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Purification and characterization of apolipophorin III from immune hemolymph of Heliothis virescens pupae
Comparative Biochemistry and Physiology Part B 132 (2002) 505–514
Purification and characterization of apolipophorin III from immune hemolymph of Heliothis virescens pupae Kyung Tae Chung1, Donald D. Ourth* Department of Microbiology and Molecular Cell Sciences, The University of Memphis, Memphis, TN 38152, USA Received 28 November 2001; received in revised form 20 February 2002; accepted 3 March 2002
Abstract Apolipophorin III (ApoLp-III) from Heliothis virescens pupae was purified by heat-treatment followed by Sephadex G-50 filtration and reverse phase-HPLC. The molecular mass of the purified ApoLp-III was determined as 17 965.9"5 Da by mass spectrometry. The N-terminal sequence confirmed the protein as ApoLp-III with homology of 56–83% to other insect ApoLp-III molecules. The amino acid spatial arrangement of the predicted a-helix 1 of Heliothis ApoLp-III was nearly identical to that of the amphipatic a-helix 1 of Manduca sexta ApoLp-III. The absorption spectrum from 240–340 nm of the Heliothis ApoLp-III was the same as the UV spectra of ApoLp-III from Manduca sexta and Galleria mellonella, showing absorption maxima at 280, 268, 264 and 259 nm. These results indicated that the primary structure of ApoLp-III is conserved in lepidopterans. The Heliothis ApoLp-III was not a glycoprotein and showed hemagglutination activity against rabbit red blood cells. This hemagglutination activity was abolished by Tween 80, but not by six different carbohydrates. Hydrophobic interaction of ApoLp-III with red blood cells agreed with structural studies since ApoLp-III binds lipid through hydrophobic interaction after conformational change. Bacterial injection apparently increased the amount of ApoLp-III in immune hemolymph when compared with normal hemolymph, and may indicate that ApoLpIII plays a role in insect immunity. 䊚 2002 Elsevier Science Inc. All rights reserved. Keywords: Apolipophorin III; N-terminal sequence; a-Helix; UV absorption spectrum; Hemagglutination; Heliothis virescens
1. Introduction Apolipophorin-III (ApoLp-III) is an 18–20-kDa amphipathic apolipoprotein (Cole et al., 1987) found in insect hemolymph and has an essential role in lipid transport (Wells et al., 1987). Unlike other insect apolipoproteins (apolipophorin I and II), which are integral non-exchangeable apolipoproteins, ApoLp-III is a water-soluble and exchangeable apolipoprotein which reversibly associates with lipophorin particles (Narayanas*Corresponding author. Tel.: q1-901-678-2950; fax: q1901-678-4457. E-mail address: [email protected] (D.D. Ourth). 1 Current address: Department of Tumor Cell Biology, St. Jude Children’s Research Hospital, 332 North Lauderdale St., Memphis, TN 38105, USA.
wami and Ryan, 1997). ApoLp-III normally exists as a monomeric, lipid-free hemolymph protein. When ApoLp-III is required for lipid transport, the protein associates with high-density lipophorin (HDLp) to form a low-density lipophorin (LDLp) by stabilizing the increment of lipid–water interface resulting from diacylglycerol uptake (Wells et al., 1987). Its function in lipid transport has been reviewed with Locusta migratoria and Manduca sexta ApoLp-III (Chino, 1985; Van der Horst, 1990; Blacklock and Ryan, 1994). The three-dimensional structure of ApoLp-III was determined by X-ray crystallography of Locusta migratoria ApoLp-III (Breiter et al., 1991) and by the NMR technique with Manduca sexta ApoLp-III (Wang et al., 1997). Although
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there is low homology (29%) in the amino acid sequences of Locusta and Manduca ApoLp-III molecules, these studies showed great similarity in structure between the two ApoLp-III proteins. Both Locusta and Manduca ApoLp-III molecules consist of five discrete anti-parallel a-helices, which are connected by short loops. The hydrophobic surfaces of each helix face inward to the center of the bundle. These structural studies supported the hypothesis that ApoLp-III undergoes a large conformational change that opens hydrophobic surfaces when binding to lipophorin (Kawooya et al., 1986; Wells et al., 1987). These hydrophobic surfaces of each helix are then interacting with lipid. Apolipophorin III has also been reported to have functions related to immune responses against bacterial infection in insects. In Galleria mellonella, intrahemocoelic injection of purified ApoLpIII into larvae increased antibacterial activity in hemolymph, and ApoLp-III enhanced phagocytic activity of isolated hemocytes in vitro (Wiesner et al., 1997). Kato et al. (1994) demonstrated that lipophorin of Bombyx mori was able to detoxify bacterial lipopolysaccharide (LPS) by forming an LPS-lipophorin complex. Hemagglutination characteristics of Galleria ApoLp-III were demonstrated (Imura et al., 1998). Antimicrobial (Tada et al., 1993) and antiviral (Srinivas et al., 1990) activities of human apolipoprotein A-1 were also reported. Heliothis virescens (tobacco budworm) is an insect pest of many important crops including cotton. We have been studying antibacterial factors from H. virescens after bacterial infection (Ourth et al., 1994; Lockey and Ourth, 1996; Chung and Ourth, 2000). We investigated the role of ApoLpIII in the immune hemolymph of H. virescens, since ApoLp-III has not before been identified or studied in association with an immune response. We describe here our study of Heliothis ApoLpIII focusing on its purification and characterization along with a comparison of the physico-chemical and molecular characteristics of Heliothis ApoLpIII with other insect ApoLp-III molecules. We also describe the up-regulation of ApoLp-III by bacterial injection and a mechanism of ApoLp-III hemagglutination activity with red blood cells, which has not been demonstrated before.
