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Ligand blot analysis of juvenile hormone esterase binding proteins
in Manduca sexta L.
Madasamy Shanmugavelu
1,a, Larysa Porubleva
b, Parag Chitnis
b, Bryony C. Bonning
a,*aDepartment of Entomology and Program in Genetics, Iowa State University, Ames, IA 50011, USA bDepartment of Biochemistry, Biophysics and Molecular Biology, Iowa State University, Ames, IA 50011, USA
Received 10 February 2000; received in revised form 22 May 2000; accepted 25 May 2000
Abstract
Biotinylated recombinant juvenile hormone esterase (JHE) was used for ligand blotting of proteins from fat body tissue and pericardial athrocytes of Manduca sexta. Proteins were separated by SDS–polyacrylamide gel electrophoresis or by two-dimensional electrophoresis. Eight putative JHE binding proteins were detected in fat body tissue and in pericardial athrocytes of both M. sexta and Heliothis virescens. The predominant bands were 29, 72, 75, 125 and 240 kDa, with minor bands at 50, 80 and 205 kDa. All putative JHE binding proteins were present from the second through to the fifth instar larvae of M. sexta. On wide-range isoelectric focusing, the 29 kDa JHE binding protein separated into three species with isoelectric points of 6.5, 6.6 and 6.8. Biotinylated-JHE did not bind recombinant M. sexta-derived juvenile hormone binding protein. The mutant JHE with mutations K29R and K524R binds weakly to the JHE binding protein P29, relative to binding of wild-type JHE [Shanmugavelu et al., J. Biol. Chem., 275 (2000) 1802–1806]. A similar reduction in binding was not seen for the 29 kDa binding protein identified here in pericardial athrocytes by ligand blot. This result is discussed.2001 Elsevier Science Ltd. All rights reserved.
Keywords: Juvenile hormone esterase binding proteins; Manduca sexta; Pericardial cells; Fat body; Ligand blot
1. Introduction
Insect growth, development and reproduction are regulated by the combined actions of juvenile hormone (JH) and ecdysteroids. Juvenile hormone esterase (JHE; EC 3.1.1.1) hydrolyzes JH to the biologically inactive JH acid. JHE is the predominant JH hydrolytic enzyme in the hemolymph while JH epoxide hydrolase (JHEH; EC 3.3.2.3), which produces JH diol, is intracellular. The titer of JHE in the hemolymph is regulated by the rate of biosynthesis in the fat body and epidermis (Wroblewski et al., 1990), and by the rate of removal from the hemolymph by pericardial athrocytes (pericardial cells; Locke and Russell, 1998). JHE is removed by receptor-mediated endocytosis and degraded
* Corresponding author. Tel: +1-515-294-1989; fax: + 1-515-294-5957.
E-mail address: [email protected] (B.C. Bonning). 1 Present address: Torrey Pines Institute for Molecular Studies,
3550 General Atomics Court, San Diego, CA 92121, USA.
0965-1748/01/$ - see front matter2001 Elsevier Science Ltd. All rights reserved. PII: S 0 9 6 5 - 1 7 4 8 ( 0 0 ) 0 0 1 0 4 - 1
in lysosomes (Bonning et al., 1997a; Booth et al., 1992; Brockhouse et al., 1999; Ichinose et al., 1992a,b).
observation is that JHE-KK displays altered binding to a protein involved in targeting JHE to the lysosome, and/or in lysosomal degradation. In a recent study, we screened a pericardial cell cDNA phage display library derived from Manduca sexta and identified a JHE bind-ing protein, P29, with reduced bindbind-ing to JHE-KK rela-tive to wild-type and control mutant enzymes (Shanmugavelu et al., 2000). In the present study, we used ligand blot to examine other JHE binding proteins in the fat body and pericardial cells of M. sexta.
