation is significant, as washed peritrophic membranes may contain up to 13% and 18% of the midgut luminal activity of amylase and trypsin, respectively (Ferreira et al., 1994). In this paper, trypsin immunolocalization in S. frugiperda larval midgut cells is presented and attempts to solubilize membrane-bound enzymes are described. The data obtained supported the hypothesis that in anterior midgut, trypsin is anchored by a peptide to the membrane of secretory vesicles, which eventually are freed by a pinching-off process followed by proteolytic processing. In posterior midgut, trypsin molecules are soluble and secreted by exocytosis.

2. Materials and methods 2.1. Animals Spodoptera frugiperda (Lepidoptera: Noctuidae) were laboratory reared according to Parra (1986). The larvae were individually contained in glass vials with a diet based on kidney bean (Phaseolus vulgaris), wheat germ, yeast and agar and were maintained under a natural photoregime (summer, 14L: 10D; winter, 10L: 14D) at 25°C. Adults were fed a 10% honey solution. Fifth (last) instar larvae of both sexes were used in the experiments. 2.2. Preparation of S. frugiperda midgut samples Larvae were immobilized by placing them on ice, after which they were rinsed in water and blotted with filter paper. Their guts were dissected in cold 125 mM NaCl, and then the peritrophic membrane with contents and the midgut tissue were pulled apart. Midgut tissue, after being rinsed thoroughly with saline, was homogenized in double distilled water. After centrifuging the homogenate at 100,000g for 60 min at 4°C, the resulting supernatant was recovered and used as source of soluble midgut proteins, whereas the corresponding pellet was resuspended in double distilled water, frozen-andthawed three times and centrifuged as before. The remaining pellet, after being suspended in double distilled water, was used as a source of midgut cell membranes. 2.3. SDS-PAGE and Western blotting SDS-PAGE of S. frugiperda midgut samples was carried out on 12% (w/v) polyacrylamide gels containing 0.1% (w/v) SDS (sodium dodecyl sulphate), on a discontinuous pH system (Laemmli, 1970), using Bio-Rad

(U.S.A.) Mini-Protean II equipment. Samples were mixed with sample buffer (2:1) containing 60 mM TrisHCl, pH 6.8, 2.5% (w/v) SDS, 0.36 mM 2-mercaptoethanol, 10% glycerol and 0.05% (w/v) bromophenol blue and heated for 2 min at 95°C in a water bath before being loaded onto the gels. Electrophoresis was carried out at 200 V until the front marker (bromophenol blue) reached the bottom of the gel. The gel was then divided into two parts, one of which was silver-stained according to Blum et al. (1987), while the corresponding proteins in the other part of the gel were electrophoretically transferred onto a nitrocellulose membrane filter (pore size 0.45 ␮m; Bio-Rad, U.S.A.) (Towbin et al., 1979). The transfer efficiency was evaluated by observing the prestained molecular weight markers (Bio-Rad or Sigma, U.S.A.). The filters were first reacted (after a blocking step) with the trypsin diluted 100-fold in TBS (Tris-buffered saline) containing 0.05% Tween 20 (TBS-T) for 2 h at room temperature. After extensive washing, the filters were reacted with anti-rabbit IgG coupled with peroxidase (Sigma) diluted 1: 1500 in TBS-T for 2 h at room temperature. After washing extensively with the same buffer, the strips were treated with ECL Western blotting kit (Amersham, U.K.) according to the manufacturer’s instructions, and then exposed to a high-performance luminescence detection film (Hyperfil-ECL, Amersham, U.K.). 2.4. Proteinase assays after SDS-polyarylamide gel electrophoresis (SDS-PAGE) Midgut samples without heating were subjected to SDS-PAGE in 12% gel. After electrophoresis, the gel slabs were transferred to a solution of 50 mM Tris buffer pH 7.5 containing 2.5% (w/v) Triton X-100 and allowed to stand for 10 min at room temperature to decrease the SDS content. The gel slabs were layered onto gelatincontaining polyacrylamide gel slabs [prepared according to Heussen and Dowdle (1980)] and the two were left in contact overnight at 33°C in a humid chamber. Finally, the original gel slabs were removed and the gelatin-containing gel slabs were stained with 0.1% Coomassie blue R-250 in water–ethanol–acetic acid (5: 5:2, v/v/v) for 6–16 h. Destaining was accomplished in ethanol–acid acetic–water (30: 7: 63, v/v/v) until clear bands could be visualized against a dark blue background. In order to abolish trypsin activity, the polyacrylamide-gelatin gel slab contained 2 mM benzamidine and a layer of a 100 mM benzamidine solution was applied at its surface immediately before the gel with the samples was placed on it. 2.5. Differential solubilization and temperature induced phase separation in Triton X-114 The procedure used is a modification of that of Hooper and Bashir (1991) as performed by Jorda˜o et al. (1996).

