The Insect Hemolymph Protein HP19 Mediates the Nongenomic Effect of Ecdysteroids on Acid Phosphatase Activity
2004; Elsevier BV; Volume: 279; Issue: 27 Linguagem: Inglês
10.1074/jbc.m402311200
ISSN1083-351X
AutoresAbul Arif, Palaniappan Vasanthi, Immo A. Hansen, Klaus Scheller, Aparna Dutta‐Gupta,
Tópico(s)Insect Resistance and Genetics
ResumoThe activity of acid phosphatase (ACP) in insect fat bodies is stimulated by the steroid hormone 20-hydoxyecdysone (20E) in vivo. However, in fat bodies kept in culture, a factor from the hemolymph is required to enhance the ACP activity. We identified the factor as a protein with a molecular mass of 19 kDa (HP19) from the hemolymph of a lepidopteran insect, the rice moth, Corcyra cephalonica. Western analysis of hemolymph proteins with denaturing and non-denaturing PAGE using antibodies raised against HP19 suggest that this protein exists as a monomer. It is synthesized by the hind gut-associated lobular fat body of the larvae and is released into the hemolymph. The stimulatory effect of HP19 on the ACP activity is developmentally regulated and exhibits its maximal effect shortly before the onset of metamorphosis. We cloned the HP19 cDNA by immunoscreening a hind gut-associated lobular fat body cDNA expression library. Analysis of the amino acid sequence shows that HP19 belongs to the family of glutathione S-transferase (GST) like proteins. However, affinity-purified GST from Corcyra failed to show any mediation effect on 20E-stimulated ACP activity, and HP19 lacks GST enzymatic activity. Notably, HP19 mediates the hormone-stimulated ACP activity in intact fat body tissue and homogenates even in the presence of inhibitors of transcription and translation, suggesting a nongenomic mode of action. In addition, we show that HP19 inhibits the 20E-induced phosphorylation of the hexamerin receptor protein. The activity of acid phosphatase (ACP) in insect fat bodies is stimulated by the steroid hormone 20-hydoxyecdysone (20E) in vivo. However, in fat bodies kept in culture, a factor from the hemolymph is required to enhance the ACP activity. We identified the factor as a protein with a molecular mass of 19 kDa (HP19) from the hemolymph of a lepidopteran insect, the rice moth, Corcyra cephalonica. Western analysis of hemolymph proteins with denaturing and non-denaturing PAGE using antibodies raised against HP19 suggest that this protein exists as a monomer. It is synthesized by the hind gut-associated lobular fat body of the larvae and is released into the hemolymph. The stimulatory effect of HP19 on the ACP activity is developmentally regulated and exhibits its maximal effect shortly before the onset of metamorphosis. We cloned the HP19 cDNA by immunoscreening a hind gut-associated lobular fat body cDNA expression library. Analysis of the amino acid sequence shows that HP19 belongs to the family of glutathione S-transferase (GST) like proteins. However, affinity-purified GST from Corcyra failed to show any mediation effect on 20E-stimulated ACP activity, and HP19 lacks GST enzymatic activity. Notably, HP19 mediates the hormone-stimulated ACP activity in intact fat body tissue and homogenates even in the presence of inhibitors of transcription and translation, suggesting a nongenomic mode of action. In addition, we show that HP19 inhibits the 20E-induced phosphorylation of the hexamerin receptor protein. Insect metamorphosis, the transition from the larval to the adult stage of insects, is controlled by ecdysteroid hormones (1Trumann J.W. Riddiford L.M. Annu. Rev. Entomol. 2002; 47: 467-500Crossref PubMed Scopus (308) Google Scholar, 2Riddiford L.M. Cherbas P. Truman J.W. Vitam. Horm. 2001; 60: 1-73Crossref Google Scholar, 3Gilbert L.I. Rybczynski R. Warren J.T. Annu. Rev. Entomol. 