2. Materials and methods
2.1. Sample preparation and polyacrylamide gel electrophoresis (PAGE)
Larvae of Manduca sexta and Heliothis virescens were cooled on ice for 30 min and fat body and pericar-dial cell complexes (pericarpericar-dial athrocytes and associa-ted dorsal aorta) removed. Tissues from three or four larvae were pooled and homogenized in 20 mM Tris– HCl pH 6.8, supplemented with 150 mM NaCl, 10 mM ethylenediamine-N,N,N9,N9-tetraacetic acid (EDTA) and 10 mM phenylmethylsuphonyl fluoride (PMSF). Hemo-lymph samples were taken and diluted 1:2 in the same buffer. Samples were centrifuged at 5200g for 5 min and protein concentrations of the supernatants determined (BioRad Protein Assay). Proteins were separated by SDS–polyacrylamide gel electrophoresis (SDS–PAGE) in 12% gels (10µg per lane). SDS–PAGE broad range standards (BioRad) were used for protein gels, while prestained SDS–PAGE broad range standards (BioRad) were used for ligand blotting experiments.
Purified samples of M. sexta-derived recombinant juv-enile hormone binding protein (JHBP; Touhara et al., 1993) and the recombinant JHE binding protein P29 (Shanmugavelu et al., 2000) were separated by SDS– PAGE (2µg per lane) for ligand blot analysis. Baculo-virus-expressed JHBP and E. coli-expressed P29 were purified as described previously (Touhara et al., 1993; Shanmugavelu et al., 2000).
2.2. Two-dimensional electrophoresis
For two-dimensional electrophoresis, pericardial cell tissue from at least 10 fifth instar M. sexta, or fat body tissue from four fourth instar larvae, were homogenized in phosphate-buffered saline (PBS; pH 7.4, sup-plemented with 10 mM EDTA and 10 mM PMSF) and then centrifuged at 5200g for 10 min. After storing the supernatant at220°C for a minimum of 24 h, centrifug-ation was repeated. Proteins in the supernatant were then precipitated by adding an equal volume of ice-cold ace-tone with 20% trichloroacetic acid (TCA) (w/v), incubat-ing on ice for 30 min, then centrifugincubat-ing for 5 min at
10,000g. The pellet was washed by resuspension in 1 ml acetone, pelleted by centrifugation (5 min at 10,000g), dried, resuspended in 100µl immobilized pH gradient (IPG) rehydration buffer (Pharmacia; 9 M urea, 2% Tri-ton X-100, 20 mM dithiothreitol (DTT)) and rehydrated for 3 h.
First dimensional separation of proteins was perfor-med in 7 cm Immobiline DryStrip IPG gels at 20°C with a pH gradient of 3 to 10 using an IPGphor IEF system (Amersham Pharmacia Biotech). Samples (100µg per strip) were separated at 500 V for 30 min, 1000 V for 30 min and 8000 V for 6–12 h. Second dimensional sep-aration was carried out using 8–16% linear gradient SDS preparative gels (BioRad). Proteins were visualized by silver staining (Harlow and Lane, 1988) and were ana-lyzed using Melanie II 2-D PAGE software (BioRad).
2.3. Preparation of biotin-labeled JHE and ligand blot analysis
Recombinant JHE and the JHE mutants 29, JHE-524 and JHE-KK (Bonning et al., 1997b) were produced in serum-free (ExCell405, JRH BioSciences) insect cell culture by infection of Sf21 cells (Vaughn et al., 1977) with recombinant baculoviruses (Bonning et al., 1992, 1997b). Recombinant JHE was purified as described (Edgar et al., 2000) using Q-sepharose ion-exchange chromatography (Amersham Pharmacia Biotech). Briefly, medium containing JHE was diluted 1:2 with Tris–phosphate buffer pH 8.5 (50 mM Tris, 5% sucrose, 2 mM EDTA, 0.02% sodium azide, titrated with phos-phoric acid), then applied to the column. JHE eluted with a sodium chloride gradient (0–200 mM, 50 mM Tris– phosphate buffer pH 7.5) was concentrated on a Cen-tricon-30 filter (Amicon). Samples of purified enzyme were visualized by separation in SDS–polyacrylamide gels and staining with Commassie Blue. Specific activi-ties were calculated from protein content (BioRad Pro-tein Assay) and enzyme activities (Hammock and Sparks, 1977) to estimate the degree of enrichment.