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A sample containing midgut cell membranes was placed in a 1.5 ml-microcentrifuge tube, and to it, Triton X-114 (pre-condensed according to Bordier, 1981) and sodium citrate-phosphate (pH 5.0 or 6.0) or sodium phosphate (pH 7.0 or 8.0) were added, so that the final volume was 0.5 ml and the final concentrations were: membrane proteins, 1 mg ml−1; buffer, 40 mM; detergent, 5% (w/v). After vortex-mixing and incubation at 4°C for 16 h, the samples were centrifuged at 11,000g for 30 min at 4°C in a fixed angle rotor, resulting in a pellet (detergentinsoluble material) and in a supernatant. The supernatant was removed to a second microcentrifuge tube, incubated at 30°C for 3 min and, following centrifugation at 15,000g for 3 min at 30°C in a fixed angle rotor, was separated into two phases. The upper detergent-poor phase was recovered and the lower detergent-rich phase was added to 200 ␮l of a washing detergent-poor phase (obtained as described above with double distilled water replacing the midgut cell membranes). After vortex-mixing and standing for 5 min on ice, the mixture was incubated at 30°C for 3 min and centrifuged for 3 min as before. The upper phase was removed and pooled with the detergent-poor phase obtained previously. This is the final detergent-poor phase. The remaining lower detergent-rich phase was made up to 0.5 ml with the corresponding buffer (40 mM sodium citrate–phosphate at pH 5.0 or 6.0, or 40 mM sodium phosphate at pH 7.0 or 8.0). The pellet was washed (not suspended) with 0.5 ml the corresponding buffer and centrifuged at 11,000g for 30 min at 4°C in a fixed angle rotor. The pellet was finally resuspended in 0.5 ml buffer. The three phases (pellet, upper- and lower-phase) were then assayed for trypsin activity at 20°C. This activity was calculated as a percentage of the trypsin activity originally found in the midgut cell membrane preparation. 2.6. Solubilization of membrane proteins by papain. To midgut cell membranes suspended in 0.1 M HEPES at pH 7.4, activated papain (by previous incubation with 50 mM cysteine) was added in a ratio of 1 mg of papain to 4 mg of membrane protein. After 45 min at 30°C, with occasional stirring, the sample was centrifuged at 100,000g for 1 h at 4°C, and the supernatant recovered. 2.7. Solubilization of membrane proteins by glycosylphosphatidylinositol-specific phospholipase C (GPIPLC). GPI-PLC from Bacillus thuringiensis (a gift from Dr Martin Low) was added (4.875 Units to 1 mg membrane protein) to a suspension of midgut cell membranes in 10 mM HEPES at pH 7.4. After 60 min at 37°C, with occasional stirring, the sample was centrifuged at 100,000g for 1 h at 4°C, and the supernatant recovered.

