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Proteolytic breakdown of the Neb-trypsin modulating oostatic factor (Neb-TMOF) in the hemolymph of different insects and its gut epithelial transport
Journal of Insect Physiology 47 (2001) 1235–1242 www.elsevier.com/locate/jinsphys
Proteolytic breakdown of the Neb-trypsin modulating oostatic factor (Neb-TMOF) in the hemolymph of different insects and its gut epithelial transport W. Zhu a, A. Vandingenen a, R. Huybrechts a, T. Vercammen a, G. Baggerman a, A. De Loof a, C.P. Poulos b, A. Velentza b, M. Breuer a,* a
Zoological Institute, Katholieke Universiteit Leuven, Naamesestraat 59, B-3000 Leuven, Belgium b Department of Chemistry, University of Patras, GR-26500 Patras, Greece Received 15 November 2000; accepted 13 February 2001
Abstract The degradation of the unblocked hexapeptide, trypsin modulating oostatic factor of the flesh fly Neobellieria (Sarcophaga) bullata (Neb-TMOF) was studied in vitro in the hemolymph of the lepidopteran Spodoptera frugiperda, the orthopteran Schistocerca gregaria and the dictyopteran Leucophaea maderae. The half-life in the different species varied from 苲3 min in L. maderae to 苲25 min in S. gregaria. Purification of the degradation products and ESI-Qq-oa-Tof mass spectrometry revealed the fragments AsnPro-Thr-Asn, Leu-His and Asn-Pro, which were the same in the hemolymph of all species. Except in Leucophaea, Neb-TMOF was cleaved in dipeptides starting from the C-terminus and the reaction could be, at least partially, inhibited by captopril. These observations suggest that a dipeptidase, which has very similar enzymatic properties as mammalian angiotensin converting enzyme (ACE) and which circulates in the hemolymph, apparently is involved in the breakdown of Neb-TMOF and might be a common but not a universal enzyme in insect hemolymph. The introduction of Neb-TMOF into the gut of S. gregaria with the help of a capillary tube (intubation) demonstrated that the intact peptide is able to cross the gut epithelium and to appear in the hemolymph compartment. Since [3H]-inulin, which is too large to cross cell membranes, was found to penetrate the gut walls at a measurable rate, the paracellular pathway might be also permeable to smaller peptides. There was indeed a clear correlation between the molecular weight of inulin, NebTMOF, and inositol and the rate of penetration of these compounds through the gut epithelium to the hemolymph. These are promising findings in view of a potential use of such peptides for insect control purposes. 2001 Elsevier Science Ltd. All rights reserved. Keywords: Neb-TMOF; Spodoptera frugiperda; Schistocerca gregaria; Leucophaea maderae; Hemolymph; Proteolytic breakdown; Angiotensin converting enzyme; Midgut; Epithelial transport
1. Introduction A variety of peptidic hormones control aspects of key physiological processes such as juvenile hormone and ecdysone biosynthesis, digestion, reproduction, metamorphosis, diuresis, etc. The question whether such peptides could be used for insect control purposes has been repeatedly raised. The fact that patents have been filed
* Corresponding author. Tel.: +32-16-324-260; fax: +32-16-323902. E-mail address: [email protected] (M. Breuer).
on such compounds indicates that one hopes that sooner or later proper protocols for economic use of peptidic compounds will be found. To exert its hormonal effect, an exogenously applied peptide should be able to reach the hemolymph compartment unaltered, either via the cuticle or, upon oral ingestion, by crossing intact the epithelium of the gut. There are already examples of modifications of insect peptides that enhance cuticular penetration while preserving biological activity (Nachman, R.J., personal communication). At present, the large-scale synthesis of such peptides is still very expensive. Especially for phytophagous insects, oral application of peptides, produced
0022-1910/01/$ - see front matter 2001 Elsevier Science Ltd. All rights reserved. PII: S 0 0 2 2 - 1 9 1 0 ( 0 1 ) 0 0 0 8 6 - 5
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by the methods of molecular biology (transgenic plants, modified insect or plant viruses, etc.) is an option. Here the requirements are that the peptide should be cleaved from its precursor protein by the digestive proteases present in the gut. Once released, it must be able to cross the gut epithelium without being degraded. Moreover, sufficient peptide must accumulate in the hemolymph to reach a biologically active concentration (De Loof, 1996). It has been intuitively assumed that only very short peptides, not longer than a few amino acids, can be actively transported. Indeed, in animals there are no systems known that can transport peptides longer than 4–5 amino acids. Some plants, however, do have such systems. These transporters have up to 11 hydrophobic transmembrane domains (Stacey, G., patent and personal communication). In theory, there is also the possibility that an epithelium is so leaky that passive passage of larger molecules might take place through the intercellular pathway. Contrary to the general belief, at least some peptides resist degradation when passing through the alimentary canal. In vertebrates, transport of orally fed hormones (e.g. the gonadotropin releasing hormone which is used to induce spawning) through the gut epithelium has been reported for the gold fish, chinook salmon and the carp (Suzuki et al., 1988; McLean et al., 1990; Hertz et al., 1991). In insects, the pheromone biosynthesis activating neuropeptide (PBAN; 33 amino acid residues) is still active after oral uptake (Raina et al., 1994). Upon feeding of TMOF of Aedes aegypti (AeaTMOF, H-Tyr-Asp-Pro-Ala-Pro-Pro-Pro-Pro-Pro-ProOH) trypsin biosynthesis and egg development is affected in the female mosquito (Borovsky and Mahmood, 1995), suggesting that the decapeptide does cross the gut walls. In this paper, we addressed the question whether the hexapeptidic peptide TMOF of the flesh fly Neobellieria bullata (Neb-TMOF, H-Asn-Pro-Thr-Asn-LeuHis-OH) fed to phytophagous pest insects is able to access the hemolymph via the gut wall and, if so, in what amounts and at what rate. The data were compared with the penetration of the macromolecule inulin and the small sugar inositol. We also report the in vitro stability of Neb-TMOF in the hemolymph of insects of different orders (Spodoptera frugiperda, Schistocerca gregaria and Leucophaea maderae) and characterize the sites of cleavage. We have chosen the TMOF peptide because it does not have internal cleavage sites for trypsin and chymotrypsin, ensuring a rather long half-life in the gut lumen (Zhu et al., 2001). Furthermore, in the flesh fly it controls important physiological processes such as trypsin and ecdysone biosynthesis (Bylemans et al., 1994; Hua et al., 1994).