Purified JHE and mutant JHEs were labeled with biotin (Biotin Labeling Kit, Roche Molecular Biochemicals) and column-purified to eliminate non-biotinylated protein according to the manufacturer’s directions. Biotinylated-JHE was quantified using the BioRad Protein Assay. The efficiency of biotinylation of all enzymes was quantified by colorimetric assay at 412 nm in a microtiter plate using streptavidin–horse radish peroxidase (HRP) conjugate with 2,29 -azinobis{3-ethylbenzothiazoline-6-sulfonic acid}-diammonium salt (ABTS; Amersham Pharmacia Biotech). All assays (50µl of 2µg enzyme/ml stock per well) were replicated four times, and data were analyzed by one-way analysis of variance (ANOVA).
membrane (Amersham Pharmacia Biotech). Blots were incubated with biotin-labeled recombinant JHE or mutant JHE (2µg/ml) in PBS for 4 h, followed by wash-ing with PBS–0.1% Tween-20 and blockwash-ing with skimmed milk. Bound enzyme was detected using strep-tavidin–HRP conjugate and one-step TMB (3,39,5,59 -tetramethylbenzidine; Pierce Chemical Co.). All ligand blots were repeated at least three times.
3. Results
3.1. JHE binding proteins detected in M. sexta and H. virescens by ligand blot
Proteins of fat body, pericardial athrocytes and hemo-lymph immobilized on nylon membranes were assayed for JHE binding using biotinylated-JHE. Recombinant JHE was enriched to .85% purity (based on specific activity) using Q-sepharose ion-exchange chromato-graphy, and biotinylated for use as a probe in ligand blot analysis. Fig. 1(b) and (c) shows that when proteins in fat body and pericardial athrocytes of M. sexta and H.
virescens are separated by SDS–PAGE,
biotinylated-JHE binds proteins of the same molecular mass in both tissues. The predominant putative JHE binding proteins were approximately 29, 72, 75, 125 and 240 kDa. Additional minor putative JHE binding proteins of 50, 80 and 205 kDa were also seen in tissues from both
H. virescens and M. sexta (Figs. 1 and 2). A weak band
of approximately 70 kDa was seen in hemolymph samples (data not shown). Biotin-labeled JHE did not bind the 32 kDa recombinant JHBP (data not shown).
Analysis of pericardial athrocyte proteins from second, third, fourth and fifth instar larvae of M. sexta by ligand blot with recombinant JHE showed that all predominant putative JHE binding proteins were present in all instars examined (Fig. 2).
Fig. 1. JHE binding proteins in pericardial athrocytes and fat body of H. virescens and M. sexta. Pericardial cell (lane 1; 10µg) and fat body (lane 2; 10µg) proteins from fourth instar larvae were separated by SDS–PAGE in 12% gels, transferred to Hybond-P membrane and probed with biotin-labeled recombinant JHE (2µg/ml). (a) SDS gel of proteins from M. sexta stained with Coomassie Blue; (b) ligand blot of proteins from
M. sexta; (c) ligand blot of proteins from H. virescens. The minor 50 kDa putative JHE binding protein is indicated (arrow). M, Molecular mass
markers in kDa: (a) SDS–PAGE broad range standards (BioRad); (b) and (c) prestained SDS–PAGE broad range standards (BioRad).
Fig. 2. JHE binding proteins in pericardial cell tissue during larval development of M. sexta. Pericardial athrocytes were dissected from three or four larvae at each instar, pooled and lysed. A 10µg quantity of the soluble protein was separated by SDS–PAGE (12% gel), trans-ferred to Hybond-P membrane and probed with biotin-labeled recombi-nant JHE (2µg/ml). M, Molecular mass markers in kDa; 1, second instar; 2, third instar; 3, fourth instar, day 3; 4, fifth instar, day 3.