2. Materials and methods 2.1. Apolipophorin III purification Immune and normal hemolymph samples were obtained as described before (Chung and Ourth 2000). A 5= dilution of immune hemolymph with saline was heat-treated at 100 8C for 1 min with continuous agitation. Hemolymph samples (0.5 ml) were heat-treated at one time. After heat treatment, the samples were cooled immediately on ice and then centrifuged for 5 min at 14 000 rev.ymin using an Eppendorf centrifuge. The supernatant was applied to a Sephadex G-50 gel filtration column (1=50 cm) equilibrated with 0.1 M ammonium acetate buffer, pH 6.4. The column was eluted with the same buffer at 15 mlyh, and 1 ml per tube was collected. The fractions containing ApoLp-III were then pooled. The sample was concentrated using a 10-kDa molecular weight cutoff ultrafiltration membrane (10 MWCO Centricon, Amicon, Beverly, MA, USA). The concentrated sample was adjusted to contain 0.1% trifluoroacetic acid (TFA) as a final concentration and applied to a 4.6=250-mm Microsorb-MW C18 reverse-phase high pressure liquid chromatography (RP-HPLC), particle size 5 m, pore size 100 ˚ (Rainin, Woburn, MA, USA) having a Perisorb A C18 pre-column (Upchurch Scientific, Oak Harbor, WA, USA), using an ISCO HPLC system (ISCO, Lincoln, NE, USA). Proteins were eluted with a linear gradient (0–60%) of acetonitrile containing 0.1% TFA over a 120-min period. The flow rate was 1 mlymin with optical density being monitored at 214 nm. Protein peak fractions were vacuum dried by SpeedVac and reconstituted with 50 ml of saline or 0.1 M ammonium acetate buffer, pH 6.4, depending on subsequent experiments. Protein samples were analyzed on SDS-PAGE gels according to the method of Schagger and von Jagow (1987). Protein concentration of samples was determined by the Bicinchoninic acid (BCA) protein assay according to instructions (Pierce Chemical Co., Rockford, IL, USA). Bovine serum albumin was used as the standard protein. 2.2. N-terminal amino acid sequence The N-terminal amino acid sequence of the purified ApoLp-III was determined by the Center for Biotechnology at St. Jude Children’s Research
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Hospital, Memphis, TN, USA. The instrument used was a Perkin-Elmer Applied Biosystems 494 cLC protein sequencer with standard cycles supplied by the manufacturer. 2.3. Mass spectrometry Molecular mass of the purified Heliothis ApoLp-III was determined by electrospray ionization mass spectrometry (ESI-MS) using VG Platform II (Micromass, Beverly, MA, USA). 2.4. Schiffer–Edmundson helical wheel projection A helical wheel of the potential helix domain from the N-terminus of Heliothis ApoLp-III was generated by using the Wisconsin Package software version 9.1 from Genetics Computer Group (GCG), Madison, WI, USA. The helical wheel of the predicted a-helix domain from the N-terminus of Heliothis ApoLp-III was compared with that from the a-helix 1 domain of Manduca ApoLpIII. 2.5. Ultraviolet absorption spectrum of ApoLp-III The absorption spectrum of ApoLp-III scanned from 240 to 340 nm with a Shimazu 1601 spectrophotometer. A protein sample ml) in 0.85% saline (approx. 400 mgyml) used.
was UV(0.5 was
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ApoLp-III protein (5.6 mM) were mixed together in a U-bottom microtiter plate to give a final concentration of 200 mM of six different carbohydrates. The mixture was incubated for 10 min at room temperature prior to the addition of trypsin-treated RBC. Twenty-five microliters of 4% trypsin-treated rabbit RBC were added and incubated for 30 min at 37 8C. The mixture was continuously incubated for 18 h at 4 8C. Saline and the carbohydrate solutions listed below without ApoLp-III were used as negative controls. The following six carbohydrates were used for the competition assay: L-fucose, D-galactose, D-glucose, D-mannose, N-acetyl-D-galactosamine and Nacetyl-D-glucosamine. 2.8. Effect of Tween 80 on interaction of ApoLpIII with rabbit red blood cells Tween 80 was used to study the mechanism of interaction between ApoLp-III and rabbit RBC. The final concentration of Tween 80 was 0.02% in the hemagglutination mixture that was tested. Fifty microliters of the assay mixture contained the purified ApoLp-III, 2% red blood cell suspension and Tween 80. Buffer solutions were 0.85% NaCl saline, 50 mM Tris–HCl buffered saline, pH 7.2. The mixture was incubated as previously done for the hemagglutination assay. Wheat germ agglutinin (1 mgyml) was used as the positive control, and bovine serum albumin (1 mgyml) was used as the negative control.
2.6. Hemagglutination assay 2.9. Lectin blotting Serial two-fold dilutions of the purified ApoLpIII were prepared with 0.85% saline in a microtiter U-bottom plate with a final volume of 25 mlywell. To each well was added an equal volume of rabbit red blood cell (RBC) suspension (108 cellsyml). The mixture was incubated for 30 min at 37 8C and then incubated overnight at 4 8C. Positive hemagglutination was judged by matt formation at the bottom of the plate well and by microscopic observation. 2.7. Hemagglutination competition assay Trypsin-treated rabbit RBC were used for hemagglutination competition. Trypsin-treated rabbit RBC were basically prepared according to Nowak et al. (1976). Twenty microliters of
Lectin blotting was used to test if Heliothis ApoLp-III is a glycoprotein. This was done with minor modification according to Bar-Nun and Gershoni (1994). Approximately 4 mg of ApoLp-III in saline was applied directly onto a nitrocellulose membrane, and the nitrocellulose membrane then blocked with 2% non-fat dry milk for 2 h. The membrane was washed three times for 1 h with washing buffer (25 mM Tris–HCl, pH 7.4, 150 mM NaCl, 1 mM CaCl2, 0.5 mM MgCl2) and incubated with rhodamine-conjugated concanavalin A and rhodamine-conjugated wheat germ agglutinin. Under UV light, fluorescence of glycoprotein on the membrane was examined. A positive control for lectin blotting was 10 mg of ovalbumin in MilliQ water.
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Fig. 1. (a) Reverse phase-HPLC of the pooled fraction sample from Sephadex G-50 gel filtration column after 10 MWCO concentration. The sample was applied to a Microsorb-MW C18 column equilibrated in 0.1% TFA. A linear gradient of acetonitrile (0–60%) containing 0.1% TFA was applied at a flow rate of 1 mlymin over a period of 120 min. Each peak fraction was dried by SpeedVac and resuspended in 50 ml of sterile water. The bar represents fractions (118–120) of ApoLp-III. (b) SDS gel electrophoresis of purified ApoLp-III protein. The fraction No. 120 from RP-HPLC was loaded onto a SDS-PAGE gel. Lane 1 contained standard proteins; Lane 2 and Lane 3 contained samples of fraction No. 120 under reducing and non-reducing conditions, respectively. The gel was visualized by silver staining.
