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Effects of two hemolymph proteins on humoral defense reactions in the wax moth, Galleria mellonella
Developmental and Comparative Immunology 29 (2005) 43–51 www.elsevier.com/locate/devcompimm
Effects of two hemolymph proteins on humoral defense reactions in the wax moth, Galleria mellonella Shin Yong Parka, Chong Han Kima, Woo Hyuk Jeonga, Joon Ha Leea, Sook Jae Seob, Yeon Soo Hanc, In Hee Leea,* a
Department of Life Science, Hoseo University, Sechul-Ri, Baebang-Myun, Asan City, Chungnam-Do 336-795, South Korea b Division of Life Science, College of Natural Sciences, Gyeongsang National University, Chinju, South Korea c Department of Agricultural Biology, Division of Applied Plant Science, Chonnam National University, 300 Yongbongdong Buk-gu, Kwangju 500-757, South Korea Received 30 July 2003; revised 26 May 2004; accepted 2 June 2004 Available online 2 July 2004
Abstract Two hemolymph proteins were isolated from the wax moth, Galleria mellonella, larvae by a two-step procedure consisting of acid extraction and reversed phase (RP)-HPLC. One was an apolipophorin III (apoLp-III) previously characterized as a lipopolysaccharide (LPS) binding protein in the hemolymph of G. mellonella. The other was confirmed to be a new protein with a molecular mass of 23,768.69 Da, referred to as Gm protein-24. The full-length cDNA of Gm protein-24 was cloned from the fat body. The cDNA structure showed that it is a 219-residues protein derived from the precursor of 236 amino acids. The effects of apoLp-III and Gm protein-24 have been tested on the insect humoral immunity. ApoLp-III enhanced the activity of antibacterial peptide such as cecropin but Gm protein-24 had no effect on cecropin activity. On the other hand, Gm protein-24 and apoLp-III were both involved in the activation of prophenoloxidase (PPO) cascade, which has been regarded as a critical immune reaction in insect hemolymph. Of note, the Gm protein-24 was a significantly stronger activator of PPO cascade than apoLp-III. q 2004 Elsevier Ltd. All rights reserved. Keywords: Apolipophorin III; Gm protein-24; Antibacterial peptide; Prophenoloxidase cascade; Hemolymph; Galleria mellonella
1. Introduction Defense systems in insect hemocoels are comprised of cellular and humoral components. These two immune systems cooperatively function Abbreviations: LPS, Lipopolysaccharide; PPO, Prophenoloxidase; Gm, Galleria mellonella; apoLp-III, Apolipophorin III; TBS, Tris buffered saline; AU-PAGE, Acid urea PAGE. * Corresponding author. Tel.: þ 82-41-5405626; fax: þ 82-415486231. E-mail address: [email protected] (I.H. Lee). 0145-305X/$ - see front matter q 2004 Elsevier Ltd. All rights reserved. doi:10.1016/j.dci.2004.06.001
in clearance of invading pathogenic microbes from the hemolymph. Antimicrobial peptides such as cecropins or insect defensins have been known to be key factors in insect humoral immunity [1]. They appear in the hemolymph in response to microbial infection and kill bacteria at the initial phase of immune reactions. The activation of a prophenoloxidase (PPO) cascade is also considered to be a critical humoral defense reaction against microbial infection and parasitization [2]. The reaction is usually linked to the induced synthesis
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of antimicrobial peptides and the stimulation of cellular immune responses such as nodule formation and encapsulation [3]. Insect hemolymph contains a number of proteins, which are involved in diverse developmental, physiological or immunological processes. Some of these proteins have been confirmed as having dual functions in hemolymph. For instance, apoLp-III has been known as a hemolymph protein that plays a role in lipid metabolism in insects [4]. Also, the protein was found to participate in immune reactions as an LPS-binding protein (LBP) or as a potentiator of bacteriolytic activity of hemolymph other than lipid transport [5,6]. Insect PPO cascade systems appear to be similar to the complement system of mammals. As in mammals, pathogens invading insect hemocoel are recognized as foreign by hemolymph proteins. This recognition is often followed by activation of the PPO cascade for self-defense against microbial infection [7]. To date, several hemolymph proteins have been described as activators of PPO cascades in a variety of insects. Most of them were found to be recognition proteins that have binding affinity to foreign pattern molecules such as lipopolysaccharides (LPS), peptidoglycan and b-1,3-glucan, which are cell wall components of Gram-negative, Gram-positive bacteria and fungi, respectively [8,9,10]. LBPs isolated from the hemolymph of Bombyx mori [9], Manduca sexta [11], Drosophila melanogaster [12] and Periplaneta americana [13] have been well characterized as immune factors to prevent infection by Gram-negative bacteria. However, more information on immune proteins in hemolymph is still needed to understand the detailed defense mechanisms of insects. Recently, we purified two proteins from the hemolymph of Galleria mellonella larvae, which may be related to defense reactions. Matrixassisted laser desorption ionization – mass spectrometry (MALDI – MS), N-terminal amino acid sequence analysis and cDNA cloning revealed that one protein is an apoLp-III previously isolated from hemolymph of G. mellonella larvae [6] and the other was a new protein, referred to as Gm protein-24, which had homology in the amino acid sequence to a 29 kDa glycoprotein from an adult male of M. sexta [14] and to a tissue specific protein from B. mori [15]. This study has been performed to investigate
the immune functions of two hemolymph proteins (apoLp-III and Gm protein-24) in hemolymph of G. mellonella larvae.
2. Materials and methods 2.1. Insect and hemolymph collection Larvae of G. mellonella were grown on an artificial diet (Quaker Oats Co., USA) at 30 8C in a dark incubator. Last instar larvae (155 – 170 mg/larva) were used. For immunization of G. mellonella, log phase E. coli (2 £ 103 colony forming units) was injected into the larvae hemocoel 24 h before hemolymph collection. Hemolymph was directly collected into sterile tubes containing a few crystals of phenylthiourea (PTU), an inhibitor of phenoloxidase. Hemolymph used in assays for the activation of the PPO cascade was also collected into a tube without PTU at 4 8C to prevent the spontaneous activation of PPO. Hemolymph samples were centrifuged at 10,000g for 10 min to remove hemocytes and cell debris.