2002; 47: 883-916Crossref PubMed Scopus (387) Google Scholar). The ecdysteroids, like the steroids in vertebrates, regulate gene transcription by binding to the nuclear receptors, which are ligand-activated transcription factors that convert the hormonal stimulus into a transcription response (4White R. Parker M.G. Endocr. Relat. Cancer. 1998; 5: 1-14Crossref Scopus (128) Google Scholar, 5Scheller K. Sekeris C.E. Exp. Physiol. 2003; 88: 129-140Crossref PubMed Scopus (84) Google Scholar, 6Henrich V.C. Rybczynski R. Gilbert L.I. Vitam. Horm. 1999; 55: 73-125Crossref PubMed Scopus (123) Google Scholar, 7Mangelsdorf D.J. Thummel C. Beato M. Herrlich P. Schutz G. Umesono K. Blumberg B. Kastner P. Mark M. Chambon P. Evans R.M. Cell. 1995; 83: 835-839Abstract Full Text PDF PubMed Scopus (6000) Google Scholar). Metamorphosis involves the breakdown of larval structures and the formation of new tissues (1Trumann J.W. Riddiford L.M. Annu. Rev. Entomol. 2002; 47: 467-500Crossref PubMed Scopus (308) Google Scholar). As a part of cell remodeling during metamorphosis, acidic autophagic vacuoles accumulate in the cells of the fat body, and the activity of several lysosomal enzymes such as acid phosphatases increase and cause the lysis of larval tissues (8Thummel C.S. BioEssays. 2001; 23: 677-682Crossref PubMed Scopus (67) Google Scholar, 9Lee C.Y. Baehriecke E.H. Development. 2001; 128: 1443-1455PubMed Google Scholar, 10Sass M. Kovacs J. J. Insect Physiol. 1980; 26: 569-577Crossref Scopus (21) Google Scholar, 11Verkuil E.V.P. J. Insect Physiol. 1980; 26: 91-101Crossref Scopus (12) Google Scholar). The fat body fills a large fraction of the insect, and its function has been considered equivalent to the role of the vertebrate liver in the intermediary metabolism (12Hansen I.A. Meyer S.R. Schafer I. Scheller K. Eur. J. Biochem. 2002; 269: 954-960Crossref PubMed Scopus (19) Google Scholar). It has been demonstrated that the stimulation of the lysosomal activity is governed by ecdysteroids (11Verkuil E.V.P. J. Insect Physiol. 1980; 26: 91-101Crossref Scopus (12) Google Scholar, 13Verkuil E.V.P. Van Ronger E. de Priester W. Cell Tissue Res. 1979; 203: 443-445Crossref PubMed Scopus (9) Google Scholar, 14Sass M. Komuves L. Csikos G. Kovacs J. Comp. Biochem. Physiol. A. 1989; 92: 285-289Crossref Scopus (12) Google Scholar, 15Kutuzova N.M. Filippovich IuB. Kholodova IuD. Mildera K. Kindruk N.L. Korniets G.V. Moroz N.S. Ukr. Biokhim. Zh. 1991; 63: 41-45PubMed Google Scholar, 16Ashok M. Dutta-Gupta A. Biochem. Int. 1988; 17: 1087-1091PubMed Google Scholar). There is an indication that in this case the hormone possibly acts on a nongenomic level (17Verkuil E.V.P. J. Insect Physiol. 1979; 25: 965-973Crossref Scopus (18) Google Scholar). Although the molecular mechanism of the genomic mode of steroid action is well known, the mechanism of nongenomic steroid action remains unclear to date (18Losel R. Wehling M. Nat. Rev. Mol. Cell Biol. 2003; 4: 46-56Crossref PubMed Scopus (683) Google Scholar). Earlier studies show that 20E 1The abbreviations used are: 20E, 20-hydroxyecdysone; ACP, acid phosphatase; HP19, 19-kDa hemolymph protein; CcHP19, C. cephalonica HP19; CfGST, C. fumiferana GST; GST, glutathione S-transferase; HGLFB, hind gut-associated lobular fat body; ELI, early last instar larvae; MLI, mid-last instar larvae; LLI, late-last instar larvae. 