3. Results Apolipophorin-III purification was accomplished utilizing its heat stable characteristics (Kanost et al., 1988; Wiesner et al., 1997). Heat treatment was employed as the first step of purification followed by two sequential chromatogra-
phies, Sephadex G-50 gel filtration and RP-HPLC. Fractions (118–120; Fig. 1a) obtained by RPHPLC contained a homogenous 21 kDa ApoLpIII band on an SDS gel under both reducing and non-reducing conditions, indicating that the protein does not have intra-disulfide bonds (Fig. 1b). The
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Fig. 2. (a) A comparison of the N-terminal amino acid sequences of seven lepidopteran ApoLp-III proteins with Heliothis ApoLp-III. Gap (dash line) was introduced to obtain the maximum sequence homology. X represents an unknown amino acid. The box represents a-helix 1 of Manduca ApoLp-III. All of the sequences start at the first amino acid of mature ApoLp-III proteins. Homologous residues are in bold. Percentage homologies among the ApoLp-III proteins to H. virescens are given. M. sexta: Manduca sexta, A. convolvuli: Agrius convolvuli (NIH GENBANK accession AF001632), B. mandarina: Bombyx mandarina, B. mori: Bombyx mori, D. grandiosella: Diatraea grandiosella, G. mellonella: Galleria mellonella, T. ni: Trichoplusia ni, H. virescens: Heliothis virescens. (b) Schiffer– Edmundson helical wheel projection of the potential a-helix of Heliothis ApoLp-III N-terminus. Helical wheel was projected with an angle of 1008. Hydrophobic amino acids are boxed.
molecular mass of Heliothis ApoLp-III was determined to be 17 965.9"5 Da by mass spectrometry. This result was very similar to the molecular mass of ApoLp-III from G. mellonella larvae which was determined to be 18 077"2 Da by mass spectrometry (Wiesner et al., 1997). The N-terminal amino acid sequence from the RP-HPLC fractions confirmed that the 21 kDa protein was ApoLp-III (Fig. 2a). The N-terminal amino acid sequence of the Heliothis ApoLp-III protein was 56–83% homologous with the Nterminal sequences of ApoLp-III from seven other lepidopteran insects (Cole et al., 1987; Burks et al., 1992; Ferkovich et al., 1995; Wiesner et al.,
1997; Yamauchi et al., 2000; NIH GENBANK accession AF001632), indicating that the ApoLpIII protein is highly conserved in lepidopterans. The Schiffer–Edmundson helical wheel was predicted for the sequenced N-terminal region of Heliothis ApoLp-III using the GCG computer program (Fig. 2b). The predicted Schiffer–Edmundson helical wheel of the 7–29 amino acid residues of Heliothis ApoLp-III was compared with the spatial arrangement of amino acids of a-helix 1 (amino acid residues 10–31) from Manduca ApoLp-III. The N-terminal region of Heliothis ApoLp-III corresponded to the a-helix 1 (residues 10–31) of Manduca ApoLp-III in which a struc-
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Fig. 3. Ultraviolet absorption spectrum of ApoLp-III. A solution of ApoLp-III (400 mgyml) was scanned from 240 to 340 nm.
tural study has been done (Wang et al., 1997). Helical wheels of Heliothis and Manduca ApoLpIII proteins showed nearly identical arrangements of hydrophobic amino acids. The predicted a-helix in Heliothis ApoLp-III N-terminus was an amphipathic helix with an angle of 808 on the hydrophobic face, which was exactly the same conformation as seen with Manduca ApoLp-III. The absorption spectrum (Fig. 3) from 240 to 340 nm of Heliothis ApoLp-III was nearly identical to the UV spectra of ApoLp-III from Manduca (Ryan et al., 1993) and Galleria (Wiesner et al., 1997), showing absorption maxima at 280, 268, 264 and 259 nm. These data indicate that ApoLpIII molecules of lepidopterans share common physico-chemical characteristics. Hemagglutination activity of ApoLp-III was positive with rabbit RBC (Fig. 4) even though ApoLp-III has not been reported as a lectin. The interaction of ApoLp-III with rabbit RBC was studied using Tween 80 and carbohydrate competition assays. Hemagglutination by ApoLp-III was completely inhibited by 0.02% Tween 80, whereas hemagglutination by the wheat germ agglutinin was not inhibited by 0.02% Tween 80 (Fig. 4). However, the carbohydrate competition assay for hemagglutination by ApoLp-III was negative for all the six carbohydrates tested. This indicated that the interaction between ApoLp-III and rabbit RBC apparently is a hydrophobic interaction. Heliothis
ApoLp-III, blotted onto nitrocellulose membrane, did not react with rhodamine-conjugated concanavalin A and rhodamine-conjugated wheat germ agglutinin. The amount of ApoLp-III in normal and immune hemolymph samples obtained from fifth instar larvae and 1-day-old pupae were analyzed by SDS gel electrophoresis. After bacterial infection, the ApoLp-III protein levels of both larval and pupal immune hemolymph increased when compared to normal hemolymph from both larvae and pupae (Fig. 5). Since normal and immune hemolymph samples were obtained on the same day of each larval and pupal developmental stage, this increase in ApoLp-III levels is thought to bemainly due to bacterial infection of the insects. However, the level of increase of ApoLp-III was much higher in pupal immune hemolymph than in larval immune hemolymph. 4. Discussion Apolipophorin III molecules have been isolated from insects by ultracentrifugation in a KBr density gradient (Wells et al., 1985; Kawooya et al., 1984; Wientzek et al., 1994). By taking advantage of its heat stability characteristic (Kanost et al., 1988; Wiesner et al., 1997), ApoLp-III was readily purified from Heliothis pupal hemolymph with a combination of heat treatment and two sequential
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Fig. 4. Effect of Tween 80 on hemagglutination activity of ApoLp-III. Patterns of agglutination by ApoLp-III were observed by light microscopy (100=). Bovine serum albumin (BSA) was used as the negative control; Wheat germ agglutinin (WGA) was used as the positive control. (a–c) are negative and (d–f) show positive agglutination.
chromatographies. This approach is efficient, reproducible and not time-consuming. From comparison of the N-terminal sequences and helical wheel diagram (Fig. 2), the structure of the full-length Heliothis ApoLp-III is likely to be very similar to the structure of Manduca ApoLp-III, although the entire secondary structure of Heliothis ApoLp-III was not elucidated here. All of the other six lepidopteran ApoLp-III molecules also showed great similarity in their amino acid arrangements with the a-helix1 of Manduca ApoLp-III regardless of their homology when their corresponding sequences were projected by the Schiffer–Edmundson helical wheel determination. Nearly identical absorption spectrum of the Heliothis ApoLp-III also supported a similarity in tertiary structure to the other lepidopteran ApoLp-
III molecules. The same conformations of ApoLpIII among the different lepidopterans, when compared here, indicate that the basic structure is important for the same biological function of ApoLp-III protein from different insects. This concept is supported by the striking structural similarity of data between Manduca ApoLp-III and Locusta ApoLp-III, although they share low (29%) amino acid sequence homology (Kanost et al., 1988; Breiter et al., 1991 Wang et al., 1997). Manduca ApoLp-III is known to be a nonglycosylated polypeptide (Kawooya et al., 1984), whereas Locusta ApoLp-III is a glycoprotein containing fucose, mannose and glucosamine (Chino and Yazawa, 1986). Heliothis ApoLp-III was found here not to be a glycoprotein, since it did not react with concanavalin A and wheat germ
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Fig. 5. SDS gel electrophoresis of immune and normal hemolymph samples from larvae and pupae. Lane 1, standard proteins (BioRad); Lane 2, normal larval hemolymph; Lane 3, immune larval hemolymph; Lane 4, normal pupal hemolymph; Lane 5, immune pupal hemolymph; Lane 6, purified Heliothis ApoLp-III. All of the hemolymph samples were similarly heattreated. An equal volume (8 ml) of each hemolymph sample was applied to each lane.