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2.8. Protein determination and enzyme assays Protein was determined according to Bradford (1976) using ovalbumin as a standard. Aminopeptidase was assayed in 50 mM sodium phosphate buffer (pH 8.0) using 1 mM leucine p-nitroanilide as a substrate (Erlanger et al., 1961). Amylase was assayed in 50 mM sodium citrate–phosphate (pH 6.5) containing 10 mM NaCl with 0.5% soluble starch as substrate (Noelting and Bernfeld, 1948). Trypsin was assayed in 50 mM sodium phosphate pH 7.5 with 0.8 mM ␣-N-benzoyl-DL-arginine-p-nitroanilide as a substrate (Erlanger et al., 1961). Incubations were carried out for at least four different time periods, and initial rates of hydrolysis were calculated. All assays were performed under conditions such that activity was proportional to protein concentration and to time. Controls without enzyme or without substrate were performed. One U of enzyme is defined as the amount of the enzyme that hydrolyzes 1 ␮mol of substrate (or bond) per min. 2.9. Preparation and evaluation of trypsin antiserum Soluble trypsin was purified from 12,000 Musca domestica larvae using ion-exchange and affinity chromatography as described by Lemos and Terra (1992); trypsin antiserum was isolated from rabbits previously injected with the purified enzyme as described by Jorda˜o et al. (1996). Antibody production and specificity were monitored by double immunodiffusion tests in agarose gels and Western blots (Jorda˜o et al., 1996). 2.10. Immunolocalization of trypsin in S. frugiperda midgut cells Insects were dissected in their own hemolymph. Midguts were isolated and, after being divided into three identical length sections (anterior, middle, and posterior), were fixed in 4% paraformaldehyde with 0.3% glutaraldehyde in 0.1 M phosphate buffer at pH 7.4 for 2 h at 4°C. After rinsing with phosphate buffer, the material was dehydrated in graded ethanol solutions at room temperature, and embedded in “hard grade” L.R. White acrylic resin (Electron Microscopy Sciences, Ft. Washington, USA). Ultra-thin sections were cut on the ultramicrotome and collected on 200 mesh colloidoncoated nickel grids. The grids were then floated on drops of TBS at pH 7.2 containing 1% BSA (Sigma, USA) for 5 min, and placed on NGS (Amersham, UK), diluted 1:30, for 30 min. The sections were then incubated overnight in the primary antisera diluted 1:2000 in TBS at pH 7.2 containing 1% BSA at 4°C. As controls, sections were incubated with non-immune serum using the same conditions. After rinsing 4 ⫻ 5 min in TBS at pH 7.2 with 0.2% BSA, 0.05% NaN3 and 0.1% Tween 20, the samples were placed in TBS at pH 8.2 with 1% BSA

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and 0.05% NaN3 for 30 min at room temperature, and incubated with goat anti-rabbit IgG coupled to 15 nm gold particles (Amersham, UK) diluted 1:20 in TBS at pH 8.2 plus 1% BSA and 0.05% NaN3 for 1 h at room temperature. The grids were then washed 4 ⫻ 5 min in TBS at pH 7.2 containing 0.2% BSA, 0.05% NaN3 and 0.1% Tween 20, followed by the same solution without BSA (2 ⫻ 5 min). After fixation in 2.5% glutaraldehyde in 0.1 M sodium cacodylate buffer pH 7.4 for 10 min at room temperature, the samples were finally washed in double distilled water, stained with uranyl acetate and lead citrate, and examined in a Zeiss EM 900 electron microscope.

3. Results 3.1. Specificity of M. domestica trypsin antiserum in S. frugiperda midgut samples A Western blot of soluble midgut contents from S. frugiperda after SDS-PAGE and incubated with trypsin antiserum showed only one band (Fig. 1). This band agrees with a major band in midgut content polypeptides (Fig. 1). No bands are visible in Western blots of tissue homogenates (not shown), probably due to the fact that only 1.3% of midgut trypsin is recovered from S. frugiperda midgut tissue (Ferreira et al., 1994). Gelatin slab gels showed that the recognized band in midgut contents migrates in SDS-PAGE as a benzamidine-inhibited pro-

Fig. 1. Silver-stained SDS-PAGE (lane 1) and Western blot after SDS-PAGE (lane 2) of S frugiperda soluble proteins from midgut contents. Gel used: 12% polyacrylamide gel. Amount of proteins in the lanes: 1, 3 ␮g; 2, 9 ␮g.