1The abbreviations used are: 20E, 20-hydroxyecdysone; ACP, acid phosphatase; HP19, 19-kDa hemolymph protein; CcHP19, C. cephalonica HP19; CfGST, C. fumiferana GST; GST, glutathione S-transferase; HGLFB, hind gut-associated lobular fat body; ELI, early last instar larvae; MLI, mid-last instar larvae; LLI, late-last instar larvae. stimulates ACP activity in fat bodies in vivo but not in vitro (19Caglayan S.H. Biochem. Int. 1990; 20: 511-518Google Scholar, 20Ashok M. Dutta-Gupta A. Invert. Reprod. Dev. 1991; 20: 159-165Crossref Scopus (4) Google Scholar). This result suggests that 20E, the active form of ecdysone, requires an additional factor (or factors) to enhance ACP activity. Hence, we have focused on the process of acid phosphatase activation by 20E in the fat body cells of our model insect, the rice moth Corcyra cephalonica. We report the appearance of a stage and tissue-specific-regulated protein, HP19, in the hemolymph of Corcyra responsible for the activation of the 20E-dependent stimulation of ACP activity. This hormone-triggered activation is independent of gene transcription. Insects—Larvae of the rice moth, C. cephalonica (Stainton), were reared on coarsely crushed sorghum seeds at 26 ± 1 °C, 60 ± 5% relative humidity, and a 14:10-h light:dark photo period. In the present study, the last (=Vth) instar larvae, classified into early (ELI), mid (MLI), late-last instar (LLI) larvae and prepupae on the basis of body weight and head capsule size were used (21Lakshmi M. Dutta-Gupta A. Biochem. Int. 1990; 22: 269-278Crossref PubMed Scopus (10) Google Scholar). The larvae were thorax-ligated behind the first pair of prolegs by slipping a loop of silk thread around the head of the larvae as described earlier (20Ashok M. Dutta-Gupta A. Invert. Reprod. Dev. 1991; 20: 159-165Crossref Scopus (4) Google Scholar). The fat body and other insect tissue homogenates from ligated or unligated larvae were prepared as published earlier (22Arif A. Scheller K. Dutta-Gupta A. Insect Biochem. Mol. Biol. 2003; 33: 921-928Crossref PubMed Scopus (19) Google Scholar) and used after protein estimation in an aliquot of the homogenate (23Bradford M.M. Anal. Biochem. 1976; 72: 248-254Crossref PubMed Scopus (211941) Google Scholar). ACP Assay—The enzyme assay was carried out according to the method of Henrickson and Clever (24Henrickson P.A. Clever U. J. Insect Physiol. 1972; 18: 1981-2004Crossref Scopus (44) Google Scholar). The reaction mixture contained 150 mm sodium acetate buffer (pH 5.0) and 20 μg of tissue homogenate proteins. It was incubated at 37 °C for 10 min to exclude glucose-6-phosphatase activity (25Beaufay H. Bertlet J. de Durve C. Bull. Ste. Chim. Biol. 1954; 36: 1539-1550PubMed Google Scholar). The reaction was initiated by the addition of 5 μmol of substrate, p-nitrophenyl bisodium phosphate (Sigma) to the assay mixture followed by incubation for 1 h at 37°C. The reaction was terminated by the addition of 0.5 ml of 0.1 n NaOH, and the color was measured at 420 nm against a substrate blank. The p-nitrophenol was used for the preparation of a standard curve. The activity of the enzyme was expressed as nmol of p-nitrophenol released/h/μg of fat body protein. Hemolymph Sample—Hemolymph from LLI larvae was collected into tubes pretreated with 0.025% phenylthiourea, diluted (1:20) with 10 mm Tris-HCl (pH 7.4), and spun for 3 min at 1000 × g to remove hemocytes. These hemolymph samples were used immediately after preparation. Studies on Fat Body Cultures—Ribbon-shaped visceral fat bodies from LLI larvae were dissected 24 h after ligation under sterile conditions in cold insect Ringer's solution and transferred to 100 μl of TC-100 insect culture medium (JRH Biosciences Inc.) with traces of streptomycin sulfate. After rinsing, the tissue was transferred to 200 μl of fresh culture medium, and 80 nm 20E was added while an equal volume of carrier solvent (ethanol) was added to the control cultures. The hormone 20E (Sigma) was dissolved in ethanol, the final concentration of which never exceeded 0.05% in any of the experiments. To study the hemolymph effect, the diluted (1:20) or fractionated hemolymph was added to the fat body culture in the presence or absence of 80 nm 20E. Studies with glutathione S-transferase (GST) were