agglutinin. No lepidopteran ApoLp-III has been found to be a glycoprotein. Previous investigations of ApoLp-III protein have been mainly focused on lipid transport function and structural studies of this protein from Manduca and Locusta insects (Chino, 1985; Van der Horst, 1990; Blacklock and Ryan, 1994; Breiter et al., 1991; Wang et al., 1997). However, it has recently been found that additional functions of ApoLp-III are linked to insect innate immunity. It has been demonstrated that ApoLp-III can interact with hemocytes in vitro (Ferkovich et al., 1995) and in vivo (Dettloff et al., 2001) and is able to opsonize yeast cells for phagocytosis by isolated hemocytes in vitro (Wiesner et al., 1997). Imura et al. (1998) showed that Galleria ApoLpIII agglutinated red blood cells from mammals, and that this hemagglutination was inhibited by LPS, indicating that ApoLp-III binds LPS. Detoxification of injected LPS (Kato et al. 1994) is also likely mediated by ApoLp-III, since ApoLp-III is able to bind LPS and associate with LDLp (Dettloff et al., 2001). These activities strongly suggest that ApoLp-III plays a role in the insect immune response, which has been highlighted as an additional function of ApoLp-III. We believe that the hemagglutination and LPS binding activity of
Heliothis ApoLp-III is from its lipid binding properties due partially to hydrophobic interaction which was demonstrated here. We added a nonionic detergent, Tween 80, to the hemagglutination activity assay mixture to test if hemagglutination activity was abolished. Tween 80 disrupts hydrophobic interaction. As expected, hemagglutination by Heliothis ApoLp-III was completely inhibited by 0.02% Tween 80 (Fig. 4), but was not inhibited by any of the six carbohydrates found in rabbit RBC membrane. This indicates that hydrophobic interaction of ApoLp-III with rabbit RBC is required for hemagglutination activity and is probably needed for LPS binding. The hydrophobic characteristic of insect ApoLp-III is known to be essential for the protein to lipid interaction (Wells et al., 1987). This hydrophobic characteristic of insect ApoLp-III is, therefore, important for both lipid and cell interaction. Direct evidence of an ApoLp-III link to the insect immune response is that injection of ApoLpIII into larvae was shown to simultaneously increase both anti-E. coli activity and lysozyme activity as occurs after injection of live bacteria (Wiesner et al., 1997). Our SDS gel electrophoresis profile of normal and immune larval and pupal hemolymph showed that the amount of endogenous ApoLp-III protein in immune hemolymph increased after bacterial injection during the fifth instar larval stage and early pupal stage (Fig. 5). In a previous study, the ApoLp-III mRNA in M. sexta was shown to be absent in the fat body during the late fifth instar larval stage and in the first 8 days of the pupal stage (Cole and Wells, 1990). However, we were able to detect low levels of ApoLp-III protein in earlier (1-day-old) pupal hemolymph of H. virescens (Fig. 5). In addition, the ApoLp-III level was greatly increased in Heliothis immune hemolymph. This strongly suggests that ApoLp-III is inducible and thereby involved in the insect immune response by possibly increasing the production of antibacterial proteins against bacterial infection. This hypothesis is also supported by our previous data that showed an increase of antibacterial activity in pupal immune hemolymph following bacterial injection (Chung and Ourth, 2000). Future investigations may show how ApoLp-III is regulated after bacterial infection of insects and how up-regulation of ApoLp-III is associated with induction of an antibacterial response. These stud-
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ies may provide a better understanding of the insect immune response, which could lead to enhanced control of insects in the environment. Acknowledgments We thank the Department of Microbiology and Molecular Cell Sciences at the University of Memphis for research support. Mass spectrometry analysis was done in the Department of Chemistry at the University of Memphis by Dr Chhabil Dass. We thank Dr James Davenport, St. Jude Children’s Research Hospital for assistance in analysis of the Schiffer–Edmundson helical wheel data. K.T. Chung is a scholarship recipient of the Ha-Chung Foundation, Seoul, Korea. References Bar-Nun, S., Gershoni, J., 1994. Protein-blot analysis of glycoproteins and lectin overlays. In: Celis, J.E. (Ed.), Cell Biology: A Laboratory Handbook, Vol. 3. Academic Press, Inc, San Diego, CA, pp. 326–327. Blacklock, B.J., Ryan, R.O., 1994. Hemolymph lipid transport. Insect Biochem. Mol. Biol. 24, 855–873. Breiter, D.R., Kanost, M.R., Benning, M.M., et al., 1991. Molecular structure of an apolipoprotein determined at 2.5˚ resolution. Biochemistry 30, 603–608. A Burks, C.S., Shelby, K.S., Chippendale, G.M., 1992. Characteristics of apolipophorin-III of the southwestern corn borer, Daitraea Grandiosella. Insect Biochem. Mol. Biol. 22, 905–915. Chino, H., 1985. Lipid transport: biochemistry of hemolymph lipophorin. In: Kerkut, G., Gilbert, L. (Eds.), Comprehensive Insect Physiology Biochemistry and Pharmacology, Vol. 10. Pergamon Press, Elmsford, NY, pp. 115–135. Chino, H., Yazawa, M., 1986. Apolipophorin III in locusts: purification and characterization. J. Lipid Res. 27, 377–385. Chung, K.T., Ourth, D.D., 2000. Viresin: a novel antibacterial protein from immune hemolymph of Heliothis virescens pupae. Eur. J. Biochem. 267, 677–683. Cole, K.D., Fernando-Warnakulasuriya, G.J., Boguski, M.S., et al., 1987. Primary structure and comparative sequence analysis of an insect apolipoprotein. J. Biol. Chem. 262, 11794–11800. Cole, K.D., Wells, M.A., 1990. A comparison of adult and larval Manduca sexta apolipophorin-III. Insect Biochem. 20, 373–380. Dettloff, M., Kaiser, B., Wiesner, A., 2001. Localization of injected apolipophorin III in vivo—new insights into the immune activation process directed by this protein. J. Insect Physiol. 47, 789–797. Ferkovich, S.M., Miller, S.G., Oberlander, H., 1995. Multicellular-vesicle-promoting polypeptide from Trichoplusia ni: tissue distribution and N-terminal sequence. Arch. Insect Biochem. Physiol. 29, 381–390.