teolytic activity (Fig. 2). This together with Western blotting data (Fig. 1), strongly suggests that M. domestica trypsin antibody is specifically recognizing the S. frugiperda trypsin. 3.2. Immunocytochemical localization of trypsin in S. frugiperda midgut cells The general morphology of S. frugiperda larval midgut (not shown) is similar to that of Manduca sexta (Cioffi, 1979) and Erinnyis ello (Santos et al., 1984) in having columnar and goblet cells, whose morphology change along the midgut, and few endocrine cells. Preliminary results showed that trypsin immunolabelling is restricted to columnar cells and that anterior and middle midgut cells are alike. Therefore, only results referring to anterior and posterior S. frugiperda columnar midgut cells are described here. The anterior columnar cells have microvilli with dilated tips and displaying secretory vesicles inside (Fig. 3a and corresponding inset). Trypsin immunolabelling is observed on the membranes of secretory vesicles found in the apical cytoplasm (Fig. 3d) and inside the microvilli (Fig. 3c). Labelling is also seen at the surface of microvilli (Fig. 3b). The apical cytoplasm of posterior columnar cells displays large secretory vesicles at the base of cylindrical microvilli (Fig. 4). Trypsin is randomly distributed inside the secretory vesicles, which apparently undergo exocytosis (Fig. 4). Labelling is also observed at the surface of the microvilli, although in less extent than in anterior midgut cells.

Fig. 2. Proteinase assays after SDS-PAGE 12% in the absence (a) or presence (b) of benzamidine. Clear areas result from gelatin proteolysis.

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Fig. 3. Apical surface of S. frugiperda anterior midgut columnar cell. (a) General morphology of the microvilli (Mv) showing dilated tips with secretory vesicles (SV) (arrows). Inset: detail showing a secretory vesicle in the middle of the microvillus (arrow). (b) Immunolocalization of trypsin in the microvillar surface. (c) Trypsin immunolabelling in secretory vesicles inside microvilli (arrows). (d) Immunolocalization of trypsin in the limiting membrane of the secretory vesicles (arrows) and in the microvilli. Bars = 1 ␮m (a and b); 0.1 ␮m (inset of a, c and d).

3.3. Solubilization of membrane-bound aminopeptidase, amylase, and trypsin from S. frugiperda midgut cells Soluble amylase and trypsin are thought to be derived from membrane-bound forms in S. frugiperda midguts (Ferreira et al., 1994). In order to understand the processing mechanism by which the membrane-bound forms are converted into soluble enzymes, it is necessary to know how amylase and trypsin are anchored to S. frugiperda midgut cell membranes. For this, several experiments were performed to solubilize amylase and trypsin from midgut cell membranes. Aminopeptidase was included in these studies as an example of a

microvillar enzyme (Ferreira et al., 1994; Capella et al., 1997) which is not secreted into luminal contents (Ferreira et al., 1994). Aminopeptidase is solubilized by GPI-PLC and detergents, from which those with high critical micellar concentration (CMC) (octylGlu and CHAPS) are more efficient than those with low CMC (Triton X-100) (Table 1). Membrane-bound amylase is significantly solubilized in buffer and none of the agents in Table 1 improve its solubilization remarkably (Table 1). Membrane-bound trypsin is rendered soluble by the action of papain and detergents. In contrast to aminopeptidases, the different detergents solubilize trypsin in approximately the same extent (Table 1).

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3.4. Soluble aminopeptidase, amylase and trypsin from pelletable material recovered from ectoperitrophic fluid Aminopeptidase, amylase, and trypsin has been found in fractions of the ectoperitrophic fluid obtained by differential centrifugation (Ferreira et al., 1994). To characterize further these fractions, the crude fraction was discarded and the other two were sub-divided into soluble and membrane-bound material. There is a small amount of soluble aminopeptidase associated with both large and small particles, whereas trypsin shows intermediate and amylase large amounts of soluble forms (Table 3).

4. Discussion 4.1. The nature of the anchor of S. frugiperda membrane-bound aminopeptidase, amylase and trypsin

Fig. 4. Apical cytoplasm of S. frugiperda posterior midgut columnar cell. Note immunolabelling in microvilli (Mv) and in large secretory vesicle (SV) contents. Bar = 0.1 ␮m.

The solubilization of membrane-bound aminopeptidase, amylase and trypsin in Triton X-114 increase at high pH values (Table 2). On solubilizing, aminopeptidase and trypsin accumulate in the detergent-rich phase, whereas amylase goes to the detergent-poor phase. At all pH values, negligible amounts of amylase activity can be determined in the detergent-rich phase (Table 2).