carried out by adding purified cytosolic GST from Corcyra to the fat body cultures in the presence of hormone. These cultures were then incubated for 4 h at 25 °C with gentle shaking. At the end of incubation the tissue was removed, rinsed in ice-cold insect Ringer, homogenized, and used for ACP assay. Fractionation and Purification of Hemolymph Proteins—Total hemolymph protein was loaded on a pre-equilibrated (10 mm Tris-HCl (pH 7.4)) Sephadex G-50 column and eluted with the equilibration buffer. The single fractions were checked for their ability to enhance the ACP activity in LLI fat bodies kept in culture. The apparent molecular mass of the active fraction was determined by gel electrophoresis. The purification of the active fraction was carried out by fractionating the hemolymph proteins followed by gel filtration chromatography. For fractionation we used molecular weight cut-off fractionators (Amicon Inc.). The hemolymph sample preparation (1 mg of protein/ml) was first transferred into 30-kDa cut-off fractionators (YM-30) and centrifuged at 4000 × g for 20 min at 4 °C to strip the protein fractions with mass above 100 kDa. The resultant filtrate as well as the retentate was collected separately. The filtrate with molecules <100 kDa was again subjected to fractionation using a 10-kDa cut-off fractionator (YM-10) for obtaining fraction of 109 dpm/μg. The blots were worked up following standard procedures (30Sambrook J. Fritsch E.F. Maniatis T. Molecular Cloning: A Laboratory Manual. 2nd Ed. Cold Spring Harbor Press, Cold Spring Harbor, NY1989Google Scholar). GST Assay—The GST activity was measured by the method of Habig et al. (32Habig W.H. Pabst M.J. Jakoby W.B. J. Biol. Chem. 1974; 249: 7130-7139Abstract Full Text PDF PubMed Google Scholar). A 1-ml reaction mixture contained 10 μl of 100 mm 1-chloro-2,4-dinitrobenzene, 10 μlof100 mm reduced glutathione (GSH), and 100 mm potassium phosphate buffer (pH 6.5). The reaction was initiated by the addition of the enzyme source and the product, i.e. the formation of thioether conjugate, was measured at 340 nm on a time scan of 0–60 s. Immunohistochemical Studies—The insect tissues were fixed in Carnoy's fixative (ethanol:chloroform:acetic acid, 6:3:1) for 4 h at room temperature and paraffin-embedded. Sections (5 μm) were cut and mounted on glass slides. For immunohistochemical staining the sections were deparaffinized and treated with blocking solution (2% bovine serum albumin and 1% preimmune goat serum in 50 mm Tris-buffered saline (pH 7.4) containing 0.1% Triton X-100) for 1 h at 4 °C. This was followed by anti-HP19 IgG treatment for 24 h at 4 °C with gentle shaking. The slides were then treated with anti-rabbit IgG coupled with alkaline phosphatase for 1 h. Washing after each step was done with three changes of Tris-buffered saline. These slides were finally processed for staining using nitro blue tetrazolium chloride/5-bromo-4-chloro-3-indolyl phosphate color reaction and mounted in glycerol gel (50% glycerol, 7.5% gelatin, and 0.1% azide in 0.1 m Tris-buffered saline) (33Meltzer J.C. Grimm P.C. Greenberg A.H. Nance D.M. J. Histochem. Cytochem. 1997; 45: 599-610Crossref PubMed Scopus (40) Google Scholar, 34Webster S.G. Biol. Bull. 1998; 195: 282-289Crossref PubMed Scopus (14) Google Scholar). The specificity of the antibodies was checked for all immunochemical experiments by treating parallel tissue sections using pre-immune rabbit serum. [35S]Methionine Incorporation Studies—In vitro incorporation of [35S]methionine in the fat bodies kept in culture was carried out to compare the changes in the level of total protein synthesis and ACP activity stimulation in the presence of 20E and HP19. The fat bodies kept in culture were first preincubated for 2 h with 10 μCi of [35S]methionine (∼1000 Ci/m