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versions in adult Manduca sexta. J. Biol. Chem. 262, 4172–4176. Wientzek, M., Kay, C.M., Oikawa, K., Ryan, R.O., 1994. Binding of insect apolipophorin III to dimyristoylphosphatidylcholine vesicles. J. Biol. Chem. 269, 4605–4612. Wiesner, A., Losen, S., Kopacek, P., Weise, C., Gotz, P., 1997. Isolated apolipophorin III from Galleria mellonella stimu-
lates the immune reactions of this insect. J. Insect Physiol. 43, 383–391. Yamauchi, Y., Hoeffer, C., Yamamoto, A., et al., 2000. cDNA and deduced amino acid sequences of apolipophorin-IIIs from Bombyx mori and Bombyx mandarina. Arch. Insect Biochem. Physiol. 43, 16–21.
Purification and characterization of apolipophorin III from immune hemolymph of Heliothis virescens pupae Kyung Tae Chung1, Donald D. Ourth* Department of Microbiology and Molecular Cell Sciences, The University of Memphis, Memphis, TN 38152, USA Received 28 November 2001; received in revised form 20 February 2002; accepted 3 March 2002
Abstract Apolipophorin III (ApoLp-III) from Heliothis virescens pupae was purified by heat-treatment followed by Sephadex G-50 filtration and reverse phase-HPLC. The molecular mass of the purified ApoLp-III was determined as 17 965.9"5 Da by mass spectrometry. The N-terminal sequence confirmed the protein as ApoLp-III with homology of 56–83% to other insect ApoLp-III molecules. The amino acid spatial arrangement of the predicted a-helix 1 of Heliothis ApoLp-III was nearly identical to that of the amphipatic a-helix 1 of Manduca sexta ApoLp-III. The absorption spectrum from 240–340 nm of the Heliothis ApoLp-III was the same as the UV spectra of ApoLp-III from Manduca sexta and Galleria mellonella, showing absorption maxima at 280, 268, 264 and 259 nm. These results indicated that the primary structure of ApoLp-III is conserved in lepidopterans. The Heliothis ApoLp-III was not a glycoprotein and showed hemagglutination activity against rabbit red blood cells. This hemagglutination activity was abolished by Tween 80, but not by six different carbohydrates. Hydrophobic interaction of ApoLp-III with red blood cells agreed with structural studies since ApoLp-III binds lipid through hydrophobic interaction after conformational change. Bacterial injection apparently increased the amount of ApoLp-III in immune hemolymph when compared with normal hemolymph, and may indicate that ApoLpIII plays a role in insect immunity. 䊚 2002 Elsevier Science Inc. All rights reserved. Keywords: Apolipophorin III; N-terminal sequence; a-Helix; UV absorption spectrum; Hemagglutination; Heliothis virescens
1. Introduction Apolipophorin-III (ApoLp-III) is an 18–20-kDa amphipathic apolipoprotein (Cole et al., 1987) found in insect hemolymph and has an essential role in lipid transport (Wells et al., 1987). Unlike other insect apolipoproteins (apolipophorin I and II), which are integral non-exchangeable apolipoproteins, ApoLp-III is a water-soluble and exchangeable apolipoprotein which reversibly associates with lipophorin particles (Narayanas*Corresponding author. Tel.: q1-901-678-2950; fax: q1901-678-4457. E-mail address: [email protected] (D.D. Ourth). 1 Current address: Department of Tumor Cell Biology, St. Jude Children’s Research Hospital, 332 North Lauderdale St., Memphis, TN 38105, USA.
wami and Ryan, 1997). ApoLp-III normally exists as a monomeric, lipid-free hemolymph protein. When ApoLp-III is required for lipid transport, the protein associates with high-density lipophorin (HDLp) to form a low-density lipophorin (LDLp) by stabilizing the increment of lipid–water interface resulting from diacylglycerol uptake (Wells et al., 1987). Its function in lipid transport has been reviewed with Locusta migratoria and Manduca sexta ApoLp-III (Chino, 1985; Van der Horst, 1990; Blacklock and Ryan, 1994). The three-dimensional structure of ApoLp-III was determined by X-ray crystallography of Locusta migratoria ApoLp-III (Breiter et al., 1991) and by the NMR technique with Manduca sexta ApoLp-III (Wang et al., 1997). Although
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there is low homology (29%) in the amino acid sequences of Locusta and Manduca ApoLp-III molecules, these studies showed great similarity in structure between the two ApoLp-III proteins. Both Locusta and Manduca ApoLp-III molecules consist of five discrete anti-parallel a-helices, which are connected by short loops. The hydrophobic surfaces of each helix face inward to the center of the bundle. These structural studies supported the hypothesis that ApoLp-III undergoes a large conformational change that opens hydrophobic surfaces when binding to lipophorin (Kawooya et al., 1986; Wells et al., 1987). These hydrophobic surfaces of each helix are then interacting with lipid. Apolipophorin III has also been reported to have functions related to immune responses against bacterial infection in insects. In Galleria mellonella, intrahemocoelic injection of purified ApoLpIII into larvae increased antibacterial activity in hemolymph, and ApoLp-III enhanced phagocytic activity of isolated hemocytes in vitro (Wiesner et al., 1997). Kato et al. (1994) demonstrated that lipophorin of Bombyx mori was able to detoxify bacterial lipopolysaccharide (LPS) by forming an LPS-lipophorin complex. Hemagglutination characteristics of Galleria ApoLp-III were demonstrated (Imura et al., 1998). Antimicrobial (Tada et al., 1993) and antiviral (Srinivas et al., 1990) activities of human apolipoprotein A-1 were also reported. Heliothis virescens (tobacco budworm) is an insect pest of many important crops including cotton. We have been studying antibacterial factors from H. virescens after bacterial infection (Ourth et al., 1994; Lockey and Ourth, 1996; Chung and Ourth, 2000). We investigated the role of ApoLpIII in the immune hemolymph of H. virescens, since ApoLp-III has not before been identified or studied in association with an immune response. We describe here our study of Heliothis ApoLpIII focusing on its purification and characterization along with a comparison of the physico-chemical and molecular characteristics of Heliothis ApoLpIII with other insect ApoLp-III molecules. We also describe the up-regulation of ApoLp-III by bacterial injection and a mechanism of ApoLp-III hemagglutination activity with red blood cells, which has not been demonstrated before.