2.2. Purification of Gm protein-24 and apoLp-III Gm protein-24 and apoLp-III were purified from hemolymph by a two-step procedure used to purify lysozyme from G. mellonella [16]. Cell-free hemolymph was mixed with an equal volume of 10% acetic acid and agitated overnight at 4 8C. After centrifugation for 30 min at 10,000g, the resulting supernatant was removed and directly injected to a reversed-phase (RP) C4 HPLC column (Vydac 214TP54). The RP-HPLC was performed using a continuous 1.0%/min linear gradient of acetonitrile in 0.1% trifluoroacetic acid. The homogeneity of the purified proteins was confirmed through a tricine SDS-PAGE [17] and an acid urea PAGE [18] analysis. Each protein was subjected to MALDI – MS and N-terminal amino acid sequence analysis by Edman degradation. For further experiments, the purified proteins were lyophilized in a Speed-Vac centrifugation system (Bioneer, Daejon, Korea) and stored at 2 70 8C.
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2.3. Production of antibodies against Gm protein-24 or apoLp-III and immunoblot analysis Phosphate buffered saline (0.5 ml) containing 100 mg of Gm protein-24 or apoLp-III was thoroughly mixed with Freund’s complete adjuvant (0.5 ml) and subcutaneously injected into a rabbit. For the second and the booster injections, the antigen was mixed with Freund’s incomplete adjuvant. Antiserum was taken 1 week after the booster injection and was diluted with Tris buffered saline (TBS: 50 mM Tris– HCl and 150 mM NaCl, pH 7.4). Immunoblot analyses were performed to detect Gm protein-24 and apoLp-III in the hemolymph of naı¨ve larvae and immunized larvae with E. coli as described by Towbin (1992) [19]. 2.4. cDNA cloning of Gm protein-24 The cDNA of Gm protein-24 was cloned by a combination of RT-PCR and 50 , 30 -rapid amplification of cDNA ends (RACE)-PCR. Total RNA was isolated from the fat body of the last instar larvae using the Total RNA extraction Kit (Promega, Madison, WI, USA). The first strand cDNA was synthesized using a reverse transcription system (Promega, Madison, WI, USA). Based on N-terminal amino acid sequence (K 20 AQCKK 25) obtained by Edman degradation, the degenerate primer (AARGCNCARTGYAARAAR: R ¼ A or G; Y ¼ T or C; N ¼ A, T, C or G) was designed for RT-PCR. PCR reactions were performed with two primers (a degenerate primer as a forward primer and oligo dT as a reverse primer) according to the following conditions: 5-min preheating at 95 8C, 30 cycles (95 8C 1-min; 40 8C 1-min; 72 8C 1-min) and a 5-min elongation at 72 8C. To clone the 50 end of the Gm protein-24 cDNA including the signal sequence, a 50 RACE-PCR was performed using the Marathone cDNA Amplification Kit (Clontech USA). The adaptor-ligated cDNAs were subjected to PCR under the following conditions: one cycle at 94 8C for 30 sec and 30 cycles at 94 8C for 5 sec and at 68 8C for 4 min. The forward primer provided by company was used and the reverse primer was designed on the basis of the internal sequence (underlined part in Fig. 4) of Gm protein-24 disclosed by the previous RT-PCR. Since we could not obtain the exact sequence for
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region near 30 end via RT-PCR, 30 RACE-PCR was performed with a specific forward primer (double underlined section in Fig. 3) according to the same schedule as that of 50 RACE-PCR. 2.5. Effects of Gm protein-24 and apoLp-III on the antibacterial activity of cecropin Basically, an ultrasensitive radial diffusion assay [20] was used for antibacterial tests in the present work. Cecropin A was purchased from Sigma (C6830, USA) and used as an antibacterial peptide sample. Stock cecropin solutions were prepared in acidified distilled water (0.01% acetic acid) and were two-fold serially diluted. These ranged in concentration from 3.12 to 100 mg/ml and 5 ml samples were loaded into wells (3 mm in diameter) in underlay agarose gel, in which the washed midlogarithmic phase E. coli (4 £ 106 colony forming units) were trapped. The underlay agars consisted of sterile citrate phosphate buffer (9 mM sodium phosphate, 1 mM sodium citrate, pH 7.4). 1% (w/v) Type I (low electroendosmosis) agarose (A 6013; Sigma), and 3% tryptic soy broth (TSB: Difco, USA). After allowing 3 h for diffusion of sample into underlay gels, a 10 ml nutrient-rich overlay gel containing 6% TSB and 1% agarose was poured. After the plates were incubated overnight at 37 8C to allow surviving bacteria to form colonies, the diameters of clearing zones indicating antibacterial activity were plotted against the log 10 of the peptide concentrations. To assess the effect of Gm protein-24 or apoLp-III on antibacterial activity of cecropin, we adapted two types of modified radial diffusion assays. First, after the bacteria were preincubated with diverse amounts of Gm protein24 or apoLp-III (10, 20, 40 or 80 mg) for 30 min at room temperature, they were seeded in underlay agar for radial diffusion assay. Then 5 ml samples of serially diluted cecropin were introduced into the wells. In the other test, the predetermined amounts of Gm protein-24 or apoLp-III were mixed with 5 ml of acidified water containing 0.125 mg of cecropin. The antibacterial activity of each mixture was then tested in the original radial diffusion assay as described above.
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2.6. Effects of Gm protein-24 and apoLp-III on the activation of plasma PPO Five micrograms of Gm protein-24 or apoLp-III in TBS containing 5 mM CaCl2 was separately added to 2 ml of cell-free hemolymph in wells of a 96-well plate. The total volume was adjusted to 20 ml and the mixture was incubated for 20 min at room temperature. Then 180 ml of 2 mM dopamine in 50 mM sodium phosphate (pH 6.5) was added to each well. Phenoloxidase activity in each well was assessed by measuring absorbance at 490 nm over 40 min with 5-min interval using a microtiter plate reader. The experiment was repeated five times and the mean value put into graph form.