2. Materials and methods 2.1. Insects L. maderae were taken from stock colonies kept at 27°C and fed oats and dry dog food ad libitum. S. gregaria were raised according to Ashby (1972). S. frugiperda were reared at 27°C on an artificial diet (Breuer and Schmidt, 1996). Animals were anaesthetized with CO2 prior to dissection. 2.2. Chemicals Neb-TMOF and its analogues were synthesized in the solid phase on the 2-chlorotrityl-chloride resin (Barlos et al., 1991) using the Fmoc/t-Bu strategy and purified by RP-HPLC as described in Zhu et al. (2001). The following peptides were synthesized: H-Asn-Pro-Thr-Asn-Leu-His-OH (Neb-TMOF), mol. wt. 695 Da. H-Asn-dehydroPro-Thr-Asn-Leu-His-OH, mol. wt. 693 Da. H-Asn-Pro-Thr-Asn-OH, mol. wt. 444 Da. H-Thr-Asn-OH, mol. wt. 233 Da. H-Leu-His-OH, mol. wt. 266 Da. The dipeptide H-Asn-Pro-OH was synthesized in the liquid phase and finally purified by semi-preparative RPHPLC to yield the desired peptide (mol. wt. 223 Da). The compound H-Asn-dehydroPro-Thr-Asn-Leu-HisOH was used as a precursor for tritiation. The reduction of the dehydro-Pro and removal of labile tritium was done by Cambridge Research Chemicals, England. The crude material was further purified by RP-HPLC. [3H]-inositol and [3H]-inulin as well as the protease inhibitors captopril and lisinopril were purchased from Sigma, USA. 2.3. In vitro incubation of Neb-TMOF The hemolymph of S. frugiperda larvae (5th instar), S. gregaria nymphs (4-day-old 5th instar) and adults (vitellogenic and previtellogenic females), and L. maderae (nymphs and adults) was collected by cutting a leg. Hemolymph from several individuals was pooled and stored on ice. The locust and cockroach hemolymph was centrifuged before further use. To study the breakdown of Neb-TMOF, [3H]-NebTMOF (20,000±2000 dpm) or cold Neb-TMOF (20– 40 µg) was incubated in 50 µl hemolymph. At different time intervals (0.5–120 min) aliquots (5 µl) were taken and immediately mixed with 95 µl of 0.1% TFA in water to stop enzyme activity. The samples were then stored on ice and further analyzed by HPLC. Experiments were repeated at least twice.
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2.4. Inhibition of proteolytic breakdown in the hemolymph Hemolymph (50 µl), collected from S. frugiperda, S. gregaria and L. maderae (see above), was pre-incubated with the protease inhibitors captopril and lisinopril (0.5– 1 mM), dissolved in Hepes buffer, for 20 min at 30°C. Reactions were initiated by adding 1 µl of [3H]-NebTMOF and the breakdown was studied as described before. Incubation of the radioactive peptide in hemolymph, diluted with an equal amount of Hepes buffer without such inhibitors, served as the control. 2.5. Intubation of Neb-TMOF, inositol and inulin Radioactive materials were introduced into the foregut of 3-day-old 5th instar nymphs of S. gregaria, which were starved for several hours. Therefore, the nymphs were dorsally fixed to a Styrofoam board by means of pins. The mandibles were opened with a tweezers and a Tygon Teflon tubing (0.5 mm dm) was gently inserted into the esophagus until reaching 1.5 cm of intubation. Aqueous sample solution (5 µl) containing [3H]-NebTMOF (20,000±2000 dpm),[3H]-inositol (20,000±2000 dpm) or [3H]-inulin (20,000±2000 dpm) was then applied through this tubing into the foregut using a Hamilton injector. Amaranth (1%) was routinely added to all intubation solutions. This dye is not absorbed from the gut lumen and allows detection of mechanical damage to the gut that would result in leakage into the hemolymph. At different time intervals after intubation (5– 120 min, exceptionally longer), hemolymph was collected in 4 µl capillaries from a small wound made near the hind-leg. The hemolymph of 5 nymphs was pooled and immediately mixed with 80 µl of 0.1% trifluoroacetic acid (TFA) in water to stop proteolytic breakdown. Hemolymph samples obtained after the intubation of [3H]-inositol and [3H]-inulin were assayed with a liquid scintillation counter (Beckman LS 9000). Samples from the peptide intubation were stored on ice and analyzed by HPLC. Experiments were repeated twice. The percentage of radiolabel appearing in the hemolymph was calculated considering a hemolymph volume of 200 µl for a 3-day-old last instar nymph (according to Lee, 1961). 2.6. HPLC analysis of hemolymph samples Hemolymph samples obtained after intubation or during incubation of labeled and unlabeled Neb-TMOF were centrifuged at 13,000 rpm for 5 min at 4°C and analyzed by RP-HPLC on a Microsorb-MV C18 column (4.6×520 mm, Varian, USA). Following a 5 min wash with 0.1% TFA in water, a linear gradient from 0 to 50% CH3CN containing 0.1% TFA in 30 min at a flow rate of 1 ml/min was used. The absorbance at 214 nm was
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monitored with an UV wavelength detector. Synthetic samples served as references. Fractions were collected at intervals of 0.5 min. For the analysis of [3H]-NebTMOF cleavage products, radioactivity of each fraction was assayed with a liquid scintillation counter (Beckman LS 9000). Fractions obtained from the incubation experiment with cold Neb-TMOF were used for the determination of the amino acid sequence of the degradation products. 2.7. Identification of the degradation products Nanoflow electrospray (ESI) Quadrupole collision (Qq) orthogonal acceleration (oa) Time of Flight (Tof) mass spectrometry was performed on a Q-Tof hybrid ESI-Tof system (Micromass, UK) (Mozzis et al., 1996). The HPLC fractions containing the peptides were dried and redissolved in 5 µl of acetonitrile/water/formic acid (50:49:1). Each fraction (1 µl) was loaded in a gold coated capillary (Micromass type F nanoflow needle). The samples were sprayed at a flow rate of typically 30 nl/min giving extended analysis time in which an MS spectrum as well as several MS/MS spectra were obtained. Needle voltage was set at 900 V, the cone voltage was 25 V. During MS/MS or tandem mass spectrometry fragment ions are generated from a selected precursor ion by collision-induced dissociation (CID). Argon was used as the collision gas. Since not all peptide ions fragment with the same efficiency, the collision energy is typically varied between 20 and 35 eV so that the parent ion is fragmented in a satisfying number of different daughter ions.
3. Results 3.1. In vitro degradation of Neb-TMOF in hemolymph [3H]-Neb-TMOF was incubated in hemolymph of 5th instar larvae of S. frugiperda. Samples, taken at different time intervals and analyzed by HPLC, showed that the amount of intact peptide decreased rapidly (Fig. 1). The half-life of Neb-TMOF was estimated to be 苲3 min. There was a corresponding increase of a [3H]-labeled product with a retention time of 14 min. This fragment called P1 reached high values after about 5 min of incubation. The amount of P1 decreased during further incubation, whereas that of another degradation product, which was called P2 and eluted at about 12 min, increased. After 1 h of incubation the amount of P2 averaged 53% of the radioactivity applied. The rest of the [3H]-labeled material was found back in the fractions that eluted much earlier in the “wash” (with water containing 0.1% TFA). Neb-TMOF was slowly degraded in the hemolymph of 5th instar nymphs of S. gregaria (Fig. 2). The esti-
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Fig. 1. Degradation of [3H]-Neb-TMOF in the hemolymph of 5th instar larvae of S. frugiperda. Hemolymph samples were separated by C18 RP-HPLC and fractions analyzed by liquid scintillation counting. Besides the intact peptide, the appearance of the degradation products P1 and P2 as well as radioactivity measured in the “wash” of the HPLC run (further degradation products with shorter retention time) is depicted.