3.2. Two-dimensional electrophoresis
Fig. 3. Two-dimensional electrophoresis and ligand blot of M. sexta pericardial cell proteins. Pericardial cell proteins from 10 fifth instar
M. sexta were separated on the basis of isoelectric point in the first
dimension using an IPGphor IEF system (Amersham Pharmacia Biotech) and on a linear gradient SDS–polyacrylamide gel (8–16%) in the second dimension (BioRad). Proteins were transferred to Hybond-P membrane for ligand blot with biotin-labeled recombinant JHE (2µg/ml). (a) Proteins separated by two-dimensional electrophoresis and stained with silver stain. (b) Ligand blot of separated proteins. Three 29 kDa JHE binding species, BP1 (pI 6.5), BP2 (pI 6.6) and BP3 (pI 6.8) are indicated. Molecular mass markers in kDa are shown.
Although the protein profile for fat body tissue was con-siderably different from that of pericardial cell tissue, a similar profile was seen for ligand blot of fat body tissue proteins separated by two-dimensional electrophoresis (data not shown). The JHE binding proteins with higher molecular mass were not resolved well using two-dimen-sional electrophoresis under the conditions described.
3.3. Binding of JHE binding proteins to JHE mutants
We recently isolated a cDNA that codes for a 29 kDa JHE binding protein, P29, which binds JHE-KK less efficiently than JHE (Shanmugavelu et al., 2000). To validate whether the 29 kDa protein seen in ligand blots of M. sexta tissues [Fig. 1(b)] is indistinguishable from P29, experiments were conducted to assay binding of JHE mutants to protein extracts from pericardial cells and to purified P29. In these ligand blotting experiments, blots probed with biotinylated 29, 524 or JHE-KK support previous findings that JHE-JHE-KK binds less to
purified P29 [Fig. 4(c), lane 2] relative to the other mutant enzymes. Binding of JHE-KK to proteins in peri-cardial cell extracts [Fig. 4(c), lane 1] was not reduced relative to the other mutants. In these experiments, there were no significant differences in the degree of biotinyl-ation of the enzymes (one-way ANOVA; P.0.05).
4. Discussion
We undertook a preliminary characterization of pro-teins that bind JHE in the fat body and pericardial athro-cytes of M. sexta. Because M. sexta JHE had not been cloned at the beginning of this study, we used H.
vires-cens-derived JHE for examination of JHE binding
pro-teins in M. sexta. Given its large size and ease of hand-ling, M. sexta provides a useful model for studies of endocrine regulation, and the coding sequences of sev-eral proteins from this species relating to regulation of juvenile hormone have been determined (Touhara et al., 1993; Wojtasek and Prestwich, 1996).
Analysis of binding of baculovirus-expressed, recom-binant JHE derived from H. virescens to proteins in both
H. virescens and M. sexta suggests that JHE binding
pro-teins in the two species are similar, and that recombinant
H. virescens-derived JHE can be used for examination
of M. sexta JHE binding proteins. Indeed, H. virescens JHE shares 56% identity with M. sexta JHE (Hinton and Hammock, unpublished). The profile of JHE binding
proteins detected by ligand blot in tissues of the black cutworm Agrotis ipsilon and the armyworm Pseudoletia
unipuncta were similar to those detected in H. virescens
and M. sexta (Shanmugavelu, unpublished).
We examined fat body and pericardial cell tissues from second through fifth instar larvae to address whether the putative JHE binding proteins were expressed throughout larval development. The titer of JHE fluctuates during larval development, with very low activity in the hemolymph of first and second instars and peaks of activity in the fourth and fifth instars (Jesudason et al., 1990; Sparks et al., 1983). All putative JHE bind-ing proteins were detected in all instars examined. Whether expression of these proteins is correlated with hemolymph titers of JHE remains to be established.