3. Results 3.1. Purification of Gm protein-24 and apoLp-III Two proteins were purified from the hemolymph of naı¨ve G. mellonella larvae by acid extraction and C4 RP-HPLC. As shown in an HPLC profile, Gm protein-24 and apoLp-III were consecutively eluted at 37.6 and 41.1% of acetonitrile, respectively (Fig. 1A). When two purified proteins were electrophoresed on tricine SDS-PAGE (left panel of inset) and AU-PAGE (right panel of inset) gels, they appeared to be homogeneous. 3.2. Characterization of two purified proteins and immunoblot analyses Two proteins were subjected to MALDI – MS (Voyager-DE STR, PerSeptive Biosystem, USA) and Edman degradation (Procise 491, Applied Biosystems, USA) analysis to determine molecular masses and N-terminal amino acid sequences, respectively. The molecular mass of Gm protein-24 was measured as 23,768.69 Da (Fig. 1B) and its N-terminal 30 amino acids sequence was determined. The other protein’s molecular mass and N-terminal 30 amino acids sequence (data not shown) showed complete accordance with those of apoLp-III, previously isolated from G. mellonella hemolymph [6]. Therefore we conclude that the protein is an apoLp-III. Immunoblot analysis performed with antibody to Gm protein-24 revealed that the protein
Fig. 1. Profile of reversed phase-HPLC and mass spectrometric analysis. (A) Two LBPs (Gm protein-24 and apoLp-III) were separately eluted in linear gradient (0.1% 10 min; 0– 60% 60 min) of an acetonitile concentration containing 0.1% trifluoroacetic acid (TFA). Arrows indicate peaks containing Gm protein-24 or apoLp-III. Insets show Tricine SDS-PAGE and acid urea (AU)PAGE performed with two purified proteins. Tricine SDS-PAGE: lane 1, acid extracts of naı¨ve G. mellonella hemolymph; lane 2, Gm protein-24; lane 3, apoLp-III. AU-PAGE: lane 1, commercial cecropin A (Sigma) and G. mellonella lysozyme; lane 2, Gm protein-24; lane 3, apoLp-III. Gels were stained with coomassie brilliant blue R-250. (B) MALDI mass analysis for Gm protein-24. The major peak appeared at m/z value of 23,768.69.
was present in invariable amounts in the hemolymph of naı¨ve and immunized larvae (Fig. 2). 3.3. cDNA structure of Gm protein-24 The full-length cDNA and the deduced amino acid sequence of Gm protein-24 are shown in Fig. 3. The cDNA consists of 893 nucleotides and contains an open reading frame of 708 nucleotides encoding a Gm protein-24 precursor of 236 amino acids, which includes a 17-residues signal peptide and a 219-residues mature protein. This is followed by
S.Y. Park et al. / Developmental and Comparative Immunology 29 (2005) 43–51
Fig. 2. Tricine SDS-PAGE and immunoblotting analyses with antiserums to Gm protein-24. Left panel shows a tricine SDS-PAGE gel stained with coomassie blue and right panel shows the immunoblotting analysis performed with duplicate gel: lane 1, hemolymph of naı¨ve G. mellonella larvae; lane 2, hemolymph of immunized G. mellonella larvae; lane 3, acid extraction of naı¨ve larval hemolymph; lane 4, purified Gm protein-24.
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a stop signal and a 30 untranslated stretch of 122 nucleotides containing a putative polyadenylation consensus signal (AATAAA), and 13 additional nucleotides before the poly (A) tail. Our database search revealed that the cDNA structure of Gm protein-24 was completely identical with 27 kDa gene from G. mellonella (AJ575661 in the GenBank) and the primary structure of Gm protein-24 lacks homology to immune proteins found to date in the insect hemolymph. However, it shows a 56% identity in the amino acid sequence with 29 kDa glycoprotein from M. sexta [14] and a 55% identity with a tissue specific protein in the suboesophageal body of B. mori [15] (data not shown). Based on amino acid sequence deduced from a Gm protein-24 cDNA, the molecular mass of Gm protein-24 was calculated to be
Fig. 3. cDNA structure of Gm protein-24 and the deduced amino acid sequence. The complementary oligonucleotide to the underlined region was used as a reverse primer for 5’ RACE-PCR and the double underlined part was used as a forward primer for 30 RACE-PCR. A putative signal sequence is indicated in bold-type and the polyadenylation signal is expressed in a box. Asterisk represents a stop signal.
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Fig. 4. Effects of apoLp-III and Gm protein-24 on the antibacterial activity of cecropin. Bacteria were preincubated with given amounts (10, 20, 40, and 80 mg) of apoLp-III and Gm protein-24 at room temperature for 30 min and they were used in radial diffusion assay to measure antibacterial activity of cecropin. Diameters of clearing zone were expressed in units (1 mm ¼ 10 units). Note that the activities of cecropin were improved as the amount of apoLp-III incubated was increased. Error bars are too narrow for display.
Fig. 5. Antibacterial activities of cecropin changed according to the amount of two hemolymph proteins mixed with cecropin. The fixed amount (0.125 mg) of cecropin was preincubated with diverse amounts of apoLp-III (A) or Gm protein-24 (B), and then the mixture was tested for antibacterial activity in radial diffusion assay. As in Fig. 5, activity of constant amount of cecropin was increased upon incubation with apoLp-III. Error bars are too narrow for display.
23,763.90 Da, 4.79 Da smaller than the value (23,768.69) measured by MALDI analysis. Thus it was postulated that Gm protein-24, unlike M. sexta glycoprotein, might not be glycosylated.
only apoLp-III improved the anti-E. coli activity of cecropin.
3.4. Effects of Gm protein-24 and apoLp-III on cecropin activity against E. coli The purified Gm protein-24 and apoLp-III were tested for their effects on the antibacterial activity of cecropin against E. coli by two types of antimicrobial assays. Fig. 4 shows the changes of cecropin activity against E. coli preincubated with diverse amounts of Gm protein-24 and apoLp-III. In proportion to the amount of apoLp-III preincubated with E. coli, anti-E. coli activity of cecropin became stronger (Fig. 4A). In contrast, there was no increase of cecropin activity against E. coli upon preincubation with Gm protein-24 (Fig. 4B). When the mixture of cecropin and apoLp-III or Gm protein-24 was tested for antibacterial activity in radial diffusion assay (Fig. 5), it was in good accordance with results shown in Fig. 4. The activity of constant amount (0.125 mg) of cecropin against E. coli was enhanced as the amount of apoLp-III increased in the mixture (Fig. 5A) but Gm protein-24 did not influence the activity of cecropin (Fig. 5B). Considering the results from the two antibacterial assays, it is concluded that
3.5. Activation of PPO cascade To test whether Gm protein-24 or apoLp-III may be involved in the PPO activation system, each protein was added to G. mellonella plasma and phenoloxidase activity was measured after incubation for 5 – 40 min (Fig. 6). Addition of each protein to
Fig. 6. Activation of prophenoloxidase (PPO) by Gm protein-24. Five micrograms of each sample was incubated with 2 ml of cell free hemolymph in a 96-well plate at room temperature for 20-min. Then 180 ml of 2 mM dopamine solution was added, and phenoloxidase activity was measured at 490 nm over 5 min. The points represent the mean of five individual measurements ^ SD.