Fig. 3. Degradation of [3H]-Neb-TMOF in the hemolymph of adult L. maderae. Hemolymph samples were separated by C18 RP-HPLC and fractions analyzed by liquid scintillation counting. Besides the intact peptide, the appearance of the degradation products P1 and P2 as well as radioactivity measured in the “wash” of the HPLC run (further degradation products with shorter retention time) is depicted.
ured on an ESI-Qq-oa-Tof mass spectrometer to be 445.17 and 229.97 Da. Fragmentation of the ions by CID on the same system identified the sequences as Asn-ProThr-Asn and Asn-Pro. When non-labeled Neb-TMOF was incubated in hemolymph of the different insect species, another fragment with a mass of 269.18 Da was obtained, which was identified as Leu-His. The retention times found during HPLC analysis were the same as those obtained with the synthetic Neb-TMOF peptide fragments. 3.2. Inhibition of proteolytic breakdown in the hemolymph by ACE inhibitors The hydrolysis of Neb-TMOF in the hemolymph of S. gregaria could be inhibited by 0.5 mM captopril (Fig. 4). This result indicates that an angiotensin converting
Fig. 2. Degradation of [3H]-Neb-TMOF in the hemolymph of 4-dayold 5th instar nymphs of S. gregaria. Hemolymph samples were separated by C18 RP-HPLC and fractions analyzed by liquid scintillation counting. Besides the intact peptide, the appearance of the degradation product P1 as well as radioactivity measured in the “wash” of the HPLC run (further degradation products with shorter retention time) is depicted.
mated half-life was 苲25 min. Even after 2 h, more than 10% of the intact peptide could still be found in the incubation mixture. An increasing amount of the fragment P1 could be again detected. P2 was only recorded in traces. Breakdown was similar in hemolymph of adult locusts (data not shown). Neb-TMOF was quickly cleaved in the hemolymph of both larval and adult L. maderae (Fig. 3). The halflife was similar to that found in S. frugiperda hemolymph. HPLC analysis also revealed the fragments P1 and P2. The masses of the fragments P1 and P2 were meas-
Fig. 4. Mean percentage of [3H]-Neb-TMOF found after incubation with hemolymph of 4-day-old 5th instar nymphs of S. gregaria in the presence of captopril (±SD). The hemolymph was analyzed by C18 RP-HPLC and liquid scintillation counting. *Indicates statistical significance to the control according to t-test (∗P⬍0.05, ∗∗P⬍0.01).
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enzyme (ACE)-like peptidase, of which captopril is a specific inhibitor, is present in the hemolymph of this orthopteran species. This inhibitor was also slightly inhibiting the degradation in the hemolymph of S. frugiperda (Fig. 5), but the effect was less in comparison to the situation in S. gregaria. However, breakdown of Neb-TMOF could not be inhibited by captopril nor by the potent ACE inhibitor lisinopril in the hemolymph of L. maderae, even with concentrations up to 1 mM (data not shown). 3.3. Permeability of the gut epithelium to inulin and inositol [3H]-inulin is not metabolized in the gut and is too large to cross the gut cells. Therefore, it is a good marker for a paracellular transport of larger molecules. Following application by intubation, very low levels of radioactivity appeared in the hemolymph and averaged maximally 0.01% of the total intubated inulin (data not shown). In order to have an idea about the dynamics of a small molecule, [3H]-inositol was applied by intubation. Already after 5 min a considerable amount of radiolabel could be registered in the hemolymph samples (Fig. 6). The highest radioactivity (on av. 20% of total intubated material) appeared in the hemolymph samples 1 and 2 h after intubation. The radioactivity gradually decreased afterwards, but remained rather high even after 12 h.
Fig. 6. Percentage of total radioactivity in the hemolymph of 4-dayold 5th instar nymphs of S. gregaria (±SD) found after intubation of [3H]-inositol.
3.4. Transepithelial transport of Neb-TMOF The transepithelial transport of Neb-TMOF was as well studied after intubation. HPLC analysis of the hemolymph, taken at different time intervals showed that already after 5 min on av. 1% of intact [3H]-Neb-TMOF could be detected in the hemolymph (Fig. 7). The highest amount of [3H]-peptide (5.0±2.3%) was measured
Fig. 7. Percentage of total radioactivity in the hemolymph of 4-dayold 5th instar nymphs of S. gregaria (±SD) found after intubation of [3H]-Neb-TMOF. The hemolymph was analyzed by C18 RP-HPLC and liquid scintillation counting.
30 min after intubation. Afterwards, the amount of intact Neb-TMOF decreased gradually. In contrast, the total amount of radioactivity in the hemolymph increased and reached on av. 9.1% of the total dpm intubated initially. But even 2 h after intubation, intact Neb-TMOF could still be detected in the hemolymph.
4. Discussion
Fig. 5. Mean percentage of [3H]-Neb-TMOF found after incubation with hemolymph 5th instar larvae of S. frugiperda in the presence of captopril (± SD). The hemolymph was analyzed by C18 RP-HPLC and liquid scintillation counting. *Indicates statistical significance to the control according to t-test (∗P⬍0.05, ∗∗P⬍0.01).
Although the amount of proteolytic activity varied in the hemolymph of the different insects tested, the pattern of peptide degradation was identical. Since only the proline was labeled, two major proteolytic metabolites could be seen; they were identified as Neb-TMOF-(1-4) and (1-2). Counterparts (-5-6, -3-4) could be detected after use of sufficient amounts of non-labeled peptide
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material. These results suggest that the initial proteolytic sites are at the Asn4-Leu5 and Pro2-Thr3 bonds. Apparently, Neb-TMOF is cleaved into dipeptides by a dipeptidyl-peptidase present in the hemolymph of these insects. This has already been demonstrated in earlier studies in the flesh fly N. bullata, from which NebTMOF was formerly isolated (Zhu et al., 2001). Breakdown was significantly inhibited if captopril or lisinopril was added to the flesh fly hemolymph, indicating that an ACE-like protease, of which captopril and lisinopril are specific inhibitors, was present. The present study showed also in S. gregaria and S. frugiperda hemolymph, that Neb-TMOF is cleaved in C-terminal dipeptides, and proteolysis could be at least partially inhibited by captopril. Also other investigations demonstrated the presence of ACE-like enzymes in different insect species, in particular flies (Lamango et al., 1996; Wijffels et al. 1996, 1997; Schoofs et al., 1998; Ekbote et al., 1999). These findings suggest that ACE-like enzymes are more common in insects and are involved in the regulation (hydrolysis) of signalling peptides. Insect ACE is even able to hydrolyze C-terminally amidated peptides (Lamango et al., 1997). Nevertheless, captopril or lisinopril were not capable of inhibiting the degradation of Neb-TMOF in the hemolymph of L. maderae, even though the fragments isolated from the incubation mixture were identical. Obviously, the enzyme differs from that in the hemolymph of the other insects tested. Since the degradation product NebTMOF-(1-2) (P2) could be detected in sufficient amounts right from the beginning of the incubation, Neb-TMOF might be cleaved in this case from the Nterminus as described by Martensen et al. (1998). The authors isolated a proline-specific dipeptidyl-peptidase from homogenized and soluble membrane fractions of the blow fly Calliphora vicina, releasing the fragment Asn-Pro from the N-termini. Furthermore, ACE inhibitors could not inhibit this reaction. On the other hand, ACE immunoreactivity has been localized in the neuropile regions and neurosecretory cells of the CNS of L. maderae (Schoofs et al., 1998), indicating that there is, at least, some neuronal ACE-like activity. In N. bullata, hemolymph enzymatic breakdown of Neb-TMOF was rather quick, and the half-life was less than half a minute (Zhu et al., 2001). In the hemolymph of S. frugiperda and L. maderae the half-life was found to be slightly longer (苲3 min). Neb-TMOF was more resistant to proteolysis in the hemolymph of S. gregaria. It took about 25 min until the level of intact peptide dropped to 50%. These data are comparable to the halflives of other peptides found in the hemolymph of different insects (e.g. Oudejans et al., 1996; Garside et al., 1997). Due to these findings, S. gregaria was most suitable to serve as a model insect for the in vivo study of the penetration of Neb-TMOF through the gut epithelium.