Baculovirus-expressed JHE was used as probe in all ligand blot experiments. This protein was enriched to .85% from serum-free cell culture medium by ion-exchange chromatography. Although no contaminating proteins were seen on inspection of Coomassie-Blue-stained gels, it is possible that low-abundance proteins secreted by baculovirus-infected cells co-purified with JHE in ion-exchange chromatography. Hence further work is required to confirm the JHE binding properties of the putative binding proteins identified in this study. In a previous study, H. virescens-derived JHE labeled with 35S was injected into M. sexta larvae, and
pericar-dial cell proteins analyzed (Bonning et al., 1997a). JHE and presumed degradation products of JHE were detected by autoradiogram, along with a35S-labeled
pro-tein of .200 kDa. This result suggests that the 240 kDa binding protein described here may consist of JHE or a fragment of JHE conjugated to a second protein. Oligo-mers of JHE have not been reported for the Lepidoptera, and proteins of larger molecular mass than JHE (60– 66 kDa) have not been detected by Western blot using anti-JHE antisera (Venkatesh et al., 1990; Hanzlik and Hammock, 1987).
We also examined the potential binding of JHE to M.
sexta-derived JHBP (32 kDa) by ligand blot and
determ-ined that recombinant H. virescens-derived JHE does not bind to M. sexta-derived JHBP in the absence of JH under the conditions used. Touhara et al. (1995) sug-gested binding of JHE to JHBP as a mechanism by which JHBP might facilitate degradation of JH by JHE in the hemolymph. Preliminary circular dichroism experiments with recombinant JHE and JHBP in the absence of JH were reported to support this hypothesis (Touhara et al., 1995).
Two-dimensional electrophoresis followed by ligand blotting showed that the 29 kDa protein detected by SDS–PAGE and Western blot of insect tissues (Shanmugavelu et al., 2000) resolves into three species, all of which bind JHE. The JHE binding protein P29 has multiple potential phosphorylation sites and a myristyl-ation site, and differential post-translmyristyl-ational processing
could account for the multiple 29 kDa species. Alterna-tively, the multiple species of the 29 kDa JHE binding protein may result from artefactual charge heterogeneity. Several factors, such as oxidation of cysteine residues, deamidation of asparagine residues and incomplete reduction of the protein sample, could cause charge het-erogeneity. Preliminary N-terminal amino-acid sequen-cing results suggest the presence of more than one pro-tein, although contaminating proteins may have been present in the membrane samples submitted [see Fig. 3(A)]. Further work is required to address whether BP1, BP2 and BP3 are variants of the same protein, or whether they are different proteins.
We have previously shown that binding of JHE-KK to the JHE binding protein, P29, in a microtiter plate assay is significantly reduced relative to wild-type and control mutant JHEs (Shanmugavelu et al., 2000). While binding of JHE-KK to purified P29 was reduced relative to that of the other JHE mutants in ligand blot, binding to the 29 kDa protein in the pericardial athrocytes was not reduced. One possible explanation for our inability to demonstrate decreased binding of JHE-KK to the 29 kDa protein in pericardial cell extracts is that JHE-KK has reduced binding to just one of the species present in this band. In this case, the reduction in binding of JHE-KK relative to JHE may be masked in ligand blot by binding to the other proteins of the same relative molecular mass. In conclusion, we have identified five major and three minor putative JHE binding proteins in fat body and pericardial cell tissue of M. sexta. The roles of JHE bind-ing proteins in synthesis and export, or uptake and degra-dation, of JHE remain to be elucidated. In Bombyx mori, athrocytes have been detected in fat body tissue in addition to their pericardial location (Lavenseau et al., 1981). It has also been suggested that there are athro-cytes in the fat body of M. sexta based on injection of recombinant H. virescens-derived JHE into the hemoc-oel, and subsequent detection of recombinant enzyme in the fat body (Ichinose et al., 1992a). To establish whether JHE binding proteins function in uptake and degradation and/or synthesis and export of the enzyme, it will be necessary to determine (1) whether athrocytes are present in the fat body of M. sexta; (2) whether athro-cytes synthesize JHE; and (3) the intracellular location of the JHE binding proteins.
Acknowledgements
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