2. Materials and methods 2.1. Apolipophorin III purification Immune and normal hemolymph samples were obtained as described before (Chung and Ourth 2000). A 5= dilution of immune hemolymph with saline was heat-treated at 100 8C for 1 min with continuous agitation. Hemolymph samples (0.5 ml) were heat-treated at one time. After heat treatment, the samples were cooled immediately on ice and then centrifuged for 5 min at 14 000 rev.ymin using an Eppendorf centrifuge. The supernatant was applied to a Sephadex G-50 gel filtration column (1=50 cm) equilibrated with 0.1 M ammonium acetate buffer, pH 6.4. The column was eluted with the same buffer at 15 mlyh, and 1 ml per tube was collected. The fractions containing ApoLp-III were then pooled. The sample was concentrated using a 10-kDa molecular weight cutoff ultrafiltration membrane (10 MWCO Centricon, Amicon, Beverly, MA, USA). The concentrated sample was adjusted to contain 0.1% trifluoroacetic acid (TFA) as a final concentration and applied to a 4.6=250-mm Microsorb-MW C18 reverse-phase high pressure liquid chromatography (RP-HPLC), particle size 5 m, pore size 100 ˚ (Rainin, Woburn, MA, USA) having a Perisorb A C18 pre-column (Upchurch Scientific, Oak Harbor, WA, USA), using an ISCO HPLC system (ISCO, Lincoln, NE, USA). Proteins were eluted with a linear gradient (0–60%) of acetonitrile containing 0.1% TFA over a 120-min period. The flow rate was 1 mlymin with optical density being monitored at 214 nm. Protein peak fractions were vacuum dried by SpeedVac and reconstituted with 50 ml of saline or 0.1 M ammonium acetate buffer, pH 6.4, depending on subsequent experiments. Protein samples were analyzed on SDS-PAGE gels according to the method of Schagger and von Jagow (1987). Protein concentration of samples was determined by the Bicinchoninic acid (BCA) protein assay according to instructions (Pierce Chemical Co., Rockford, IL, USA). Bovine serum albumin was used as the standard protein. 2.2. N-terminal amino acid sequence The N-terminal amino acid sequence of the purified ApoLp-III was determined by the Center for Biotechnology at St. Jude Children’s Research
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Hospital, Memphis, TN, USA. The instrument used was a Perkin-Elmer Applied Biosystems 494 cLC protein sequencer with standard cycles supplied by the manufacturer. 2.3. Mass spectrometry Molecular mass of the purified Heliothis ApoLp-III was determined by electrospray ionization mass spectrometry (ESI-MS) using VG Platform II (Micromass, Beverly, MA, USA). 2.4. Schiffer–Edmundson helical wheel projection A helical wheel of the potential helix domain from the N-terminus of Heliothis ApoLp-III was generated by using the Wisconsin Package software version 9.1 from Genetics Computer Group (GCG), Madison, WI, USA. The helical wheel of the predicted a-helix domain from the N-terminus of Heliothis ApoLp-III was compared with that from the a-helix 1 domain of Manduca ApoLpIII. 2.5. Ultraviolet absorption spectrum of ApoLp-III The absorption spectrum of ApoLp-III scanned from 240 to 340 nm with a Shimazu 1601 spectrophotometer. A protein sample ml) in 0.85% saline (approx. 400 mgyml) used.
was UV(0.5 was
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ApoLp-III protein (5.6 mM) were mixed together in a U-bottom microtiter plate to give a final concentration of 200 mM of six different carbohydrates. The mixture was incubated for 10 min at room temperature prior to the addition of trypsin-treated RBC. Twenty-five microliters of 4% trypsin-treated rabbit RBC were added and incubated for 30 min at 37 8C. The mixture was continuously incubated for 18 h at 4 8C. Saline and the carbohydrate solutions listed below without ApoLp-III were used as negative controls. The following six carbohydrates were used for the competition assay: L-fucose, D-galactose, D-glucose, D-mannose, N-acetyl-D-galactosamine and Nacetyl-D-glucosamine. 2.8. Effect of Tween 80 on interaction of ApoLpIII with rabbit red blood cells Tween 80 was used to study the mechanism of interaction between ApoLp-III and rabbit RBC. The final concentration of Tween 80 was 0.02% in the hemagglutination mixture that was tested. Fifty microliters of the assay mixture contained the purified ApoLp-III, 2% red blood cell suspension and Tween 80. Buffer solutions were 0.85% NaCl saline, 50 mM Tris–HCl buffered saline, pH 7.2. The mixture was incubated as previously done for the hemagglutination assay. Wheat germ agglutinin (1 mgyml) was used as the positive control, and bovine serum albumin (1 mgyml) was used as the negative control.
2.6. Hemagglutination assay 2.9. Lectin blotting Serial two-fold dilutions of the purified ApoLpIII were prepared with 0.85% saline in a microtiter U-bottom plate with a final volume of 25 mlywell. To each well was added an equal volume of rabbit red blood cell (RBC) suspension (108 cellsyml). The mixture was incubated for 30 min at 37 8C and then incubated overnight at 4 8C. Positive hemagglutination was judged by matt formation at the bottom of the plate well and by microscopic observation. 2.7. Hemagglutination competition assay Trypsin-treated rabbit RBC were used for hemagglutination competition. Trypsin-treated rabbit RBC were basically prepared according to Nowak et al. (1976). Twenty microliters of
Lectin blotting was used to test if Heliothis ApoLp-III is a glycoprotein. This was done with minor modification according to Bar-Nun and Gershoni (1994). Approximately 4 mg of ApoLp-III in saline was applied directly onto a nitrocellulose membrane, and the nitrocellulose membrane then blocked with 2% non-fat dry milk for 2 h. The membrane was washed three times for 1 h with washing buffer (25 mM Tris–HCl, pH 7.4, 150 mM NaCl, 1 mM CaCl2, 0.5 mM MgCl2) and incubated with rhodamine-conjugated concanavalin A and rhodamine-conjugated wheat germ agglutinin. Under UV light, fluorescence of glycoprotein on the membrane was examined. A positive control for lectin blotting was 10 mg of ovalbumin in MilliQ water.
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Fig. 1. (a) Reverse phase-HPLC of the pooled fraction sample from Sephadex G-50 gel filtration column after 10 MWCO concentration. The sample was applied to a Microsorb-MW C18 column equilibrated in 0.1% TFA. A linear gradient of acetonitrile (0–60%) containing 0.1% TFA was applied at a flow rate of 1 mlymin over a period of 120 min. Each peak fraction was dried by SpeedVac and resuspended in 50 ml of sterile water. The bar represents fractions (118–120) of ApoLp-III. (b) SDS gel electrophoresis of purified ApoLp-III protein. The fraction No. 120 from RP-HPLC was loaded onto a SDS-PAGE gel. Lane 1 contained standard proteins; Lane 2 and Lane 3 contained samples of fraction No. 120 under reducing and non-reducing conditions, respectively. The gel was visualized by silver staining.