The results demonstrate that the intact peptide can cross the midgut epithelium into the hemolymph of the fifth instar nymphs. This indicates that, in the conditions used, this peptide is not quickly degraded by gut enzymes and has the possibility to pass the gut walls. This is not totally unexpected since the sequence (HAsn-Pro-Thr-Asn-Leu-His-OH) does not contain any of the typical cleavage sites for the known trypsins and chymotrypsins. In gut luminal content of previtellogenic flesh flies, the hexapeptide was also quite stable (Zhu et al., 2001). Even though in vitro experiments had demonstrated that the peptide is quickly degraded in the hemolymph of flesh flies, it has recently been shown in our laboratory that orally administered Neb-TMOF still exerts its action and trminates trypsin biosynthesis in previtellogenic flies (Janssen, I., personal communication). These are promising results from the perspective of using such peptides in insect control. Borovsky and Mahmood (1995) reported that 28% of orally ingested Aea-TMOF could be traced back in the mosquito hemolymph. In our study, at most 5% of the initial Neb-TMOF was found in the locust hemolymph 30 min after intubation. Since we have studied this hormone using HPLC, the amount that we report here represents intact Neb-TMOF and not degradation products, which appear in the hemolymph (see above). Considering all fragments, maximally 9% of the total amount of TMOF that was administered was registered in the hemolymph. Three other peptides tested in our experiments have a lower transport rate as compared to Neb-TMOF (Zhu, W., unpublished results). It indicates that NebTMOF is either more stable in the locust gut than other peptides and/or that it is more easily transported. The rate of transport at which the peptide moves from the luminal to the haemocoel side of the gut was quite high. Within 5 min after intubation, already more than 1% of the intubated radiolabel could be recorded in the hemolymph. For free amino acids or peptide fragments it is reported that absorption occurs very rapidly in the anterior section of the midgut (Woods and Kingsolver, 1999), but absorptive sites are distributed throughout the midgut (Wolfersberger, 1996). In S. gregaria the absorption seems likewise to be dependant on a diffusion gradient across the midgut walls, created perhaps by the relative rapid movement of water into the hemolymph (Treherne, 1959). Not much is known about the mode of absorption of peptides longer than two amino acids. There are two major transport pathways across epithelia: a transcellular pathway through cells and a paracellular pathway between cells. Cell membranes are impermeable to inulin, leaving the paracellular pathway as the only route for their transepithelial permeation (Wang et al., 1996). Our experiments showed that only a small but measurable amount of [3H]-inulin (0.01% of total intubated radioactivity) could be registered in the hemolymph after
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intubation. This indicates that the intercellular junctions between the epithelial cells are tight but still allow passive penetration of macro-molecules as large as inulin (mol. wt. approx. ⬎5000). These findings suggest that the paracellular pathway might be (to a higher degree) also permeable to smaller molecules, such as peptides. Considering the results of the experiments with inositol (mol. wt. 180) and Neb-TMOF (mol. wt. 695), there is indeed an obvious correlation (r2=0.98) between the molecular weight and the percentage of radiolabeled material appearing in the hemolymph. We are, of course, aware of the fact that the way of transport of these molecules could be different. Further investigations are needed to find out whether the molecules we studied are actively or passively transported. In S. gregaria even the uptake of sugars seems to be passive (Treherne, 1958a,b). On the other hand, research on the mode of absorption of free inositol in segments of hamster small intestine has revealed that inositol is actively transported (Caspary and Crane, 1970). For numerous lepidopteran species a carriermediated transport of amino acids has been described (for further details see Sacchi and Wolfersberger, 1996).
Acknowledgements This project was undertaken as part of an EC-funded project, FAIR3-CT96-1648. Mass spectrometry was supported by the F.W.O (G. 0356.98).
References Ashby, G.J., 1972. Locusts. In: UFAW (Ed.), The UFAW Handbook of the Care and Management of Laboratory Animals. Livingstone, London, pp. 582–587. Barlos, K., Chatzi, O., Gatos, D., Stavropoulos, G., 1991. 2-Chlorotrityl chloride resin. Studies on anchoring of Fmoc-amino acids and peptide cleavage. Intenational Journal of Peptide and Protein Research 37, 513–520. Borovsky, D., Mahmood, F., 1995. Feeding the mosquito Aedes aegypti with TMOF and its analogs; effect on trypsin biosynthesis and egg development. Regulatory Peptides 57, 273–281. Breuer, M., Schmidt, G.H., 1996. Effect of Melia azedarach extract incorporated into an artificial diet on growth, development and fecundity of Spodoptera frugiperda (J.E. Smith) (Lep., Noctuidae). Journal of Plant Diseases and Protection 103, 171–194. Bylemans, D., Borovsky, D., Hunt, D.F., Shabanowitz, J., Grauwels, L., De Loof, A., 1994. Sequencing and characterisation of trypsin modulating oostatic factor (TMOF) from the ovaries of the grey flesh fly, Nebobellieria bullata. Regulatory Peptides 50, 61–72. Caspary, W.F., Crane, R.K., 1970. Active transport of myo-inositol and its relation to the sugar transport system in hamster small intestine. Biochimica Biophysica Acta 203, 308–316. De Loof, A., 1996. Endogenous insect regulatory peptides: a novel lead towards insect control? Agro-food-Industry 7 (6), 23–25. Ekbote, U., Coates, D., Isaac, R.E., 1999. A mosquito (Anopheles stephensi) angiotensin I-converting enzyme (ACE) is induced by a
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blood meal and accumulates in the developing ovary. FEBS Letters 455 (3), 219–222. Garside, C.S., Hayes, T.K., Tobe, S.S., 1997. Degradation of Dip-allatostatins by hemolymph from the cockroach, Diploptera punctata. Peptides 18, 17–25. Hertz, Y., Tchelet, A., Madar, Z., Gertler, A., 1991. Absorption of bioactive human growth hormone after oral administration in the common carp (Cyprinus carpio) and its enhancement by deoxycholbeote. Journal of Comparative Physiology B 161, 159–163. Hua, Y.J., Bylemans, D., De Loof, A., Koolman, J., 1994. Ecdysone biosynthesis in flies is inhibited by a hexapeptide. Molecular and Cellular Endocrinology 104, R1–R4. Lamango, N.S., Nachman, R.J., Hayes, T.K., Strey, A., Isaac, R.E., 1997. Hydrolysis of insect neuropeptides by an angiotensin-converting enzyme from the housefly, Musca domestica. Peptides 18, 47–52. Lamango, N.S., Sajid, M., Isaac, R.E., 1996. Purification and properties of an angiotensin-converting enzyme from the housefly, Musca domestica. Biochemical Journal 314, 