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Laboratory for Developmental Physiology and Molecular Biology (E.C., J.H., G.B., A.D.L., L.S.), Katholieke Universiteit Leuven, 3000 Leuven, Belgium; and Department of Biochemical Physiology (J.V.D., D.V.d.H.), Utrecht University, 3584 CH Utrecht, The Netherlands
Address all correspondence and requests for reprints to: Elke Clynen, Zoological Institute, Naamsestraat 59, B-3000 Leuven, Belgium. E-mail: Elke.Clynen{at}bio.kuleuven.ac.be.
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Abstract |
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Introduction |
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The IRPs are found in several invertebrate orders and display sequence similarity to vertebrate insulin (5). In locusts, IRP is produced in the A2-type neurosecretory cells of the PI and is stored in the CCs (6, 7). The overall organization of the IRP precursor is signal peptide/B-chain/C- or connecting peptide/A-chain. After processing, the A- and B-chain are joined by disulfide bonds to form the mature insulin. The C-peptide is believed to aid the formation of the appropriate conformation of the mature insulin, thus creating the correct pattern of disulfide bonds.
In this study, we unequivocally prove that the in vivo processing of the Locusta IRP precursor yields an additional decapeptide. The effect of this peptide on lipid and carbohydrate metabolism was investigated. It displays no effect on the hemolymph lipid concentration in vivo. However, it has an effect on the activity of glycogen phosphorylase in vitro, opposite to that of the adipokinetic hormones (AKHs).
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Materials and Methods |
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The CCs of adult locusts were carefully dissected in a Ringer solution [Schistocerca Ringer: 8.77 g/liter NaCl, 0.19 g/liter CaCl2, 0.75 g/liter KCl, 0.41 g/liter MgCl2, 0.34 g/liter NaHCO3, 30.81 g/liter sucrose, 1.89 g/liter trehalose (pH 7.2); Locusta Ringer: 9.82 g/liter NaCl, 0.32 g/liter CaCl2, 0.48 g/liter KCl, 0.73 g/liter MgCl2, 0.25 g/liter NaHCO3, 0.19 g/liter NaH2PO4 (pH 6.5)] and transferred to a 0.5-ml Eppendorf tube on ice, containing 50 µl of methanol/water/acetic acid (vol/vol/vol; 90/9/1). The content of the tubes was sonicated for 3 min and after centrifugation (10 min at 13,000 rpm), the supernatant was dried in a vacuum centrifuge (SpeedVac Concentrator SVC200H, Savant).
For localization in the central nervous system, five equivalents of abdominal ganglia, abdominal perisympathetic organs, thoracic ganglia, suboesophageal ganglion, pars intercerebralis, corpora allata, hypocerebral ganglion, and frontal ganglion of L. migratoria and S. gregaria were dissected in Ringer and extracted in the same way as the CCs.
Mass spectrometry
Before mass analysis, the samples were concentrated and desalted using a ZiptipC18 (Millipore, 15 µm). For this purpose, the sample was reconstituted in 50 µl of water/trifluoroacetic acid (TFA) (99.9/0.1). The ZiptipC18 was preequilibrated for sample binding using an acetonitrile/water/TFA (50/49.9/0.1) solution followed by water/TFA (99.9/0.1). The sample was loaded on the ZiptipC18 and after flushing with water/TFA (99.9/0.1), eluted in 4 µl acetonitrile/water/formic acid (70/29.9/0.1).
Matrix-assisted laser desorption/ionization (MALDI) time of flight (TOF) mass spectrometry (MS)
MALDI-TOF MS was performed on a Reflex IV (Bruker Daltonics GmbH, Bremen, Germany), equipped with an N2 laser and pulsed ion extraction accessory. In each case, 1 µl of the sample solution was transferred to a steel target, mixed with 1 µl of a 50-mM solution of 
-cyano-4-hydroxycinnamic acid in acetone and air-dried. The instrument was calibrated using a standard peptide mixture (Bruker Daltonics GmbH). Positive ion spectra were recorded in the reflectron mode within a mass range from mass to charge (m/z) 0–3000.
Quadrupole (Q)-TOF MS
Nanospray Q-TOF MS was performed on a Q-TOF system (Micromass, Manchester, UK). One microliter of the sample solution was loaded into a gold-coated capillary (Proxeon, Odense, Denmark; L/Q nanoflow needle). The sample was sprayed at a flow rate of approximately 30 nl/min giving extended analysis time in which MS spectra as well as several MS-MS spectra were acquired. During MS-MS or tandem mass spectrometry, fragment ions are generated from a selected precursor ion by collision-induced dissociation. Because not all peptide ions fragment with the same efficiency, the collision energy is typically varied between 20 and 35 V so that the parent ion is fragmented in a satisfying number of different daughter ions.