mol, BRIT) at 25 °C followed by incubations either with 20E (80 nm) alone, HP19 (40 ng) alone, or both along with actinomycin D (1 mm) or cycloheximide (1 mm) for 2 or 4 h. After incubation, the fat bodies were removed and homogenized as described above. Equal amounts of protein were used for radiolabel quantitation after trichloroacetic acid precipitation. In Vitro Phosphorylation of Fat Body Proteins—Fat body homogenate from LLI larvae was incubated with 80 nm 20E and 40 ng of HP19 for 30 s, and phosphorylation was initiated by the addition of 4 μCi of [λ-32P] ATP (∼3000 Ci/m mol, BRIT) as described earlier (22Arif A. Scheller K. Dutta-Gupta A. Insect Biochem. Mol. Biol. 2003; 33: 921-928Crossref PubMed Scopus (19) Google Scholar). The labeled proteins were separated by 10% SDS-PAGE. The gel was vacuum-dried and exposed to Kodak X-Omat AR film at –70 °C for autoradiography. Statistical Analysis—The mean and S.D. were calculated for the variables studied. The data were statistically analyzed by one way analysis of variance followed by comparisons of means by Tukey multiple comparison tests using Sigma Stat software (Jandel Corp.). The values were considered significantly different from each other when p < 0.05. Identification of a Protein in the Hemolymph of Corcyra That Mediates the 20E-stimulated ACP Activity in the Fat Bodies of Thorax-ligated, Hormone-deprived Larvae—When insect larvae are ligated behind the first pair of prolegs, i.e. behind the hormone producing glands, the posterior part of the animal is known to be relatively free of endogenous ecdysteroids (35Priester D.W. Verkuil E.V.P. de Leeuw G. Cell Tissue Res. 1979; 200: 435-442Crossref PubMed Scopus (24) Google Scholar, 36Dutta-Gupta A. Ashok M. Entomon. 1998; 23: 245-250Google Scholar, 37Burmester T. Scheller K. Eur. J. Biochem. 1997; 247: 695-702Crossref PubMed Scopus (27) Google Scholar). Fig. 1, a and b, shows the effect of thorax ligation and injection of exogenous 20E on the fat body ACP activity in LLI larvae. The ACP activity declined gradually from 6 to 72 h after ligation. Because the ACP activity in the fat body was significantly lower after 24 h of ligation, this time period was used for all hormone manipulation studies. Hormone injections of 80 nm 20E, i.e. the physiological concentration (22Arif A. Scheller K. Dutta-Gupta A. Insect Biochem. Mol. Biol. 2003; 33: 921-928Crossref PubMed Scopus (19) Google Scholar, 36Dutta-Gupta A. Ashok M. Entomon. 1998; 23: 245-250Google Scholar), to 24 h post-ligated LLI larvae caused a significant increase in the ACP activity in fat bodies after 24 h compared with the solvent-treated larvae (Fig. 1b). To study the effect of hormone on the ACP activity of fat bodies kept in culture, the tissue was dissected from 24-h post-ligated larvae and cultured for 4 h in the presence of 80 nm 20E. The results show that 20E did not elicit any stimulatory effect, and the activity was more or less the same as in the controls (Fig. 1c). However, the addition of hemolymph from LLI larvae together with 20E caused a significant increase in the ACP activity (Fig. 1c). This observation suggests that the hemolymph contains a factor (or factors) required by 20E to stimulate the ACP activity in fat body cultures. When the hemolymph was treated with alcohol, heat, acid, alkali, or protease, no stimulation of the ACP activity by 20E could be observed, suggesting the proteinaceous nature of the factor (Fig. 1d). Purification and Characterization of the Hemolymph Factor as a Protein Mediating 20E-stimulated ACP Activity—After loading total hemolymph protein on a Sephadex G-50 column, we eluted several fractions (Fig. 2) and checked their ability to mediate the 20E-stimulated ACP activity. We found an active protein fraction with a molecular mass of ∼22 kDa, calculated