639–646. Lee, R.M., 1961. The variation of blood volume with age in the desert locust (Schistocerca gregaria Forsk.). Journal of Insect Physiology 6, 36–51. Martensen, I., Koolman, J., Mentlein, R., 1998. Proline-specific dipeptidyl peptidase from the blue blowfly Calliphora vicina hydrolyzes in vitro the ecdysiostatic peptride Trypsin-Modulating Oostatic Factor (Neb-TMOF). Archives of Insect Biochemistry and Physiology 37, 146–157. McLean, E., Vonder Meden, A.C., Donaldson, E.M., 1990. Direct and indirect evidence for polypeptide absorption by the teleost gastrointestinal tract. Journal of Fish Biology 36, 489–498. Mozzis, H.R., Paxton, T., Dell, A., Langhavre, J., Bag, M., Bardolic, R.S., Hoger, J., Bateman, R.H., 1996. High sensitivity collisionally activated decomposition tandem mass spectrometry on a novel quadropole/orthogonal-acceleration time-of-flight mass spectrometer. Rapid communications in Mass Spectrometry 10, 889– 896. Oudejans, R.C., Vroemen, S.F., Jansen, R.F., Van der Horst, D.J., 1996. Locust adipokinetic hormones: carrier-independent transport and differential inactivation at physiological concentrations during rest and flight. Proceedings of the National Academy of Sciences of the United States of America 93 (16), 8654–8659. Sacchi, V.F, Wolfersberger, M.G., 1996. Amino acid absorption. In: Lehane, M.J., Billingsley, P.F. (Eds.), Biology of the Insect Midgut. Chapman and Hall, London, pp. 265-292. Raina, A.K., Rafaeli, A., Kingan, T.G., 1994. Pheromonotropic activity of orally administered PBAN and its analogues in Helicoverpa zea. Journal of Insect Physiology 40, 393–397. Schoofs, L., Veelaert, D., De Loof, A., Huybrechts, R., Isaac, E., 1998. Immunocytochemical distribution of angiotensin I-converting enzyme-like immunoreactivity in the brain and testis of insects. Brain Research 785, 215–227. Suzuki, Y., Kobayashi, M., Aida, K., Hanyu, I., 1988. Transport of physiologically active salmon gonadotropin into the circulation in goldfish, following oral administration of salmon pituitary extract. Journal of Comparative Physiology B 157, 753–758. Treherne, J.E., 1958a. The absorption of glucose from the alimentary canal of the locust Schistocerca gregaria (Forsk.). Journal of Experimental Biology 35, 297–303. Treherne, J.E., 1958b. The absorption and metabolism of some sugars in the locust Schistocerca gregaria (Forsk.). Journal of Experimental Biology 35, 611–625. Treherne, J.E., 1959. Amino acid absorption in the locust (Schistocerca gregaria Forsk.). Journal of Experimental Biology 36, 533–545. Wang, S., Rubenfeld, A.B., Hayes, T.K., Beyenbach, K.W., 1996. Leucokinin increases paracellular permeability in insect malpighian tubules. Journal of Experimental Biology 199, 2537–2542. Wijffels, G., Fitzgerald, C., Gough, J., Riding, G., Elvin, C., Kemp, D.,
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Willadsen, P., 1996. Cloning and characterisation of angiotensinconverting enzyme from the dipteran species, Haematobia irritans exigua, and its expression in the maturing male reproductive system. European Journal of Biochemistry 237, 414–423. Wijffels, G., Gough, J., Muharsini, S., Donaldson, A., Eisemann, C., 1997. Expression of angiotensin-converting enzyme-related carboxydipeptidases in the larvae of four species of fly. Insect Biochemistry and Molecular Biology 27, 451–460. Wolfersberger, M.G., 1996. Localization of amino acid absorption systems in the larval midgut of tobacco hornworm Manduca sexta. Journal of Insect Physiology 42, 975–982.
Woods, H.A., Kingsolver, J.G., 1999. Feeding rate and the structure of protein digestion and absorption in lepidopteran midguts. Archives of Insect Biochemistry and Physiology 42, 74–87. Zhu, W., Vandingenen, A., Huybrechts, R., Baggerman, G., De Loof, A., Poulos, C.P., Velentza, A., Breuer, M., 2001. In vitro degradation of the Neb-Trypsin Modulating Oostatic Factor (NebTMOF) in gut luminal content and hemolymph of the grey flesh fly, Neobielleria bullata. Insect Biochemistry and Molecular Biology 31, 87–95.
Proteolytic breakdown of the Neb-trypsin modulating oostatic factor (Neb-TMOF) in the hemolymph of different insects and its gut epithelial transport W. Zhu a, A. Vandingenen a, R. Huybrechts a, T. Vercammen a, G. Baggerman a, A. De Loof a, C.P. Poulos b, A. Velentza b, M. Breuer a,* a
Zoological Institute, Katholieke Universiteit Leuven, Naamesestraat 59, B-3000 Leuven, Belgium b Department of Chemistry, University of Patras, GR-26500 Patras, Greece Received 15 November 2000; accepted 13 February 2001
Abstract The degradation of the unblocked hexapeptide, trypsin modulating oostatic factor of the flesh fly Neobellieria (Sarcophaga) bullata (Neb-TMOF) was studied in vitro in the hemolymph of the lepidopteran Spodoptera frugiperda, the orthopteran Schistocerca gregaria and the dictyopteran Leucophaea maderae. The half-life in the different species varied from 苲3 min in L. maderae to 苲25 min in S. gregaria. Purification of the degradation products and ESI-Qq-oa-Tof mass spectrometry revealed the fragments AsnPro-Thr-Asn, Leu-His and Asn-Pro, which were the same in the hemolymph of all species. Except in Leucophaea, Neb-TMOF was cleaved in dipeptides starting from the C-terminus and the reaction could be, at least partially, inhibited by captopril. These observations suggest that a dipeptidase, which has very similar enzymatic properties as mammalian angiotensin converting enzyme (ACE) and which circulates in the hemolymph, apparently is involved in the breakdown of Neb-TMOF and might be a common but not a universal enzyme in insect hemolymph. The introduction of Neb-TMOF into the gut of S. gregaria with the help of a capillary tube (intubation) demonstrated that the intact peptide is able to cross the gut epithelium and to appear in the hemolymph compartment. Since [3H]-inulin, which is too large to cross cell membranes, was found to penetrate the gut walls at a measurable rate, the paracellular pathway might be also permeable to smaller peptides. There was indeed a clear correlation between the molecular weight of inulin, NebTMOF, and inositol and the rate of penetration of these compounds through the gut epithelium to the hemolymph. These are promising findings in view of a potential use of such peptides for insect control purposes. 2001 Elsevier Science Ltd. All rights reserved. Keywords: Neb-TMOF; Spodoptera frugiperda; Schistocerca gregaria; Leucophaea maderae; Hemolymph; Proteolytic breakdown; Angiotensin converting enzyme; Midgut; Epithelial transport
1. Introduction A variety of peptidic hormones control aspects of key physiological processes such as juvenile hormone and ecdysone biosynthesis, digestion, reproduction, metamorphosis, diuresis, etc. The question whether such peptides could be used for insect control purposes has been repeatedly raised. The fact that patents have been filed
* Corresponding author. Tel.: +32-16-324-260; fax: +32-16-323902. E-mail address: [email protected] (M. Breuer).