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G. mellonella larval plasma resulted in activation of phenoloxidase. In both cases, phenoloxidase activity continually increased up to 40 min. Gm protein-24 was much more effective in PPO activation than apoLp-III.
4. Discussion We have described two hemolymph proteins, apoLp-III and Gm protein-24, which participate in humoral defense reactions of G. mellonella larvae. Two proteins were purified from hemolymph of naı¨ve larvae by a concise two-step procedure consisting of acid extraction and RP-HPLC. Primarily, this work was undertaken to investigate if two proteins have roles in the immune systems of G. mellonella although putative immuno-competent domains for recognizing foreign invaders or exerting antimicrobial activity were not identified in the primary structure of Gm protein-24 deduced from cDNA sequence. ApoLp-III has been recently reported to take part in the immune reactions occurred in hemolymph. In particular, we examined effects of two proteins on the humoral immune reactions mediated by antimicrobial peptides and the PPO cascade system in hemolymph of G. mellonella. ApoLp-III is a hemolymph protein that associates hydrophobically with lipoproteins to facilitate lipid transport upon high-energy consumption in insects. This protein exists in two conformational states, one of which is a non-lipid associated globular form and the other is a lipid-associated form [21]. Recently, Wiesner (1997) [22] reported that a conformational change of apoLp-III could be a signal for the activation or the potentiation of the insect immune response. Moreover, it was suggested that apoLp-III might contribute to immune reactions only upon formation of a lipid – apoLp-III complex. ApoLp-III of G. mellonella was previously isolated from the larval hemolymph and its cDNA was also cloned from the fat body [6]. G. mellonella apoLp-III was found to bind to LPS derived from the surface of Gram-negative bacteria [23], and improve the bacteriolytic activity of hemolymph [24]. However, it has not been demonstrated that the activity of bacteriolytic protein in hemolymph could be strengthen by apoLp-III.
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To date, defense reactions mediated by an inducible antibacterial peptide against bacterial infection have been well characterized in a broad range of animals including mammals and insects [25]. Generally, antibacterial peptides exert their activities by binding to the cell surface of pathogens and changing membrane permeability of target cells [26]. In the present study, we examined the synergistic effect of two proteins on the antibacterial activity of cecropin, a well-known antibacterial peptide in lepidopteran and dipteran insects. Our antibacterial assays revealed that E. coli preincubated with apoLp-III became more vulnerable toward attack by cecropin. Moreover, cecropin mixed with apoLp-III exhibited stronger anti-E. coli activity than cecropin alone or in combination with Gm protein-24. In contrast, the antibacterial activity of lysozyme, a known bacteriolytic protein in G. mellonella hemolymph [16], was not affected by the two proteins (data not shown). Therefore, it was suggested that apoLp-III might facilitate the binding of cecropin to bacterial cell walls and enhance the antibacterial activity of cecropin. Consequently, apoLp-III contributes to the augmentation of bacteriolytic activity of hemolymph although it has no independent antibacterial capacity. The PPO cascade is known to be a critical humoral immune system against invading pathogens into insect hemolymph. The activation of PPO cascade system is associated with the recognition of microbes by hemolymph proteins with binding affinity to the surface molecules of invading microbes. However, the exact mechanism remains to be explained. Recently, apoLp-III was found to be associated with the activation of PPO cascades in locust hemolymph, Locusta migratoria [27]. The activation of the L. migratoria PPO cascade in response to the injection of immunogens such as laminarin was highly dependent on the hemolymph apoLp-III concentration. Testing to see if Gm protein-24 and apoLpIII could induce the activation of a PPO cascade showed that they contribute to the activation of PPO cascades in G. mellonella hemolymph. The addition of Gm protein-24 to G. mellonella hemolymph strongly led to activation of PPO cascade, even in the absence of components from microbial cell wall. Although this paper has not presented data showing the detailed immune function of Gm protein-24, we
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surmise that the protein may play a crucial role as an immune mediator between invading pathogens and endogenous PPO cascades in self-defense mechanisms of G. mellonella. In addition, apoLp-III is also thought to be involved in the activation of PPO cascades in G. mellonella hemolymph. A recent paper describing the role of an apoLp-III of G. mellonella in b-1,3-glucan pattern recognition and cellular immune reactions [28] strongly supports our results on the immune function of apoLp-III. In conclusion, this paper has described the purification and the immunological function of two proteins present in the hemolymph of naı¨ve G. mellonella larvae. Whereas apoLp-III improves antibacterial activity of cecropin against E. coli, Gm protein-24 has no effect on cecropin activity. In contrast, Gm protein-24 and apoLp-III play a role in the activation of the PPO cascades in G. mellonella hemolymph. Future research will be done to understand the mechanism by which Gm protein-24 activates PPO cascade systems in G. mellonella hemolymph, which is expected to allow critical extrapolation for the further elucidation of insect immune mechanisms.
[5]
[6]
[7] [8]
[9]
[10]
[11]
[12]
Acknowledgements [13]
This work was made possible by a grant from Advanced Basic Research Lab (ABRL Grant No. 2002-056-01003-0) from the Korean Science and Engineering Foundation (KOSEF). We also thank Yong Pyo Shin, Hong Ki Kim and Sun Ri Kim who are undergraduate students at Hoseo University for their careful rearing insects.