Nanoflow-liquid chromatography (LC)-MS
Nanoflow-LC-MS was conducted on an Ultimate HPLC (LC Packings, Amsterdam, The Netherlands) coupled to a Q-TOF instrument as described before (8). Ten microliters of sample (corresponding to half a CC) were loaded. The separation was performed using a linear gradient from 95% solvent A, 5% solvent B to 30% solvent A, 70% solvent B in 55 min, followed by a gradient to 5% solvent A, 95% solvent B in 2 min, with a constant flow rate of 150 nl/min [solvent A, water/formic acid (99.9/0.1); solvent B, acetonitrile/formic acid (99.9/0.1)].
Bioassays
The synthetic IRP copeptide was purchased from Invitrogen (Huntsville, AL) and AKH-I from Peninsula Laboratories (San Carlos, CA).
Determination of hemolymph lipid concentration
A 10-µl solution of, respectively, 1.77 pmol, 17.7 pmol, and 177 pmol IRP copeptide in 10% methanol, was injected into the abdominal cavity. As a positive control 2 pmol AKH-I was used and control animals were injected with a 10% methanol solution in water. For each condition, eight male adult locusts (11–12 d after adult ecdysis) were tested. For the control condition, two separate experiments were pooled. After 45 min of incubation, 10 µl of hemolymph was taken, and total lipids were quantified by the modified vanillin method (9). For this purpose, samples were immediately added to 1.5 ml concentrated sulfuric acid and 0.45 ml of the sample was heated for 10 min at 95 C and then chilled in ice-water. Vanillin reagent (3.0 ml) was added and this mixture was heated for 15 min at 40 C. Extinction was read at 536 nm against a reagent blank.
Determination of phosphorylase activity
Fat bodies from 11- to 12-d-old adult males were dissected and separated in four different parts under physiological saline containing 10 mM HEPES (pH 7.2), 150 mM NaCl, and 15 mM KCl. The different parts were preincubated in 1 ml of the same saline with 4 mM MgCl2, 2 mM CaCl2, and 20 mM glucose twice for 60 min each to obtain a low phosphorylase activity ratio. Subsequently, they were preincubated in 1 ml of the same solution containing 80 mM trehalose instead of glucose. After 30 min, the effectors (IRP copeptide and AKH-I), or in the controls their solvents, were added to the medium. After incubation, the fat body was homogenized in 960 µl of ice-cold 50 mM HEPES buffer (pH 7.0) containing 5 mM EDTA and 2 mM dithiothreitol, using a chilled glass-Teflon homogenizer. Subsequently, 40 µl of 500 mM NaF was added and the homogenates were centrifuged twice at 16,000 x g. The infranatant was used for the phosphorylase assay according to Van Marrewijk et al. (10). Glycogen phosphorylase activity was measured spectrophotometrically in the direction of glycogen degradation with a coupled enzyme system. The assay system contained, in a volume of 1 ml, 40 mM phosphate buffer (pH 7.0), 5 mM imidazol, 2 mM EDTA, 1.4 mM dithioerythritol, 5 mM magnesium acetate, 4 µM glucose-1,6-diphosphate, 0.6 mM NADP, 2 mg of glycogen, 4 U of phosphoglucomutase, and 0.8 U of glucose-6-phosphate dehydrogenase. Reduction of NADP was measured at 340 nm. Active phosphorylase was assayed in the absence of AMP, whereas total phosphorylase activity was measured in the presence of 2 mM AMP.
The extent of activation of the enzyme is expressed by the activity ratio between active and total phosphorylase. Seven different concentrations of the IRP copeptide were tested. Fat body parts were incubated for 15 min with, respectively, 0.2 nM, 0.5 nM, 2 nM, 5 nM, 20 nM, 50 nM, and 177 nM IRP copeptide (for each concentration n = 6–9) and were compared with a 15-min incubation with methanol (control, n = 22). As a positive control 4 nM AKH-I (15 min incubation, n = 7) was used. In another experiment, the fat bodies were incubated for 5 min with 0.5 nM AKH-I followed by a 15-min incubation with 2 nM IRP copeptide (n = 12). These results were compared with those of a 5-min incubation with 0.5 nM AKH-I solely (n = 6).
Myotropic bioassay
Myotropic activity was evaluated by the Leucophaea maderae hindgut muscle preparation as described previously (11). Concentrations of IRP copeptide ranging between 3 nM and 3 µM were tested.
Data analysis
All results are presented as means with SD. Statistical analysis was performed using Excel 2000 and consisted of an unpaired Student’s t test for comparing test with control data. A level of P < 0.05 was considered significant (*, P < 0.05; **, P < 0.01; ***, P < 0.001).