on such compounds indicates that one hopes that sooner or later proper protocols for economic use of peptidic compounds will be found. To exert its hormonal effect, an exogenously applied peptide should be able to reach the hemolymph compartment unaltered, either via the cuticle or, upon oral ingestion, by crossing intact the epithelium of the gut. There are already examples of modifications of insect peptides that enhance cuticular penetration while preserving biological activity (Nachman, R.J., personal communication). At present, the large-scale synthesis of such peptides is still very expensive. Especially for phytophagous insects, oral application of peptides, produced
0022-1910/01/$ - see front matter 2001 Elsevier Science Ltd. All rights reserved. PII: S 0 0 2 2 - 1 9 1 0 ( 0 1 ) 0 0 0 8 6 - 5
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by the methods of molecular biology (transgenic plants, modified insect or plant viruses, etc.) is an option. Here the requirements are that the peptide should be cleaved from its precursor protein by the digestive proteases present in the gut. Once released, it must be able to cross the gut epithelium without being degraded. Moreover, sufficient peptide must accumulate in the hemolymph to reach a biologically active concentration (De Loof, 1996). It has been intuitively assumed that only very short peptides, not longer than a few amino acids, can be actively transported. Indeed, in animals there are no systems known that can transport peptides longer than 4–5 amino acids. Some plants, however, do have such systems. These transporters have up to 11 hydrophobic transmembrane domains (Stacey, G., patent and personal communication). In theory, there is also the possibility that an epithelium is so leaky that passive passage of larger molecules might take place through the intercellular pathway. Contrary to the general belief, at least some peptides resist degradation when passing through the alimentary canal. In vertebrates, transport of orally fed hormones (e.g. the gonadotropin releasing hormone which is used to induce spawning) through the gut epithelium has been reported for the gold fish, chinook salmon and the carp (Suzuki et al., 1988; McLean et al., 1990; Hertz et al., 1991). In insects, the pheromone biosynthesis activating neuropeptide (PBAN; 33 amino acid residues) is still active after oral uptake (Raina et al., 1994). Upon feeding of TMOF of Aedes aegypti (AeaTMOF, H-Tyr-Asp-Pro-Ala-Pro-Pro-Pro-Pro-Pro-ProOH) trypsin biosynthesis and egg development is affected in the female mosquito (Borovsky and Mahmood, 1995), suggesting that the decapeptide does cross the gut walls. In this paper, we addressed the question whether the hexapeptidic peptide TMOF of the flesh fly Neobellieria bullata (Neb-TMOF, H-Asn-Pro-Thr-Asn-LeuHis-OH) fed to phytophagous pest insects is able to access the hemolymph via the gut wall and, if so, in what amounts and at what rate. The data were compared with the penetration of the macromolecule inulin and the small sugar inositol. We also report the in vitro stability of Neb-TMOF in the hemolymph of insects of different orders (Spodoptera frugiperda, Schistocerca gregaria and Leucophaea maderae) and characterize the sites of cleavage. We have chosen the TMOF peptide because it does not have internal cleavage sites for trypsin and chymotrypsin, ensuring a rather long half-life in the gut lumen (Zhu et al., 2001). Furthermore, in the flesh fly it controls important physiological processes such as trypsin and ecdysone biosynthesis (Bylemans et al., 1994; Hua et al., 1994).
2. Materials and methods 2.1. Insects L. maderae were taken from stock colonies kept at 27°C and fed oats and dry dog food ad libitum. S. gregaria were raised according to Ashby (1972). S. frugiperda were reared at 27°C on an artificial diet (Breuer and Schmidt, 1996). Animals were anaesthetized with CO2 prior to dissection. 2.2. Chemicals Neb-TMOF and its analogues were synthesized in the solid phase on the 2-chlorotrityl-chloride resin (Barlos et al., 1991) using the Fmoc/t-Bu strategy and purified by RP-HPLC as described in Zhu et al. (2001). The following peptides were synthesized: H-Asn-Pro-Thr-Asn-Leu-His-OH (Neb-TMOF), mol. wt. 695 Da. H-Asn-dehydroPro-Thr-Asn-Leu-His-OH, mol. wt. 693 Da. H-Asn-Pro-Thr-Asn-OH, mol. wt. 444 Da. H-Thr-Asn-OH, mol. wt. 233 Da. H-Leu-His-OH, mol. wt. 266 Da. The dipeptide H-Asn-Pro-OH was synthesized in the liquid phase and finally purified by semi-preparative RPHPLC to yield the desired peptide (mol. wt. 223 Da). The compound H-Asn-dehydroPro-Thr-Asn-Leu-HisOH was used as a precursor for tritiation. The reduction of the dehydro-Pro and removal of labile tritium was done by Cambridge Research Chemicals, England. The crude material was further purified by RP-HPLC. [3H]-inositol and [3H]-inulin as well as the protease inhibitors captopril and lisinopril were purchased from Sigma, USA. 2.3. In vitro incubation of Neb-TMOF The hemolymph of S. frugiperda larvae (5th instar), S. gregaria nymphs (4-day-old 5th instar) and adults (vitellogenic and previtellogenic females), and L. maderae (nymphs and adults) was collected by cutting a leg. Hemolymph from several individuals was pooled and stored on ice. The locust and cockroach hemolymph was centrifuged before further use. To study the breakdown of Neb-TMOF, [3H]-NebTMOF (20,000±2000 dpm) or cold Neb-TMOF (20– 40 µg) was incubated in 50 µl hemolymph. At different time intervals (0.5–120 min) aliquots (5 µl) were taken and immediately mixed with 95 µl of 0.1% TFA in water to stop enzyme activity. The samples were then stored on ice and further analyzed by HPLC. Experiments were repeated at least twice.
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2.4. Inhibition of proteolytic breakdown in the hemolymph Hemolymph (50 µl), collected from S. frugiperda, S. gregaria and L. maderae (see above), was pre-incubated with the protease inhibitors captopril and lisinopril (0.5– 1 mM), dissolved in Hepes buffer, for 20 min at 30°C. Reactions were initiated by adding 1 µl of [3H]-NebTMOF and the breakdown was studied as described before. Incubation of the radioactive peptide in hemolymph, diluted with an equal amount of Hepes buffer without such inhibitors, served as the control. 2.5. Intubation of Neb-TMOF, inositol and inulin Radioactive materials were introduced into the foregut of 3-day-old 5th instar nymphs of S. gregaria, which were starved for several hours. Therefore, the nymphs were dorsally fixed to a Styrofoam board by means of pins. The mandibles were opened with a tweezers and a Tygon Teflon tubing (0.5 mm dm) was gently inserted into the esophagus until reaching 1.5 cm of intubation. Aqueous sample solution (5 µl) containing [3H]-NebTMOF (20,000±2000 dpm),[3H]-inositol (20,000±2000 dpm) or [3H]-inulin (20,000±2000 dpm) was then applied through this tubing into the foregut using a Hamilton injector. Amaranth (1%) was routinely added to all intubation solutions. This dye is not absorbed from the gut lumen and allows detection of mechanical damage to the gut that would result in leakage into the hemolymph. At different time intervals after intubation (5– 120 min, exceptionally longer), hemolymph was collected in 4 µl capillaries from a small wound made near the hind-leg. The hemolymph of 5 nymphs was pooled and immediately mixed with 80 µl of 0.1% trifluoroacetic acid (TFA) in water to stop proteolytic breakdown. Hemolymph samples obtained after the intubation of [3H]-inositol and [3H]-inulin were assayed with a liquid scintillation counter (Beckman LS 9000). Samples from the peptide intubation were stored on ice and analyzed by HPLC. Experiments were repeated twice. The percentage of radiolabel appearing in the hemolymph was calculated considering a hemolymph volume of 200 µl for a 3-day-old last instar nymph (according to Lee, 1961). 2.6. HPLC analysis of hemolymph samples Hemolymph samples obtained after intubation or during incubation of labeled and unlabeled Neb-TMOF were centrifuged at 13,000 rpm for 5 min at 4°C and analyzed by RP-HPLC on a Microsorb-MV C18 column (4.6×520 mm, Varian, USA). Following a 5 min wash with 0.1% TFA in water, a linear gradient from 0 to 50% CH3CN containing 0.1% TFA in 30 min at a flow rate of 1 ml/min was used. The absorbance at 214 nm was
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monitored with an UV wavelength detector. Synthetic samples served as references. Fractions were collected at intervals of 0.5 min. For the analysis of [3H]-NebTMOF cleavage products, radioactivity of each fraction was assayed with a liquid scintillation counter (Beckman LS 9000). Fractions obtained from the incubation experiment with cold Neb-TMOF were used for the determination of the amino acid sequence of the degradation products. 2.7. Identification of the degradation products Nanoflow electrospray (ESI) Quadrupole collision (Qq) orthogonal acceleration (oa) Time of Flight (Tof) mass spectrometry was performed on a Q-Tof hybrid ESI-Tof system (Micromass, UK) (Mozzis et al., 1996). The HPLC fractions containing the peptides were dried and redissolved in 5 µl of acetonitrile/water/formic acid (50:49:1). Each fraction (1 µl) was loaded in a gold coated capillary (Micromass type F nanoflow needle). The samples were sprayed at a flow rate of typically 30 nl/min giving extended analysis time in which an MS spectrum as well as several MS/MS spectra were obtained. Needle voltage was set at 900 V, the cone voltage was 25 V. During MS/MS or tandem mass spectrometry fragment ions are generated from a selected precursor ion by collision-induced dissociation (CID). Argon was used as the collision gas. Since not all peptide ions fragment with the same efficiency, the collision energy is typically varied between 20 and 35 eV so that the parent ion is fragmented in a satisfying number of different daughter ions.