The binding of enzymes in cell membranes usually occurs through a hydrophobic peptide or through a GPIanchor. Enzymes bound by a hydrophobic peptide are, as a rule, well solubilized by detergents with high (e.g. octylGlu, CHAPS) or low (e.g. Triton X-100) CMC and at least partially released from membranes by the action of papain or trypsin (Hooper and Turner, 1988). Enzymes anchored to membranes via a GPI-anchor are well released into solution with detergents with high CMC or by treatment with GPI-PLC (Hooper and Turner, 1988). S. frugiperda microvillar aminopeptidase is solubilized by OctylGlu, CHAPS, and GPI-PLC, but is not affected by incubations with papain. Once solubilized in Triton X-114 at 4°C, S. frugiperda aminopeptidase partitions predominantly at 30°C into the detergent-rich phase, as expected for amphipathic proteins (Hooper and

Table 1 Solubilization of membrane-bound aminopeptidase, amylase and trypsin from S. fugiperda midgut cell membranes by different agents* Aminopeptidase Agent None GPI-PLC Papain Octylglu CHAPS Triton X-100 *

% Solubilization 11 ⫾ 5 42 ⫾ 3 9⫾1 93 ⫾ 1 68 ⫾ 6 43 ⫾ 2

Amylase

% Recovery 104 84 102 84 91 105

⫾ ⫾ ⫾ ⫾ ⫾ ⫾

7 2 1 9 5 2

% Solubilization 31 ⫾ 7 24 ⫾ 5 39 ⫾ 1 44 ⫾ 6 46 ⫾ 15 35 ⫾ 1

Trypsin

% Recovery 92 ⫾ 7 82 ⫾ 4 97 ⫾ 6 84 ⫾ 2 80 ⫾ 6 90 ⫾ 10

% Solubilization 11 11 39 48 49 39

⫾ ⫾ ⫾ ⫾ ⫾ ⫾

2 1 3 6 4 4

% Recovery 100 ⫾ 10 88 ⫾ 7 94 ⫾ 7 110 ⫾ 10 100 ⫾ 7 94 ⫾ 8

Cell membranes were maintained in the absence or presence of GPI-PLC (60 min at 37°C), papain (30 min at 37°C) or, detergents (16 h at 4°C) in 10 mM (for papain, 100 mM) HEPES buffer pH 7.4 for the indicated times, before being centrifuged at 100,000 g for 1 h at 4°C. Aminopeptidase, amylase, and trypsin were determined in the resulting supernatants and referred to in relation to the original preparation of cell membranes. Figures are means ⫾ SEM calculated from determinations in each of four different experiments.

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Table 2 Differential solubilization of membrane-bound aminopeptidase, amylase and trypsin in Triton X-114 at different pH values* % Activity in each phase pH Aminopeptidase 5.0 6.0 7.0 8.0 Amylase 5.0 6.0 7.0 8.0 Trypsin 5.0 6.0 7.0 8.0

Detergent-poor phase

Detergent-rich phase

Detergent-insoluble pellet

% Recovery

8.4 ⫾ 0.5 15 ⫾ 1 20 ⫾ 2 22 ⫾ 2

29 48 59 59

⫾ ⫾ ⫾ ⫾

1 1 2 2

62 37 21 19

⫾ ⫾ ⫾ ⫾

1 1 2 2

9⫾1 18 ⫾ 1 24 ⫾ 1 45 ⫾ 3

3 2 6 4

⫾ ⫾ ⫾ ⫾

1 1 1 1

88 80 70 51

⫾ ⫾ ⫾ ⫾

1 2 1 2

100 ⫾ 10 100 ⫾ 10 120 ⫾ 10 115 ⫾ 5

5⫾2 7⫾3 25 ⫾ 5 42 ⫾ 5

91 87 63 38

⫾ ⫾ ⫾ ⫾

2 3 5 3

100 ⫾ 10 92 ⫾ 8 112 ⫾ 4 140 ⫾ 2

3.7 ⫾ 0.3 6⫾1 12 ⫾ 1 20 ⫾ 3

71 83 82 76

⫾ ⫾ ⫾ ⫾

3 3 2 2

*

Cell membranes were subjected to differential solubilization and temperature-induced phase separation in Triton X-114 at different pH values as described in Materials and Methods section. The resultant phases were then assayed for aminopeptidase, amylase, and trypsin activity. Results are means ⫾ SEM for three separate phase separations.