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Results |
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).
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). A basic local alignment search tool (BLAST) database search revealed 100% sequence identity with amino acids 23–32 of the insulin-related peptide precursor (QSDLFLLSPK) (Fig. 3).
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Distribution of the IRP copeptide throughout the nervous system
To investigate the distribution of the IRP copeptide, several parts of the nervous system were dissected and their peptidome determined by MALDI-TOF MS. The IRP copeptide is present in the PI, the CCs, and the hypocerebral ganglion (Table 1). The latter could be due to contamination, as the hypocerebral ganglion is located close to the CCs. The neural structures in the thoracic and abdominal region were devoid of the IRP copeptide.
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). Further, we examined whether the IRP copeptide can abolish the effect of AKH. Therefore, we incubated 1/4 fat body parts for 5 min with AKH-I (0.5 nM). As expected, this yielded a significant increase of the activity ratio compared with the control. The other 1/4 fat body parts were incubated for 5 min with AKH-I, followed by a 15-min incubation with IRP copeptide (2 nM). The additional incubation with IRP copeptide did not significantly alter the activity ratio, compared with the incubation with AKH-I solely (Fig. 4).
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). The IRP copeptide did not show myotropic activity on the Leucophaea hindgut.
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Discussion |
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(1
1) glucose ] is the major circulatory sugar. It is mostly derived from glycogen stores in the fat body. So far, only hypertrehalosaemic factors, namely the AKHs, have been characterized in locusts. Here we report the identification of a novel peptide in the CC of locusts, which exerts the opposite effect of the AKHs on glycogen phosphorylase in vitro and therefore possibly represents a glycogenolysis-inhibiting peptide.
This novel peptide corresponds to the part of the Locusta IRP precursor (QSDLFLLSPK) N-terminally flanked by the signal peptide and C-terminally flanked by the B-chain. This is the first report on the purification of a peptide, derived from an insulin-like precursor gene, other than the A- and B-chain and C-peptide. In most insulins and IRPs, the B-chain is cleaved from the signal peptide by a signal peptidase. Cloning and sequencing of the cDNA of the Locusta IRP showed that the B-chain, which starts at position 34, is preceded by a dibasic doublet and according to the von Heijne algorithm the most probable cleaving site for the signal peptidase is at position 22 (12, 13) (Fig. 3
). This means that besides the A- and B-chain and the C-peptide, the processing of the Locusta IRP precursor could yield an additional peptide (6). Here we unequivocally prove that this IRP copeptide is present in the CCs.
The ion corresponding to the IRP copeptide lacking the C-terminal lysine residue was also occasionally present in the CCs, meaning that sometimes the lysine is removed by carboxypeptidases. At the N-terminal side, the IRP copeptide is protected from general aminopeptidases by the presence of a pyroglutamic acid. In addition to the blocked peptide, we also found different N-terminal fragments of the IRP copeptide in our extracts. The role of these fragments is unclear.
The IRP copeptide is identical in L. migratoria and S. gregaria. So far, nothing is known about an IRP in S. gregaria. The sequence of the IRP copeptide can be useful in future cloning experiments of the Schistocerca IRP gene.
In insects, IRPs have also been identified in Lepidoptera and Diptera, which belong to evolutionary divergent orders (holometabolic) in comparison to locusts (hemimetabolic) (14, 15, 16, 17). In contrast to locusts, these holometabolic species lack an IRP copeptide in their IRP genes, except maybe for Anopheles gambiae. One of the mosquito IRP precursors (agCP11852) might also contain a copeptide.
When we look at other insect neuropeptide precursors, we see that the presence of such a copeptide is not unusual. In locusts, four types of neuropeptide precursors have been fully identified: the AKH precursors, the neuroparsin precursors, the ion transport peptide (ITP) precursor, and the pacifastin precursors (18, 19, 20, 21, 22). The overall organization of the ITP and AKH precursors is comparable to that of the IRP precursor (Fig. 6). The ITP precursors of Locusta and Schistocerca have, respectively, a 42- and 44-amino-acid signal peptide, followed by a 9-amino-acid sequence (copeptide), a lysine-arginine cleaving site and a 75- to 79-amino-acid ITP. Recently, this copeptide was found in aqueous extracts of the CC of L. migratoria (23). The AKH-precursors consist of a 22-amino-acid signal peptide, followed by a decapeptide (AKH-I) or octapeptide (AKH-II or AKH-III), a lysine-arginine or arginine-arginine cleaving site and a 28- (for AKH-I and AKH-II) or 44- (for AKH-III) amino-acid AKH-associated peptide. Here, the sequences corresponding to the copeptides are the AKHs. In locusts, the AKHs are neurohormones that play an important role in energy metabolism (24). Based on the similarity in the overall organization of these neuropeptide precursors, we can presume that, like the AKHs, the IRP and ITP copeptides also play a significant role in locust physiology.