3. Results 3.1. In vitro degradation of Neb-TMOF in hemolymph [3H]-Neb-TMOF was incubated in hemolymph of 5th instar larvae of S. frugiperda. Samples, taken at different time intervals and analyzed by HPLC, showed that the amount of intact peptide decreased rapidly (Fig. 1). The half-life of Neb-TMOF was estimated to be 苲3 min. There was a corresponding increase of a [3H]-labeled product with a retention time of 14 min. This fragment called P1 reached high values after about 5 min of incubation. The amount of P1 decreased during further incubation, whereas that of another degradation product, which was called P2 and eluted at about 12 min, increased. After 1 h of incubation the amount of P2 averaged 53% of the radioactivity applied. The rest of the [3H]-labeled material was found back in the fractions that eluted much earlier in the “wash” (with water containing 0.1% TFA). Neb-TMOF was slowly degraded in the hemolymph of 5th instar nymphs of S. gregaria (Fig. 2). The esti-
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Fig. 1. Degradation of [3H]-Neb-TMOF in the hemolymph of 5th instar larvae of S. frugiperda. Hemolymph samples were separated by C18 RP-HPLC and fractions analyzed by liquid scintillation counting. Besides the intact peptide, the appearance of the degradation products P1 and P2 as well as radioactivity measured in the “wash” of the HPLC run (further degradation products with shorter retention time) is depicted.
Fig. 3. Degradation of [3H]-Neb-TMOF in the hemolymph of adult L. maderae. Hemolymph samples were separated by C18 RP-HPLC and fractions analyzed by liquid scintillation counting. Besides the intact peptide, the appearance of the degradation products P1 and P2 as well as radioactivity measured in the “wash” of the HPLC run (further degradation products with shorter retention time) is depicted.
ured on an ESI-Qq-oa-Tof mass spectrometer to be 445.17 and 229.97 Da. Fragmentation of the ions by CID on the same system identified the sequences as Asn-ProThr-Asn and Asn-Pro. When non-labeled Neb-TMOF was incubated in hemolymph of the different insect species, another fragment with a mass of 269.18 Da was obtained, which was identified as Leu-His. The retention times found during HPLC analysis were the same as those obtained with the synthetic Neb-TMOF peptide fragments. 3.2. Inhibition of proteolytic breakdown in the hemolymph by ACE inhibitors The hydrolysis of Neb-TMOF in the hemolymph of S. gregaria could be inhibited by 0.5 mM captopril (Fig. 4). This result indicates that an angiotensin converting
Fig. 2. Degradation of [3H]-Neb-TMOF in the hemolymph of 4-dayold 5th instar nymphs of S. gregaria. Hemolymph samples were separated by C18 RP-HPLC and fractions analyzed by liquid scintillation counting. Besides the intact peptide, the appearance of the degradation product P1 as well as radioactivity measured in the “wash” of the HPLC run (further degradation products with shorter retention time) is depicted.
mated half-life was 苲25 min. Even after 2 h, more than 10% of the intact peptide could still be found in the incubation mixture. An increasing amount of the fragment P1 could be again detected. P2 was only recorded in traces. Breakdown was similar in hemolymph of adult locusts (data not shown). Neb-TMOF was quickly cleaved in the hemolymph of both larval and adult L. maderae (Fig. 3). The halflife was similar to that found in S. frugiperda hemolymph. HPLC analysis also revealed the fragments P1 and P2. The masses of the fragments P1 and P2 were meas-
Fig. 4. Mean percentage of [3H]-Neb-TMOF found after incubation with hemolymph of 4-day-old 5th instar nymphs of S. gregaria in the presence of captopril (±SD). The hemolymph was analyzed by C18 RP-HPLC and liquid scintillation counting. *Indicates statistical significance to the control according to t-test (∗P⬍0.05, ∗∗P⬍0.01).
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enzyme (ACE)-like peptidase, of which captopril is a specific inhibitor, is present in the hemolymph of this orthopteran species. This inhibitor was also slightly inhibiting the degradation in the hemolymph of S. frugiperda (Fig. 5), but the effect was less in comparison to the situation in S. gregaria. However, breakdown of Neb-TMOF could not be inhibited by captopril nor by the potent ACE inhibitor lisinopril in the hemolymph of L. maderae, even with concentrations up to 1 mM (data not shown). 3.3. Permeability of the gut epithelium to inulin and inositol [3H]-inulin is not metabolized in the gut and is too large to cross the gut cells. Therefore, it is a good marker for a paracellular transport of larger molecules. Following application by intubation, very low levels of radioactivity appeared in the hemolymph and averaged maximally 0.01% of the total intubated inulin (data not shown). In order to have an idea about the dynamics of a small molecule, [3H]-inositol was applied by intubation. Already after 5 min a considerable amount of radiolabel could be registered in the hemolymph samples (Fig. 6). The highest radioactivity (on av. 20% of total intubated material) appeared in the hemolymph samples 1 and 2 h after intubation. The radioactivity gradually decreased afterwards, but remained rather high even after 12 h.
Fig. 6. Percentage of total radioactivity in the hemolymph of 4-dayold 5th instar nymphs of S. gregaria (±SD) found after intubation of [3H]-inositol.
3.4. Transepithelial transport of Neb-TMOF The transepithelial transport of Neb-TMOF was as well studied after intubation. HPLC analysis of the hemolymph, taken at different time intervals showed that already after 5 min on av. 1% of intact [3H]-Neb-TMOF could be detected in the hemolymph (Fig. 7). The highest amount of [3H]-peptide (5.0±2.3%) was measured
Fig. 7. Percentage of total radioactivity in the hemolymph of 4-dayold 5th instar nymphs of S. gregaria (±SD) found after intubation of [3H]-Neb-TMOF. The hemolymph was analyzed by C18 RP-HPLC and liquid scintillation counting.