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[24] Halwani AE, Dunphy GB. Apolipophorin-III in Galleria mellonella potentiates hemolymph lytic activity. Dev Comp Immunol 1999;23:563 –70. [25] Andra J, Berninghausen O, Leippe M. Cecropins, antibacterial peptides from insects and mammals, are potently fungicidal against Candida albicans. Med Microbiol Immunol 2001;189: 169 –73. [26] Silvestro L, Weiser JN, Axelsen PH. Antibacterial and antimembrane activities of cecropin A in Escherichia coli. Antimicrob Agents Chemother 2000;44:602– 7. [27] Mullen L, Goldsworthy G. Changes in lipophorins are related to the activation of phenoloxidase in the haemolymph of Locusta migratoria in response to injection of immunogens. Insect Biochem Mol Biol 2003; 33:661–70. [28] Whitten MM, Tew IF, Lee BL, Ratcliffe NA. A novel role for an insect apolipoprotein (apolipophorin III) in beta-1,3-Glucan pattern recognition and cellular encapsulation reactions. J Immunol 2004;172:2177–85.
Effects of two hemolymph proteins on humoral defense reactions in the wax moth, Galleria mellonella Shin Yong Parka, Chong Han Kima, Woo Hyuk Jeonga, Joon Ha Leea, Sook Jae Seob, Yeon Soo Hanc, In Hee Leea,* a
Department of Life Science, Hoseo University, Sechul-Ri, Baebang-Myun, Asan City, Chungnam-Do 336-795, South Korea b Division of Life Science, College of Natural Sciences, Gyeongsang National University, Chinju, South Korea c Department of Agricultural Biology, Division of Applied Plant Science, Chonnam National University, 300 Yongbongdong Buk-gu, Kwangju 500-757, South Korea Received 30 July 2003; revised 26 May 2004; accepted 2 June 2004 Available online 2 July 2004
Abstract Two hemolymph proteins were isolated from the wax moth, Galleria mellonella, larvae by a two-step procedure consisting of acid extraction and reversed phase (RP)-HPLC. One was an apolipophorin III (apoLp-III) previously characterized as a lipopolysaccharide (LPS) binding protein in the hemolymph of G. mellonella. The other was confirmed to be a new protein with a molecular mass of 23,768.69 Da, referred to as Gm protein-24. The full-length cDNA of Gm protein-24 was cloned from the fat body. The cDNA structure showed that it is a 219-residues protein derived from the precursor of 236 amino acids. The effects of apoLp-III and Gm protein-24 have been tested on the insect humoral immunity. ApoLp-III enhanced the activity of antibacterial peptide such as cecropin but Gm protein-24 had no effect on cecropin activity. On the other hand, Gm protein-24 and apoLp-III were both involved in the activation of prophenoloxidase (PPO) cascade, which has been regarded as a critical immune reaction in insect hemolymph. Of note, the Gm protein-24 was a significantly stronger activator of PPO cascade than apoLp-III. q 2004 Elsevier Ltd. All rights reserved. Keywords: Apolipophorin III; Gm protein-24; Antibacterial peptide; Prophenoloxidase cascade; Hemolymph; Galleria mellonella
1. Introduction Defense systems in insect hemocoels are comprised of cellular and humoral components. These two immune systems cooperatively function Abbreviations: LPS, Lipopolysaccharide; PPO, Prophenoloxidase; Gm, Galleria mellonella; apoLp-III, Apolipophorin III; TBS, Tris buffered saline; AU-PAGE, Acid urea PAGE. * Corresponding author. Tel.: þ 82-41-5405626; fax: þ 82-415486231. E-mail address: [email protected] (I.H. Lee). 0145-305X/$ - see front matter q 2004 Elsevier Ltd. All rights reserved. doi:10.1016/j.dci.2004.06.001
in clearance of invading pathogenic microbes from the hemolymph. Antimicrobial peptides such as cecropins or insect defensins have been known to be key factors in insect humoral immunity [1]. They appear in the hemolymph in response to microbial infection and kill bacteria at the initial phase of immune reactions. The activation of a prophenoloxidase (PPO) cascade is also considered to be a critical humoral defense reaction against microbial infection and parasitization [2]. The reaction is usually linked to the induced synthesis
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of antimicrobial peptides and the stimulation of cellular immune responses such as nodule formation and encapsulation [3]. Insect hemolymph contains a number of proteins, which are involved in diverse developmental, physiological or immunological processes. Some of these proteins have been confirmed as having dual functions in hemolymph. For instance, apoLp-III has been known as a hemolymph protein that plays a role in lipid metabolism in insects [4]. Also, the protein was found to participate in immune reactions as an LPS-binding protein (LBP) or as a potentiator of bacteriolytic activity of hemolymph other than lipid transport [5,6]. Insect PPO cascade systems appear to be similar to the complement system of mammals. As in mammals, pathogens invading insect hemocoel are recognized as foreign by hemolymph proteins. This recognition is often followed by activation of the PPO cascade for self-defense against microbial infection [7]. To date, several hemolymph proteins have been described as activators of PPO cascades in a variety of insects. Most of them were found to be recognition proteins that have binding affinity to foreign pattern molecules such as lipopolysaccharides (LPS), peptidoglycan and b-1,3-glucan, which are cell wall components of Gram-negative, Gram-positive bacteria and fungi, respectively [8,9,10]. LBPs isolated from the hemolymph of Bombyx mori [9], Manduca sexta [11], Drosophila melanogaster [12] and Periplaneta americana [13] have been well characterized as immune factors to prevent infection by Gram-negative bacteria. However, more information on immune proteins in hemolymph is still needed to understand the detailed defense mechanisms of insects. Recently, we purified two proteins from the hemolymph of Galleria mellonella larvae, which may be related to defense reactions. Matrixassisted laser desorption ionization – mass spectrometry (MALDI – MS), N-terminal amino acid sequence analysis and cDNA cloning revealed that one protein is an apoLp-III previously isolated from hemolymph of G. mellonella larvae [6] and the other was a new protein, referred to as Gm protein-24, which had homology in the amino acid sequence to a 29 kDa glycoprotein from an adult male of M. sexta [14] and to a tissue specific protein from B. mori [15]. This study has been performed to investigate
the immune functions of two hemolymph proteins (apoLp-III and Gm protein-24) in hemolymph of G. mellonella larvae.
2. Materials and methods 2.1. Insect and hemolymph collection Larvae of G. mellonella were grown on an artificial diet (Quaker Oats Co., USA) at 30 8C in a dark incubator. Last instar larvae (155 – 170 mg/larva) were used. For immunization of G. mellonella, log phase E. coli (2 £ 103 colony forming units) was injected into the larvae hemocoel 24 h before hemolymph collection. Hemolymph was directly collected into sterile tubes containing a few crystals of phenylthiourea (PTU), an inhibitor of phenoloxidase. Hemolymph used in assays for the activation of the PPO cascade was also collected into a tube without PTU at 4 8C to prevent the spontaneous activation of PPO. Hemolymph samples were centrifuged at 10,000g for 10 min to remove hemocytes and cell debris.