3. Results Apolipophorin-III purification was accomplished utilizing its heat stable characteristics (Kanost et al., 1988; Wiesner et al., 1997). Heat treatment was employed as the first step of purification followed by two sequential chromatogra-
phies, Sephadex G-50 gel filtration and RP-HPLC. Fractions (118–120; Fig. 1a) obtained by RPHPLC contained a homogenous 21 kDa ApoLpIII band on an SDS gel under both reducing and non-reducing conditions, indicating that the protein does not have intra-disulfide bonds (Fig. 1b). The
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Fig. 2. (a) A comparison of the N-terminal amino acid sequences of seven lepidopteran ApoLp-III proteins with Heliothis ApoLp-III. Gap (dash line) was introduced to obtain the maximum sequence homology. X represents an unknown amino acid. The box represents a-helix 1 of Manduca ApoLp-III. All of the sequences start at the first amino acid of mature ApoLp-III proteins. Homologous residues are in bold. Percentage homologies among the ApoLp-III proteins to H. virescens are given. M. sexta: Manduca sexta, A. convolvuli: Agrius convolvuli (NIH GENBANK accession AF001632), B. mandarina: Bombyx mandarina, B. mori: Bombyx mori, D. grandiosella: Diatraea grandiosella, G. mellonella: Galleria mellonella, T. ni: Trichoplusia ni, H. virescens: Heliothis virescens. (b) Schiffer– Edmundson helical wheel projection of the potential a-helix of Heliothis ApoLp-III N-terminus. Helical wheel was projected with an angle of 1008. Hydrophobic amino acids are boxed.
molecular mass of Heliothis ApoLp-III was determined to be 17 965.9"5 Da by mass spectrometry. This result was very similar to the molecular mass of ApoLp-III from G. mellonella larvae which was determined to be 18 077"2 Da by mass spectrometry (Wiesner et al., 1997). The N-terminal amino acid sequence from the RP-HPLC fractions confirmed that the 21 kDa protein was ApoLp-III (Fig. 2a). The N-terminal amino acid sequence of the Heliothis ApoLp-III protein was 56–83% homologous with the Nterminal sequences of ApoLp-III from seven other lepidopteran insects (Cole et al., 1987; Burks et al., 1992; Ferkovich et al., 1995; Wiesner et al.,
1997; Yamauchi et al., 2000; NIH GENBANK accession AF001632), indicating that the ApoLpIII protein is highly conserved in lepidopterans. The Schiffer–Edmundson helical wheel was predicted for the sequenced N-terminal region of Heliothis ApoLp-III using the GCG computer program (Fig. 2b). The predicted Schiffer–Edmundson helical wheel of the 7–29 amino acid residues of Heliothis ApoLp-III was compared with the spatial arrangement of amino acids of a-helix 1 (amino acid residues 10–31) from Manduca ApoLp-III. The N-terminal region of Heliothis ApoLp-III corresponded to the a-helix 1 (residues 10–31) of Manduca ApoLp-III in which a struc-
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Fig. 3. Ultraviolet absorption spectrum of ApoLp-III. A solution of ApoLp-III (400 mgyml) was scanned from 240 to 340 nm.
tural study has been done (Wang et al., 1997). Helical wheels of Heliothis and Manduca ApoLpIII proteins showed nearly identical arrangements of hydrophobic amino acids. The predicted a-helix in Heliothis ApoLp-III N-terminus was an amphipathic helix with an angle of 808 on the hydrophobic face, which was exactly the same conformation as seen with Manduca ApoLp-III. The absorption spectrum (Fig. 3) from 240 to 340 nm of Heliothis ApoLp-III was nearly identical to the UV spectra of ApoLp-III from Manduca (Ryan et al., 1993) and Galleria (Wiesner et al., 1997), showing absorption maxima at 280, 268, 264 and 259 nm. These data indicate that ApoLpIII molecules of lepidopterans share common physico-chemical characteristics. Hemagglutination activity of ApoLp-III was positive with rabbit RBC (Fig. 4) even though ApoLp-III has not been reported as a lectin. The interaction of ApoLp-III with rabbit RBC was studied using Tween 80 and carbohydrate competition assays. Hemagglutination by ApoLp-III was completely inhibited by 0.02% Tween 80, whereas hemagglutination by the wheat germ agglutinin was not inhibited by 0.02% Tween 80 (Fig. 4). However, the carbohydrate competition assay for hemagglutination by ApoLp-III was negative for all the six carbohydrates tested. This indicated that the interaction between ApoLp-III and rabbit RBC apparently is a hydrophobic interaction. Heliothis
ApoLp-III, blotted onto nitrocellulose membrane, did not react with rhodamine-conjugated concanavalin A and rhodamine-conjugated wheat germ agglutinin. The amount of ApoLp-III in normal and immune hemolymph samples obtained from fifth instar larvae and 1-day-old pupae were analyzed by SDS gel electrophoresis. After bacterial infection, the ApoLp-III protein levels of both larval and pupal immune hemolymph increased when compared to normal hemolymph from both larvae and pupae (Fig. 5). Since normal and immune hemolymph samples were obtained on the same day of each larval and pupal developmental stage, this increase in ApoLp-III levels is thought to bemainly due to bacterial infection of the insects. However, the level of increase of ApoLp-III was much higher in pupal immune hemolymph than in larval immune hemolymph. 4. Discussion Apolipophorin III molecules have been isolated from insects by ultracentrifugation in a KBr density gradient (Wells et al., 1985; Kawooya et al., 1984; Wientzek et al., 1994). By taking advantage of its heat stability characteristic (Kanost et al., 1988; Wiesner et al., 1997), ApoLp-III was readily purified from Heliothis pupal hemolymph with a combination of heat treatment and two sequential
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Fig. 4. Effect of Tween 80 on hemagglutination activity of ApoLp-III. Patterns of agglutination by ApoLp-III were observed by light microscopy (100=). Bovine serum albumin (BSA) was used as the negative control; Wheat germ agglutinin (WGA) was used as the positive control. (a–c) are negative and (d–f) show positive agglutination.
chromatographies. This approach is efficient, reproducible and not time-consuming. From comparison of the N-terminal sequences and helical wheel diagram (Fig. 2), the structure of the full-length Heliothis ApoLp-III is likely to be very similar to the structure of Manduca ApoLp-III, although the entire secondary structure of Heliothis ApoLp-III was not elucidated here. All of the other six lepidopteran ApoLp-III molecules also showed great similarity in their amino acid arrangements with the a-helix1 of Manduca ApoLp-III regardless of their homology when their corresponding sequences were projected by the Schiffer–Edmundson helical wheel determination. Nearly identical absorption spectrum of the Heliothis ApoLp-III also supported a similarity in tertiary structure to the other lepidopteran ApoLp-
III molecules. The same conformations of ApoLpIII among the different lepidopterans, when compared here, indicate that the basic structure is important for the same biological function of ApoLp-III protein from different insects. This concept is supported by the striking structural similarity of data between Manduca ApoLp-III and Locusta ApoLp-III, although they share low (29%) amino acid sequence homology (Kanost et al., 1988; Breiter et al., 1991 Wang et al., 1997). Manduca ApoLp-III is known to be a nonglycosylated polypeptide (Kawooya et al., 1984), whereas Locusta ApoLp-III is a glycoprotein containing fucose, mannose and glucosamine (Chino and Yazawa, 1986). Heliothis ApoLp-III was found here not to be a glycoprotein, since it did not react with concanavalin A and wheat germ
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Fig. 5. SDS gel electrophoresis of immune and normal hemolymph samples from larvae and pupae. Lane 1, standard proteins (BioRad); Lane 2, normal larval hemolymph; Lane 3, immune larval hemolymph; Lane 4, normal pupal hemolymph; Lane 5, immune pupal hemolymph; Lane 6, purified Heliothis ApoLp-III. All of the hemolymph samples were similarly heattreated. An equal volume (8 ml) of each hemolymph sample was applied to each lane.