30 min after intubation. Afterwards, the amount of intact Neb-TMOF decreased gradually. In contrast, the total amount of radioactivity in the hemolymph increased and reached on av. 9.1% of the total dpm intubated initially. But even 2 h after intubation, intact Neb-TMOF could still be detected in the hemolymph.
4. Discussion
Fig. 5. Mean percentage of [3H]-Neb-TMOF found after incubation with hemolymph 5th instar larvae of S. frugiperda in the presence of captopril (± SD). The hemolymph was analyzed by C18 RP-HPLC and liquid scintillation counting. *Indicates statistical significance to the control according to t-test (∗P⬍0.05, ∗∗P⬍0.01).
Although the amount of proteolytic activity varied in the hemolymph of the different insects tested, the pattern of peptide degradation was identical. Since only the proline was labeled, two major proteolytic metabolites could be seen; they were identified as Neb-TMOF-(1-4) and (1-2). Counterparts (-5-6, -3-4) could be detected after use of sufficient amounts of non-labeled peptide
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material. These results suggest that the initial proteolytic sites are at the Asn4-Leu5 and Pro2-Thr3 bonds. Apparently, Neb-TMOF is cleaved into dipeptides by a dipeptidyl-peptidase present in the hemolymph of these insects. This has already been demonstrated in earlier studies in the flesh fly N. bullata, from which NebTMOF was formerly isolated (Zhu et al., 2001). Breakdown was significantly inhibited if captopril or lisinopril was added to the flesh fly hemolymph, indicating that an ACE-like protease, of which captopril and lisinopril are specific inhibitors, was present. The present study showed also in S. gregaria and S. frugiperda hemolymph, that Neb-TMOF is cleaved in C-terminal dipeptides, and proteolysis could be at least partially inhibited by captopril. Also other investigations demonstrated the presence of ACE-like enzymes in different insect species, in particular flies (Lamango et al., 1996; Wijffels et al. 1996, 1997; Schoofs et al., 1998; Ekbote et al., 1999). These findings suggest that ACE-like enzymes are more common in insects and are involved in the regulation (hydrolysis) of signalling peptides. Insect ACE is even able to hydrolyze C-terminally amidated peptides (Lamango et al., 1997). Nevertheless, captopril or lisinopril were not capable of inhibiting the degradation of Neb-TMOF in the hemolymph of L. maderae, even though the fragments isolated from the incubation mixture were identical. Obviously, the enzyme differs from that in the hemolymph of the other insects tested. Since the degradation product NebTMOF-(1-2) (P2) could be detected in sufficient amounts right from the beginning of the incubation, Neb-TMOF might be cleaved in this case from the Nterminus as described by Martensen et al. (1998). The authors isolated a proline-specific dipeptidyl-peptidase from homogenized and soluble membrane fractions of the blow fly Calliphora vicina, releasing the fragment Asn-Pro from the N-termini. Furthermore, ACE inhibitors could not inhibit this reaction. On the other hand, ACE immunoreactivity has been localized in the neuropile regions and neurosecretory cells of the CNS of L. maderae (Schoofs et al., 1998), indicating that there is, at least, some neuronal ACE-like activity. In N. bullata, hemolymph enzymatic breakdown of Neb-TMOF was rather quick, and the half-life was less than half a minute (Zhu et al., 2001). In the hemolymph of S. frugiperda and L. maderae the half-life was found to be slightly longer (苲3 min). Neb-TMOF was more resistant to proteolysis in the hemolymph of S. gregaria. It took about 25 min until the level of intact peptide dropped to 50%. These data are comparable to the halflives of other peptides found in the hemolymph of different insects (e.g. Oudejans et al., 1996; Garside et al., 1997). Due to these findings, S. gregaria was most suitable to serve as a model insect for the in vivo study of the penetration of Neb-TMOF through the gut epithelium.
The results demonstrate that the intact peptide can cross the midgut epithelium into the hemolymph of the fifth instar nymphs. This indicates that, in the conditions used, this peptide is not quickly degraded by gut enzymes and has the possibility to pass the gut walls. This is not totally unexpected since the sequence (HAsn-Pro-Thr-Asn-Leu-His-OH) does not contain any of the typical cleavage sites for the known trypsins and chymotrypsins. In gut luminal content of previtellogenic flesh flies, the hexapeptide was also quite stable (Zhu et al., 2001). Even though in vitro experiments had demonstrated that the peptide is quickly degraded in the hemolymph of flesh flies, it has recently been shown in our laboratory that orally administered Neb-TMOF still exerts its action and trminates trypsin biosynthesis in previtellogenic flies (Janssen, I., personal communication). These are promising results from the perspective of using such peptides in insect control. Borovsky and Mahmood (1995) reported that 28% of orally ingested Aea-TMOF could be traced back in the mosquito hemolymph. In our study, at most 5% of the initial Neb-TMOF was found in the locust hemolymph 30 min after intubation. Since we have studied this hormone using HPLC, the amount that we report here represents intact Neb-TMOF and not degradation products, which appear in the hemolymph (see above). Considering all fragments, maximally 9% of the total amount of TMOF that was administered was registered in the hemolymph. Three other peptides tested in our experiments have a lower transport rate as compared to Neb-TMOF (Zhu, W., unpublished results). It indicates that NebTMOF is either more stable in the locust gut than other peptides and/or that it is more easily transported. The rate of transport at which the peptide moves from the luminal to the haemocoel side of the gut was quite high. Within 5 min after intubation, already more than 1% of the intubated radiolabel could be recorded in the hemolymph. For free amino acids or peptide fragments it is reported that absorption occurs very rapidly in the anterior section of the midgut (Woods and Kingsolver, 1999), but absorptive sites are distributed throughout the midgut (Wolfersberger, 1996). In S. gregaria the absorption seems likewise to be dependant on a diffusion gradient across the midgut walls, created perhaps by the relative rapid movement of water into the hemolymph (Treherne, 1959). Not much is known about the mode of absorption of peptides longer than two amino acids. There are two major transport pathways across epithelia: a transcellular pathway through cells and a paracellular pathway between cells. Cell membranes are impermeable to inulin, leaving the paracellular pathway as the only route for their transepithelial permeation (Wang et al., 1996). Our experiments showed that only a small but measurable amount of [3H]-inulin (0.01% of total intubated radioactivity) could be registered in the hemolymph after
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intubation. This indicates that the intercellular junctions between the epithelial cells are tight but still allow passive penetration of macro-molecules as large as inulin (mol. wt. approx. ⬎5000). These findings suggest that the paracellular pathway might be (to a higher degree) also permeable to smaller molecules, such as peptides. Considering the results of the experiments with inositol (mol. wt. 180) and Neb-TMOF (mol. wt. 695), there is indeed an obvious correlation (r2=0.98) between the molecular weight and the percentage of radiolabeled material appearing in the hemolymph. We are, of course, aware of the fact that the way of transport of these molecules could be different. Further investigations are needed to find out whether the molecules we studied are actively or passively transported. In S. gregaria even the uptake of sugars seems to be passive (Treherne, 1958a,b). On the other hand, research on the mode of absorption of free inositol in segments of hamster small intestine has revealed that inositol is actively transported (Caspary and Crane, 1970). For numerous lepidopteran species a carriermediated transport of amino acids has been described (for further details see Sacchi and Wolfersberger, 1996).
Acknowledgements This project was undertaken as part of an EC-funded project, FAIR3-CT96-1648. Mass spectrometry was supported by the F.W.O (G. 0356.98).
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