Table 3 Percentage of soluble hydrolases present in pelletable material from the ectoperitrophic fluida Ectoperitrophic fractionb Enzyme

Large particles

Small particles

Aminopeptidase Amylase Trypsin

3.4 ⫾ 0.4 60 ⫾ 10 21 ⫾ 6

13 ⫾ 6 60 ⫾ 10 40 ⫾ 10

a Results are relative activities displayed as percentage of the sum of soluble and pelletable activities in each ectoperitrophic fraction. Data are expressed as means ⫾ SEM calculated from four assays performed in each of three different preparations. b Ectoperitrophic contents were collected by rinsing the luminal surface of the midgut with saline. The rinsing saline was then centrifuged and the following fractions were collected: crude pellet, pellet resulting from centrifuging at 600 g for 10 min; large particles, pellet from 10,000 g for 10 min; small particles, pellet from 25,000 g for 30 min. The crude pellet was discarded and the other pellets were submitted to three freezingthawing cycles before centrifuging at 100,000 g for 60 min. The resulting supernatants and pellets were then assayed.

Bashir, 1991). These properties indicate that S. frugiperda aminopeptidae is bound to the microvillar membrane by a GPI-anchor. This kind of anchor has been described before for other insect midgut aminopeptidases (e.g. Bombyx mori, Takesue et al., 1992), although the occurrence of aminopeptidases bound via a peptide has also been described (Terra and Ferreira, 1994). S. frugiperda membrane-bound trypsin is solubilized by papain, detergents and is not affected by GPI-PLC. Furthermore, in the Triton X-114 experiment, it partitions into the detergent-rich phase. Thus, S. frugiperda trypsin is probably bound to cell membranes via a hydrophobic peptide. S. frugiperda membrane-bound amylase is signifi-

cantly solubilized in buffer and, except for GPI-PLC, the other agents increase in a certain extent its solubilization. The data favor the hypothesis that amylase is anchored to the membrane by a hydrophobic peptide which is not solubilized by papain. Nevertheless, S. frugiperda amylase molecules, once solubilized in Triton X-114 at 4°C, partition predominantly at 30°C into the detergent-poor phase, as if they are hydrophilic proteins. Thus, S. frugiperda membrane-bound amylase molecules might change in conformation during solubilization, hindering part of the hydrophobic peptide and becoming more hydrophilic. The hypothetical amylase change in conformation probably depends on pH, as amylase solubilization is greatly affected by the medium pH. Thus, the

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behavior of S. frugiperda membrane-bound amylase is similar to Musca domestica membrane-bound trypsin (Jorda˜o et al., 1996). 4.2. Trypsin and amylase secretion in S. frugiperda larval midguts Trypsin immunolabelling in S. frugiperda anterior midgut cells is observed on the membranes of secretory vesicles, both in the apical cytoplasm and inside the microvilli, which display dilated tips. Labelling is also seen at the microvilli surface (Fig. 3b,c,d). The same observations are true for middle midgut cells (not shown). About 82% of S. frugiperda midgut tissue trypsin is recovered from anterior plus middle midgut (Ferreira et al., 1994). Therefore, most of S. frugiperda trypsin is likely to be secreted according to the model illustrated in Fig. 5. According to this model, trypsin is synthesized bound to membranes through a hydrophobic peptide anchor. In this form it is processed in the Golgi complex and trans-

Fig. 5. General diagram showing putative microapocrine process occurring in S. frugiperda anterior columnar midgut cell. (A) Secretory vesicles migrating inside microvilli (Mv). (B) Pinching-off microvilli tip with a secretory vesicle inside. (C) Secretory vesicle limiting membranes fused with microvilli membrane before pinching-off. (D) The pinched-off vesicles become single-membrane vesicles, which probably dissociate in the highly alkaline medium. (E) The remaining vesicle membranes may be incorporated into the peritrophic membrane (PM).