2.2. Purification of Gm protein-24 and apoLp-III Gm protein-24 and apoLp-III were purified from hemolymph by a two-step procedure used to purify lysozyme from G. mellonella [16]. Cell-free hemolymph was mixed with an equal volume of 10% acetic acid and agitated overnight at 4 8C. After centrifugation for 30 min at 10,000g, the resulting supernatant was removed and directly injected to a reversed-phase (RP) C4 HPLC column (Vydac 214TP54). The RP-HPLC was performed using a continuous 1.0%/min linear gradient of acetonitrile in 0.1% trifluoroacetic acid. The homogeneity of the purified proteins was confirmed through a tricine SDS-PAGE [17] and an acid urea PAGE [18] analysis. Each protein was subjected to MALDI – MS and N-terminal amino acid sequence analysis by Edman degradation. For further experiments, the purified proteins were lyophilized in a Speed-Vac centrifugation system (Bioneer, Daejon, Korea) and stored at 2 70 8C.
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2.3. Production of antibodies against Gm protein-24 or apoLp-III and immunoblot analysis Phosphate buffered saline (0.5 ml) containing 100 mg of Gm protein-24 or apoLp-III was thoroughly mixed with Freund’s complete adjuvant (0.5 ml) and subcutaneously injected into a rabbit. For the second and the booster injections, the antigen was mixed with Freund’s incomplete adjuvant. Antiserum was taken 1 week after the booster injection and was diluted with Tris buffered saline (TBS: 50 mM Tris– HCl and 150 mM NaCl, pH 7.4). Immunoblot analyses were performed to detect Gm protein-24 and apoLp-III in the hemolymph of naı¨ve larvae and immunized larvae with E. coli as described by Towbin (1992) [19]. 2.4. cDNA cloning of Gm protein-24 The cDNA of Gm protein-24 was cloned by a combination of RT-PCR and 50 , 30 -rapid amplification of cDNA ends (RACE)-PCR. Total RNA was isolated from the fat body of the last instar larvae using the Total RNA extraction Kit (Promega, Madison, WI, USA). The first strand cDNA was synthesized using a reverse transcription system (Promega, Madison, WI, USA). Based on N-terminal amino acid sequence (K 20 AQCKK 25) obtained by Edman degradation, the degenerate primer (AARGCNCARTGYAARAAR: R ¼ A or G; Y ¼ T or C; N ¼ A, T, C or G) was designed for RT-PCR. PCR reactions were performed with two primers (a degenerate primer as a forward primer and oligo dT as a reverse primer) according to the following conditions: 5-min preheating at 95 8C, 30 cycles (95 8C 1-min; 40 8C 1-min; 72 8C 1-min) and a 5-min elongation at 72 8C. To clone the 50 end of the Gm protein-24 cDNA including the signal sequence, a 50 RACE-PCR was performed using the Marathone cDNA Amplification Kit (Clontech USA). The adaptor-ligated cDNAs were subjected to PCR under the following conditions: one cycle at 94 8C for 30 sec and 30 cycles at 94 8C for 5 sec and at 68 8C for 4 min. The forward primer provided by company was used and the reverse primer was designed on the basis of the internal sequence (underlined part in Fig. 4) of Gm protein-24 disclosed by the previous RT-PCR. Since we could not obtain the exact sequence for
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region near 30 end via RT-PCR, 30 RACE-PCR was performed with a specific forward primer (double underlined section in Fig. 3) according to the same schedule as that of 50 RACE-PCR. 2.5. Effects of Gm protein-24 and apoLp-III on the antibacterial activity of cecropin Basically, an ultrasensitive radial diffusion assay [20] was used for antibacterial tests in the present work. Cecropin A was purchased from Sigma (C6830, USA) and used as an antibacterial peptide sample. Stock cecropin solutions were prepared in acidified distilled water (0.01% acetic acid) and were two-fold serially diluted. These ranged in concentration from 3.12 to 100 mg/ml and 5 ml samples were loaded into wells (3 mm in diameter) in underlay agarose gel, in which the washed midlogarithmic phase E. coli (4 £ 106 colony forming units) were trapped. The underlay agars consisted of sterile citrate phosphate buffer (9 mM sodium phosphate, 1 mM sodium citrate, pH 7.4). 1% (w/v) Type I (low electroendosmosis) agarose (A 6013; Sigma), and 3% tryptic soy broth (TSB: Difco, USA). After allowing 3 h for diffusion of sample into underlay gels, a 10 ml nutrient-rich overlay gel containing 6% TSB and 1% agarose was poured. After the plates were incubated overnight at 37 8C to allow surviving bacteria to form colonies, the diameters of clearing zones indicating antibacterial activity were plotted against the log 10 of the peptide concentrations. To assess the effect of Gm protein-24 or apoLp-III on antibacterial activity of cecropin, we adapted two types of modified radial diffusion assays. First, after the bacteria were preincubated with diverse amounts of Gm protein24 or apoLp-III (10, 20, 40 or 80 mg) for 30 min at room temperature, they were seeded in underlay agar for radial diffusion assay. Then 5 ml samples of serially diluted cecropin were introduced into the wells. In the other test, the predetermined amounts of Gm protein-24 or apoLp-III were mixed with 5 ml of acidified water containing 0.125 mg of cecropin. The antibacterial activity of each mixture was then tested in the original radial diffusion assay as described above.
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2.6. Effects of Gm protein-24 and apoLp-III on the activation of plasma PPO Five micrograms of Gm protein-24 or apoLp-III in TBS containing 5 mM CaCl2 was separately added to 2 ml of cell-free hemolymph in wells of a 96-well plate. The total volume was adjusted to 20 ml and the mixture was incubated for 20 min at room temperature. Then 180 ml of 2 mM dopamine in 50 mM sodium phosphate (pH 6.5) was added to each well. Phenoloxidase activity in each well was assessed by measuring absorbance at 490 nm over 40 min with 5-min interval using a microtiter plate reader. The experiment was repeated five times and the mean value put into graph form.