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It has been suggested that insulin in insects, as in vertebrates, also controls the anabolic processes involved in the storage of energy, whereas the insect adipokinetic hormone, like glucagon, controls the catabolic processes linked to mobilization of energy (27). The adipokinetic hormones do show some sequence similarity with glucagon and as well glucagon as the AKHs induce a hypertrehalosaemic effect in locusts (27, 28). Neck-ligated locusts show a clear hyperglycaemic response to injection of a CCg extract, the storage site of the AKHs, but a similar treatment has no response in whole insects (29). This suggests the presence of a hypoglycaemic factor in the insect endocrine system. Furthermore, Orchard and Loughton (30) have provided evidence for the presence in the CCs of a hypolipaemic factor that seems to have common molecular features with insulin. Preliminary characterization of this factor, whose action is mimicked by the N-terminal part of the B-chain of bovine insulin, indicated a molecular mass of about 1 kDa (30, 31).
Therefore, in this study, we looked at the role of the IRP copeptide (
1 kDa) in lipid and carbohydrate metabolism. The IRP copeptide seems to have no effect on the hemolymph lipid level. It does, however, induces a significant decrease of the activity of glycogen phosphorylase. In the in vitro assay, the IRP copeptide is active in the nanomolar range, and this activity is dose dependent. High concentrations (>5 nM) showed no effect.
The rate of glycogenolysis depends on the activity of phosphorylase, which catalyzes the degradation of glycogen by the cleavage of glucosyl residues. There seems to be no functional difference between the insect phosphorylase and the vertebrate enzyme. Both exist in two interconvertible forms, the dephosphorylated b form, which displays catalytic activity only in the presence of AMP, and the phosphorylated a form, which is largely AMP independent for activity (32). AKH stimulates the conversion of inactive to active fat body glycogen phosphorylase, whereas our IRP copeptide displays the reverse effect. However, the IRP copeptide cannot abolish the effect of AKH.
In vertebrates, glycogen synthesis and glycogen breakdown are regulated by different enzymes. The distinct regulatory cascades involve AMP-dependent protein kinase and reversible protein phosphorylations. Whereas the cascade controlling glycogenolysis leads to activation of glycogen phosphorylase, the cascade controlling glycogen synthesis leads to inhibition of glycogen synthase. So far, there is little information available on glycogen synthase in insects. If the mechanism is comparable to that in vertebrates, we can suspect that binding of the IRP copeptide on the fat body receptor induces a decrease of cAMP, thereby inactivating the protein kinases, which leads to a shift toward dephosphorylated glycogen phosphorylase (inactive) and dephosphorylated glycogen synthase (active). However, further research will be necessary to prove this mechanism and unravel the exact function of the IRP copeptide.
In this paper, it is once again proven that valuable information about prohormone processing can be gained from the sequencing of peptide products isolated from tissues. Sensitive methods like MALDI-TOF MS and Q-TOF MS make it possible to gain sequence information from a very limited amount of material. Here, we were able to de novo sequence a novel peptide from a single CC. Although an increasing number of putative neuropeptide sequences may be gathered from sequenced genomes, peptide identification studies remain indispensable to reveal physiologically relevant structures. Further, our MS method shows to be an elegant tool to investigate the distribution of a neuropeptide throughout the nervous system. The IRP copeptide is present in the brain and associated structures, but not in the ganglia located in the thorax and abdomen or their neurohemal organs. The presence in the PI and the CCs can be expected because the A- and B-chain and the C-peptide of the IRP precursor were also shown to be present in these structures (7).
In sum, we have identified, for the first time, in insects a peptide that most likely inhibits glycogenolysis. Furthermore, the identification of this novel peptide yields more insight in the processing of the IRP-precursor.
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Footnotes |
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Abbreviations: AKH, Adipokinetic hormone; CC, corpora cardiaca; CCg, glandular part of CC; CCs, storage (or neurohemal) part of CC; IRP, insulin-related peptide; ITP, ion transport peptide; LC-MS, liquid chromatography-mass spectrometry; MALDI-TOF MS, matrix-assisted laser desorption/ionization time of flight mass spectrometry; MS-MS, tandem mass spectrometry; MT, myotropin; m/z, mass to charge; PK, pyrokinin; PI, pars intercerebralis; pQ, pyroglutamic acid; Q-TOF MS, quadrupole time of flight mass spectrometry; TFA, trifluoroacetic acid.
Received December 4, 2002.
Accepted for publication April 25, 2003.
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