ported in secretory vesicles. These vesicles migrate through the cell microvilli and, before or after being fused with the microvillar membrane at the tips of the microvilli, are discharged into the lumen by a pinchingoff process, resulting in double-and single-membrane vesicles, respectively. Double-membrane vesicles become single-membrane vesicles by membrane-fusing processes. Part of the trypsin on the luminal surface of single-membrane vesicles becomes soluble by limited proteolysis or by partial dissolution of the vesicles in the highly alkaline midgut lumen. Double membrane vesicles hinder trypsin from solubilization processes. This explains why large particles in the ectoperitrophic fluid, which probably correspond to double membrane vesicles, have a small percentage of associated soluble trypsin (Table 3). The remaining vesicle membrane, with part of the trypsin molecules still bound, is finally incorporated into the forming external layer of the peritrophic membrane. In addition to immunolabelling and solubilization data, the following observations support the secretory model for trypsin in S. frugiperda anterior and middle midgut: (1) The specific activity of trypsin in trypsincarrying membranes from S. frugiperda ectoperitrophic contents is much higher than in microvillar membranes, whereas the specific activity of the microvillar aminopeptidase is approximately the same in both membranes (Ferreira et al., 1994). This suggested that ectoperitrophic trypsin-carrying membranes are produced from a specific region of the columnar cell microvillar membrane (to account for the differences found in specific activities). (2) About 18% of the midgut luminal activity of trypsin was found incorporated in the peritrophic membrane (Ferreira et al., 1994). As the vesicles migrate upwards within the microvillus, the cystoskeleton must assemble and disassemble, which suggests it is not rigid. In agreement with this, lepidopteran anterior midgut cell microvilli lack a well organized cytoskeleton (Cioffi, 1979; Santos et al., 1984), which is easier to remove from the microvillar membranes than those from usual microvilli (Capella et al., 1997). Amylase secretion in S. frugiperda larval midguts probably follows a route similar to that described for trypsin in the anterior midgut. This is supported by the same kind of evidence discussed above for trypsin, except for immunocytochemical data, which are lacking. Nevertheless, in contrast to trypsin, large particles in the ectoperitrophic fluid have a large percentage of associated soluble amylase (Table 3). Thus, membrane-bound amylase starts to solubilize as soon as the pH increases inside their containing vesicles. This solubilization is probably enhanced by the exposure to the high luminal pH. As a consequence, a smaller amount of amylase than trypsin is recovered in peritrophic membranes (13% instead of 18% of total midgut luminal activity).

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In S. frugiperda posterior midgut cells, trypsin is immunolocalized in secretory vesicles, and in the microvillar surface (Fig. 4). Thus, the data suggest that in this midgut region trypsin is synthesized in soluble form and secreted by exocytosis. Up to the moment, it is not known how different are the molecules of trypsin secreted by the two midgut regions. It is tempting to speculate that posterior midgut trypsin is secreted in small amounts and, after being transported to the anterior midgut by the counter-current flux of fluid (Terra, 1990), is able to solubilize by limited proteolysis the membrane-bound trypsin from anterior and middle midgut.

Acknowledgements This work was supported by the Brazilian research agencies FAPESP and CNPq. We are indebted to Dr. J.R.P. Parra for his support in relation to S. frugiperda laboratory rearing and to the technicians L.Y. Nakabayashi, M.V. Cruz and W. Caldeira. A.N. Capella is a graduate fellow of FAPESP. W.R. Terra, A.F. Ribeiro, and C. Ferreira are staff members of their respective departments and research fellows of CNPq.

References Blum, H., Beier, H., Gross, H.J., 1987. Improved silver staining of plant proteins, RNA and DNA in polyacrylamide gels. Electrophoresis 8, 93–99. Bordier, C., 1981. Phase separation of integral membrane proteins in Triton X-114 solution. Journal of Biological Chemistry 256, 1604–1607. Bradford, M.M., 1976. A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding. Analytical Biochemistry 72, 248–254. Capella, A.N., Terra, W.R., Ribeiro, A.F., Ferreira, C., 1997. Cytoskeleton removal and characterization of the microvillar membranes isolated from two midgut regions of Spodoptera frugiperda (Lepidoptera). Insect Biochemistry and Molecular Biology 27, 793–801. Cioffi, M., 1979. The morphology and fine structure of the larval midgut of a moth (Manduca sexta) in relation to active ion transport. Tissue and Cell 11, 467–479. Eguchi, M., Iwamoto, A., Yamauchi, K., 1982. Interrelation of proteases from the midgut lumen, epithelia and peritrophic membrane of the silkworm Bombyx mori. Comparative Biochemistry and Physiology 72A, 359–363. Erlanger, B.F., Kokowsky, N., Cohen, W., 1961. The preparation and properties of two new chromogenic substrates of trypsin. Archives of Biochemistry and Biophysics 95, 271–278. Ferreira, C., Capella, A.N., Sitnik, R., Terra, W.R., 1994. Digestive enzymes in midgut cells, endo-and ectoperitrophic contents and