3. Results 3.1. Purification of Gm protein-24 and apoLp-III Two proteins were purified from the hemolymph of naı¨ve G. mellonella larvae by acid extraction and C4 RP-HPLC. As shown in an HPLC profile, Gm protein-24 and apoLp-III were consecutively eluted at 37.6 and 41.1% of acetonitrile, respectively (Fig. 1A). When two purified proteins were electrophoresed on tricine SDS-PAGE (left panel of inset) and AU-PAGE (right panel of inset) gels, they appeared to be homogeneous. 3.2. Characterization of two purified proteins and immunoblot analyses Two proteins were subjected to MALDI – MS (Voyager-DE STR, PerSeptive Biosystem, USA) and Edman degradation (Procise 491, Applied Biosystems, USA) analysis to determine molecular masses and N-terminal amino acid sequences, respectively. The molecular mass of Gm protein-24 was measured as 23,768.69 Da (Fig. 1B) and its N-terminal 30 amino acids sequence was determined. The other protein’s molecular mass and N-terminal 30 amino acids sequence (data not shown) showed complete accordance with those of apoLp-III, previously isolated from G. mellonella hemolymph [6]. Therefore we conclude that the protein is an apoLp-III. Immunoblot analysis performed with antibody to Gm protein-24 revealed that the protein
Fig. 1. Profile of reversed phase-HPLC and mass spectrometric analysis. (A) Two LBPs (Gm protein-24 and apoLp-III) were separately eluted in linear gradient (0.1% 10 min; 0– 60% 60 min) of an acetonitile concentration containing 0.1% trifluoroacetic acid (TFA). Arrows indicate peaks containing Gm protein-24 or apoLp-III. Insets show Tricine SDS-PAGE and acid urea (AU)PAGE performed with two purified proteins. Tricine SDS-PAGE: lane 1, acid extracts of naı¨ve G. mellonella hemolymph; lane 2, Gm protein-24; lane 3, apoLp-III. AU-PAGE: lane 1, commercial cecropin A (Sigma) and G. mellonella lysozyme; lane 2, Gm protein-24; lane 3, apoLp-III. Gels were stained with coomassie brilliant blue R-250. (B) MALDI mass analysis for Gm protein-24. The major peak appeared at m/z value of 23,768.69.
was present in invariable amounts in the hemolymph of naı¨ve and immunized larvae (Fig. 2). 3.3. cDNA structure of Gm protein-24 The full-length cDNA and the deduced amino acid sequence of Gm protein-24 are shown in Fig. 3. The cDNA consists of 893 nucleotides and contains an open reading frame of 708 nucleotides encoding a Gm protein-24 precursor of 236 amino acids, which includes a 17-residues signal peptide and a 219-residues mature protein. This is followed by
S.Y. Park et al. / Developmental and Comparative Immunology 29 (2005) 43–51
Fig. 2. Tricine SDS-PAGE and immunoblotting analyses with antiserums to Gm protein-24. Left panel shows a tricine SDS-PAGE gel stained with coomassie blue and right panel shows the immunoblotting analysis performed with duplicate gel: lane 1, hemolymph of naı¨ve G. mellonella larvae; lane 2, hemolymph of immunized G. mellonella larvae; lane 3, acid extraction of naı¨ve larval hemolymph; lane 4, purified Gm protein-24.
47
a stop signal and a 30 untranslated stretch of 122 nucleotides containing a putative polyadenylation consensus signal (AATAAA), and 13 additional nucleotides before the poly (A) tail. Our database search revealed that the cDNA structure of Gm protein-24 was completely identical with 27 kDa gene from G. mellonella (AJ575661 in the GenBank) and the primary structure of Gm protein-24 lacks homology to immune proteins found to date in the insect hemolymph. However, it shows a 56% identity in the amino acid sequence with 29 kDa glycoprotein from M. sexta [14] and a 55% identity with a tissue specific protein in the suboesophageal body of B. mori [15] (data not shown). Based on amino acid sequence deduced from a Gm protein-24 cDNA, the molecular mass of Gm protein-24 was calculated to be
Fig. 3. cDNA structure of Gm protein-24 and the deduced amino acid sequence. The complementary oligonucleotide to the underlined region was used as a reverse primer for 5’ RACE-PCR and the double underlined part was used as a forward primer for 30 RACE-PCR. A putative signal sequence is indicated in bold-type and the polyadenylation signal is expressed in a box. Asterisk represents a stop signal.
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Fig. 4. Effects of apoLp-III and Gm protein-24 on the antibacterial activity of cecropin. Bacteria were preincubated with given amounts (10, 20, 40, and 80 mg) of apoLp-III and Gm protein-24 at room temperature for 30 min and they were used in radial diffusion assay to measure antibacterial activity of cecropin. Diameters of clearing zone were expressed in units (1 mm ¼ 10 units). Note that the activities of cecropin were improved as the amount of apoLp-III incubated was increased. Error bars are too narrow for display.
Fig. 5. Antibacterial activities of cecropin changed according to the amount of two hemolymph proteins mixed with cecropin. The fixed amount (0.125 mg) of cecropin was preincubated with diverse amounts of apoLp-III (A) or Gm protein-24 (B), and then the mixture was tested for antibacterial activity in radial diffusion assay. As in Fig. 5, activity of constant amount of cecropin was increased upon incubation with apoLp-III. Error bars are too narrow for display.
23,763.90 Da, 4.79 Da smaller than the value (23,768.69) measured by MALDI analysis. Thus it was postulated that Gm protein-24, unlike M. sexta glycoprotein, might not be glycosylated.
only apoLp-III improved the anti-E. coli activity of cecropin.