agglutinin. No lepidopteran ApoLp-III has been found to be a glycoprotein. Previous investigations of ApoLp-III protein have been mainly focused on lipid transport function and structural studies of this protein from Manduca and Locusta insects (Chino, 1985; Van der Horst, 1990; Blacklock and Ryan, 1994; Breiter et al., 1991; Wang et al., 1997). However, it has recently been found that additional functions of ApoLp-III are linked to insect innate immunity. It has been demonstrated that ApoLp-III can interact with hemocytes in vitro (Ferkovich et al., 1995) and in vivo (Dettloff et al., 2001) and is able to opsonize yeast cells for phagocytosis by isolated hemocytes in vitro (Wiesner et al., 1997). Imura et al. (1998) showed that Galleria ApoLpIII agglutinated red blood cells from mammals, and that this hemagglutination was inhibited by LPS, indicating that ApoLp-III binds LPS. Detoxification of injected LPS (Kato et al. 1994) is also likely mediated by ApoLp-III, since ApoLp-III is able to bind LPS and associate with LDLp (Dettloff et al., 2001). These activities strongly suggest that ApoLp-III plays a role in the insect immune response, which has been highlighted as an additional function of ApoLp-III. We believe that the hemagglutination and LPS binding activity of
Heliothis ApoLp-III is from its lipid binding properties due partially to hydrophobic interaction which was demonstrated here. We added a nonionic detergent, Tween 80, to the hemagglutination activity assay mixture to test if hemagglutination activity was abolished. Tween 80 disrupts hydrophobic interaction. As expected, hemagglutination by Heliothis ApoLp-III was completely inhibited by 0.02% Tween 80 (Fig. 4), but was not inhibited by any of the six carbohydrates found in rabbit RBC membrane. This indicates that hydrophobic interaction of ApoLp-III with rabbit RBC is required for hemagglutination activity and is probably needed for LPS binding. The hydrophobic characteristic of insect ApoLp-III is known to be essential for the protein to lipid interaction (Wells et al., 1987). This hydrophobic characteristic of insect ApoLp-III is, therefore, important for both lipid and cell interaction. Direct evidence of an ApoLp-III link to the insect immune response is that injection of ApoLpIII into larvae was shown to simultaneously increase both anti-E. coli activity and lysozyme activity as occurs after injection of live bacteria (Wiesner et al., 1997). Our SDS gel electrophoresis profile of normal and immune larval and pupal hemolymph showed that the amount of endogenous ApoLp-III protein in immune hemolymph increased after bacterial injection during the fifth instar larval stage and early pupal stage (Fig. 5). In a previous study, the ApoLp-III mRNA in M. sexta was shown to be absent in the fat body during the late fifth instar larval stage and in the first 8 days of the pupal stage (Cole and Wells, 1990). However, we were able to detect low levels of ApoLp-III protein in earlier (1-day-old) pupal hemolymph of H. virescens (Fig. 5). In addition, the ApoLp-III level was greatly increased in Heliothis immune hemolymph. This strongly suggests that ApoLp-III is inducible and thereby involved in the insect immune response by possibly increasing the production of antibacterial proteins against bacterial infection. This hypothesis is also supported by our previous data that showed an increase of antibacterial activity in pupal immune hemolymph following bacterial injection (Chung and Ourth, 2000). Future investigations may show how ApoLp-III is regulated after bacterial infection of insects and how up-regulation of ApoLp-III is associated with induction of an antibacterial response. These stud-
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ies may provide a better understanding of the insect immune response, which could lead to enhanced control of insects in the environment. Acknowledgments We thank the Department of Microbiology and Molecular Cell Sciences at the University of Memphis for research support. Mass spectrometry analysis was done in the Department of Chemistry at the University of Memphis by Dr Chhabil Dass. We thank Dr James Davenport, St. Jude Children’s Research Hospital for assistance in analysis of the Schiffer–Edmundson helical wheel data. K.T. Chung is a scholarship recipient of the Ha-Chung Foundation, Seoul, Korea. References Bar-Nun, S., Gershoni, J., 1994. Protein-blot analysis of glycoproteins and lectin overlays. In: Celis, J.E. (Ed.), Cell Biology: A Laboratory Handbook, Vol. 3. Academic Press, Inc, San Diego, CA, pp. 326–327. Blacklock, B.J., Ryan, R.O., 1994. Hemolymph lipid transport. Insect Biochem. Mol. Biol. 24, 855–873. Breiter, D.R., Kanost, M.R., Benning, M.M., et al., 1991. Molecular structure of an apolipoprotein determined at 2.5˚ resolution. Biochemistry 30, 603–608. A Burks, C.S., Shelby, K.S., Chippendale, G.M., 1992. Characteristics of apolipophorin-III of the southwestern corn borer, Daitraea Grandiosella. Insect Biochem. Mol. Biol. 22, 905–915. Chino, H., 1985. Lipid transport: biochemistry of hemolymph lipophorin. In: Kerkut, G., Gilbert, L. (Eds.), Comprehensive Insect Physiology Biochemistry and Pharmacology, Vol. 10. Pergamon Press, Elmsford, NY, pp. 115–135. Chino, H., Yazawa, M., 1986. Apolipophorin III in locusts: purification and characterization. J. Lipid Res. 27, 377–385. Chung, K.T., Ourth, D.D., 2000. Viresin: a novel antibacterial protein from immune hemolymph of Heliothis virescens pupae. Eur. J. Biochem. 267, 677–683. Cole, K.D., Fernando-Warnakulasuriya, G.J., Boguski, M.S., et al., 1987. Primary structure and comparative sequence analysis of an insect apolipoprotein. J. Biol. Chem. 262, 11794–11800. Cole, K.D., Wells, M.A., 1990. A comparison of adult and larval Manduca sexta apolipophorin-III. Insect Biochem. 20, 373–380. Dettloff, M., Kaiser, B., Wiesner, A., 2001. Localization of injected apolipophorin III in vivo—new insights into the immune activation process directed by this protein. J. Insect Physiol. 47, 789–797. Ferkovich, S.M., Miller, S.G., Oberlander, H., 1995. Multicellular-vesicle-promoting polypeptide from Trichoplusia ni: tissue distribution and N-terminal sequence. Arch. Insect Biochem. Physiol. 29, 381–390.
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