37

peritrophic membranes of Spodoptera frugiperda (Lepidoptera) larvae. Archives of Insect Biochemistry and Physiology 26, 299–313. Heussen, C., Dowdle, E.B., 1980. Electrophoretic analysis of plasminogen activators in polyacrilamide gels containing sodium dodecyl sulphate and copolymerized substrates. Analytical Biochemistry 102, 196–202. Hooper, N.M., Turner, A.J., 1988. Ectoenzymes of the kidney microvillar membrane. Differential solubilization by detergents can predict a glycosyl-phosphatidylinositol membrane anchor. Biochemical Journal 250, 865–869. Hooper, N.M., Bashir, A., 1991. Glycolsyl-phosphatidylinositol-anchored membrane proteins cam be distinguished from transmembrane polypeptide-anchored proteins by differential solubilization and temperature-induced phase separation in Triton X-114. Biochemical Journal 280, 745–751. Jorda˜o, B.P., Terra, W.R., Ribeiro, A.F., Lehane, M.J., Ferreira, C., 1996. Trypsin secretion in Musca domestica larval midguts: A biochemical and immunicytochemical study. Insect Biochemistry and Molecular Biology 26, 337–346. Kuriyama, K., Eguchi, M., 1985. Conversion of the molecular form by alkaline treatment of gut protease from the silkworm Bombyx mori. Comparative Biochemistry and Physiology 82B, 575–579. Laemmli, U.K., 1970. Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature 227, 680–685. Lemos, F.J.A., Terra, W.R., 1992. Soluble and membrane-bound forms of trypsin-like enzymes in Musca domestica larval midguts. Insect Biochemistry and Molecular Biology 22, 613–619. Noelting, G., Bernfeld, P., 1948. Sur les enzymes amylolytiques. III. La ␤-amylase: dosage d’ activite´ et controˆle de l’absence d’␣-amylase. Helvetia Chimica Acta 31, 286–290. Parra, J.R.P., 1986. Criac¸a˜o de insetos para estudos com pato´genos. In: Alves, S.B. (Ed.), Controle Microbiano de Insetos. Editora Manole, Sa˜o Paulo, pp. 348–373. Santos, C.D., Terra, W.R., 1986. Distribution and characterization of oligomeric digestive enzymes from Erinnyis ello caterpillars and inferences concerning secretory mechanisms and the permeability of the peritrophic membrane. Insect Biochemistry 16, 691–700. Santos, C.D., Ribeiro, A.F., Ferreira, C., Terra, W.R., 1984. The larval midgut of the cassava hornworm (Erinnyis ello). Ultrastructure, fluid fluxes and the secretory activity in relation to the organization of digestion. Cell and Tissue Research 237, 565–574. Santos, C.D., Ribeiro, A.F., Terra, W.R., 1986. Differential centrifugation, calcium precipitation and ultrasonic disruption of midgut cells of Erinnyis ello caterpillars. Purification of cell microvilli and inferences concerning secretory mechanisms. Canadian Journal of Zoology 64, 490–500. Takesue, S., Yokota, K., Miyajima, S., Taguchi, R., Ikezawa, H., Takesue, Y., 1992. Partial release of aminopeptidase N from larval midgut cell membranes of the silkworm, Bombyx mori, by phosphatidylinositol-specific phospholipase C. Comparative Biochemistry and Physiology B 102, 7–11. Terra, W.R., 1990. Evolution of digestive systems of insects. Annual Review of Entomology 35, 181–200. Terra, W.R., Ferreira, C., 1994. Insect digestive enzymes: properties, compartmentalization and function. Comparative Biochemistry and Physiology B 109, 1–62. Towbin, H., Staehelin, T., Gordon, J., 1979. Electrophoretic transfer of proteins from polyacrilamide gels to nitrocellulose sheets: procedure and some applications. Proceedings of the National Academy of Sciences (Washington) 76, 4350–4354.