3.4. Effects of Gm protein-24 and apoLp-III on cecropin activity against E. coli The purified Gm protein-24 and apoLp-III were tested for their effects on the antibacterial activity of cecropin against E. coli by two types of antimicrobial assays. Fig. 4 shows the changes of cecropin activity against E. coli preincubated with diverse amounts of Gm protein-24 and apoLp-III. In proportion to the amount of apoLp-III preincubated with E. coli, anti-E. coli activity of cecropin became stronger (Fig. 4A). In contrast, there was no increase of cecropin activity against E. coli upon preincubation with Gm protein-24 (Fig. 4B). When the mixture of cecropin and apoLp-III or Gm protein-24 was tested for antibacterial activity in radial diffusion assay (Fig. 5), it was in good accordance with results shown in Fig. 4. The activity of constant amount (0.125 mg) of cecropin against E. coli was enhanced as the amount of apoLp-III increased in the mixture (Fig. 5A) but Gm protein-24 did not influence the activity of cecropin (Fig. 5B). Considering the results from the two antibacterial assays, it is concluded that
3.5. Activation of PPO cascade To test whether Gm protein-24 or apoLp-III may be involved in the PPO activation system, each protein was added to G. mellonella plasma and phenoloxidase activity was measured after incubation for 5 – 40 min (Fig. 6). Addition of each protein to
Fig. 6. Activation of prophenoloxidase (PPO) by Gm protein-24. Five micrograms of each sample was incubated with 2 ml of cell free hemolymph in a 96-well plate at room temperature for 20-min. Then 180 ml of 2 mM dopamine solution was added, and phenoloxidase activity was measured at 490 nm over 5 min. The points represent the mean of five individual measurements ^ SD.
S.Y. Park et al. / Developmental and Comparative Immunology 29 (2005) 43–51
G. mellonella larval plasma resulted in activation of phenoloxidase. In both cases, phenoloxidase activity continually increased up to 40 min. Gm protein-24 was much more effective in PPO activation than apoLp-III.
4. Discussion We have described two hemolymph proteins, apoLp-III and Gm protein-24, which participate in humoral defense reactions of G. mellonella larvae. Two proteins were purified from hemolymph of naı¨ve larvae by a concise two-step procedure consisting of acid extraction and RP-HPLC. Primarily, this work was undertaken to investigate if two proteins have roles in the immune systems of G. mellonella although putative immuno-competent domains for recognizing foreign invaders or exerting antimicrobial activity were not identified in the primary structure of Gm protein-24 deduced from cDNA sequence. ApoLp-III has been recently reported to take part in the immune reactions occurred in hemolymph. In particular, we examined effects of two proteins on the humoral immune reactions mediated by antimicrobial peptides and the PPO cascade system in hemolymph of G. mellonella. ApoLp-III is a hemolymph protein that associates hydrophobically with lipoproteins to facilitate lipid transport upon high-energy consumption in insects. This protein exists in two conformational states, one of which is a non-lipid associated globular form and the other is a lipid-associated form [21]. Recently, Wiesner (1997) [22] reported that a conformational change of apoLp-III could be a signal for the activation or the potentiation of the insect immune response. Moreover, it was suggested that apoLp-III might contribute to immune reactions only upon formation of a lipid – apoLp-III complex. ApoLp-III of G. mellonella was previously isolated from the larval hemolymph and its cDNA was also cloned from the fat body [6]. G. mellonella apoLp-III was found to bind to LPS derived from the surface of Gram-negative bacteria [23], and improve the bacteriolytic activity of hemolymph [24]. However, it has not been demonstrated that the activity of bacteriolytic protein in hemolymph could be strengthen by apoLp-III.
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To date, defense reactions mediated by an inducible antibacterial peptide against bacterial infection have been well characterized in a broad range of animals including mammals and insects [25]. Generally, antibacterial peptides exert their activities by binding to the cell surface of pathogens and changing membrane permeability of target cells [26]. In the present study, we examined the synergistic effect of two proteins on the antibacterial activity of cecropin, a well-known antibacterial peptide in lepidopteran and dipteran insects. Our antibacterial assays revealed that E. coli preincubated with apoLp-III became more vulnerable toward attack by cecropin. Moreover, cecropin mixed with apoLp-III exhibited stronger anti-E. coli activity than cecropin alone or in combination with Gm protein-24. In contrast, the antibacterial activity of lysozyme, a known bacteriolytic protein in G. mellonella hemolymph [16], was not affected by the two proteins (data not shown). Therefore, it was suggested that apoLp-III might facilitate the binding of cecropin to bacterial cell walls and enhance the antibacterial activity of cecropin. Consequently, apoLp-III contributes to the augmentation of bacteriolytic activity of hemolymph although it has no independent antibacterial capacity. The PPO cascade is known to be a critical humoral immune system against invading pathogens into insect hemolymph. The activation of PPO cascade system is associated with the recognition of microbes by hemolymph proteins with binding affinity to the surface molecules of invading microbes. However, the exact mechanism remains to be explained. Recently, apoLp-III was found to be associated with the activation of PPO cascades in locust hemolymph, Locusta migratoria [27]. The activation of the L. migratoria PPO cascade in response to the injection of immunogens such as laminarin was highly dependent on the hemolymph apoLp-III concentration. Testing to see if Gm protein-24 and apoLpIII could induce the activation of a PPO cascade showed that they contribute to the activation of PPO cascades in G. mellonella hemolymph. The addition of Gm protein-24 to G. mellonella hemolymph strongly led to activation of PPO cascade, even in the absence of components from microbial cell wall. Although this paper has not presented data showing the detailed immune function of Gm protein-24, we
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surmise that the protein may play a crucial role as an immune mediator between invading pathogens and endogenous PPO cascades in self-defense mechanisms of G. mellonella. In addition, apoLp-III is also thought to be involved in the activation of PPO cascades in G. mellonella hemolymph. A recent paper describing the role of an apoLp-III of G. mellonella in b-1,3-glucan pattern recognition and cellular immune reactions [28] strongly supports our results on the immune function of apoLp-III. In conclusion, this paper has described the purification and the immunological function of two proteins present in the hemolymph of naı¨ve G. mellonella larvae. Whereas apoLp-III improves antibacterial activity of cecropin against E. coli, Gm protein-24 has no effect on cecropin activity. In contrast, Gm protein-24 and apoLp-III play a role in the activation of the PPO cascades in G. mellonella hemolymph. Future research will be done to understand the mechanism by which Gm protein-24 activates PPO cascade systems in G. mellonella hemolymph, which is expected to allow critical extrapolation for the further elucidation of insect immune mechanisms.
[5]
[6]
[7] [8]
[9]
[10]
[11]
[12]
Acknowledgements [13]
This work was made possible by a grant from Advanced Basic Research Lab (ABRL Grant No. 2002-056-01003-0) from the Korean Science and Engineering Foundation (KOSEF). We also thank Yong Pyo Shin, Hong Ki Kim and Sun Ri Kim who are undergraduate students at Hoseo University for their careful rearing insects.
[14]
[15]
[16]
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