from the elution profile (Fig. 2, inset), or 19 kDa, calculated from the mobility on a SDS-PAGE (Fig 3b). On the basis of these results we purified the active hemolymph protein first by fractionating the total hemolymph protein followed by gel filtration chromatography. The fractionation was carried out using 30- and 10-kDa cut-off filters. The filtrate from the 10-kDa cut-off filter gave a protein fraction that mediated the 20E-stimulated ACP activity of fat body cultures by an increase from 0.7 to 1.2 nmol of p-nitrophenol release/h/μg of protein (Fig. 3a). However, the protein yield in the filtrate obtained from the 10-kDa cut-off filter was insufficient to proceed for further purification. Therefore, the filtrate from the 30-kDa cut-off filters, in which the HP19 was contained, was used for gel filtration. The protein fraction eluted from the Sephadex column that mediated the 20E-dependent ACP activity resulted in a contaminant-free pure polypeptide band of 19 kDa (Fig. 3b). Hence, the active hemolymph protein was named as HP19. Starting with 50 mg of total hemolymph protein, we obtained a 98.5-fold purification with 0.05% yield.Fig. 3Purification of a hemolymph protein enhancing the 20E-stimulated ACP activity in Corcyra fat body cultures.a, effect of different hemolymph fractions obtained using a 30- and 10-kDa fractionator on the ACP activity in fat body tissue culture in the presence of 20E. The fat bodies from two 24-h post-ligated LLI larvae were incubated with 80 nm 20E and 10 μl of fractionated hemolymph for 4 h. At the end of the incubation the fat bodies were assayed for ACP activity. Each value is the mean ± S.D. of four independent determinations. *, significantly different from all other values (p < 0.05). PNP, p-nitrophenol. b, SDS-PAGE to show purification profile of HP19. The hemolymph protein was fractionated using a 30-kDa fractionator (Amicon), and the filtrate was applied on Sephadex G-50 for column purification. Crude hemolymph (1), proteins from filtrate of 30-kDa filter (2), protein markers in kDa (3), and active fractions eluted from G-50 column (4–8). Lanes 1–3: 10 μg; lanes 4–6, 5 μg; lanes 7 and 8, total lyophilized protein was loaded. c, Western blot showing the specificity for HP19 (filled arrows) both in denatured (1) and non-denatured (2) PAGE. 20 μg of total hemolymph protein was loaded in each lane. d, Southern blot analysis showing single gene copy (filled arrows) of HP19. The genomic DNA (30 μg) from total larval body was digested with EcoRI (1Trumann J.W. Riddiford L.M. Annu. Rev. Entomol. 2002; 47: 467-500Crossref PubMed Scopus (308) Google Scholar) or HinfI (2Riddiford L.M. Cherbas P. Truman J.W. Vitam. Horm. 2001; 60: 1-73Crossref Google Scholar) and probed with CcHP19 cDNA.View Large Image Figure ViewerDownload (PPT) The Western blots presented in Fig. 3c show the specificity of the HP19 antibody both on denatured (lane 1) and non-denatured (lane 2) PAGE. A single protein band of 19 kDa suggested a monomeric structure of HP19. Southern analysis of genomic DNA (Fig. 3d) digested with EcoRI or HinfI probed with HP19-cDNA revealed HP19 as a single copy gene. cDNA Cloning and Sequence Analysis of HP19 —To identify the cDNA encoding the HP19 protein, a cDNA expression library, prepared from the RNA of HGLFB of LLI larvae, was immunoscreened. We picked 10 positive cDNA clones for detailed examination. The restriction analysis revealed 6 of the 10 clones to be of identical size. Initial sequencing study demonstrated significant sequence similarity among these clones. Furthermore, they showed homology with invertebrate GSTs. One of our clones was sub-cloned and totally sequenced (GenBank™ accession number AY369240). This HP19 cDNA was 634 nucleotides long, with an open reading frame of 585 bp, which encodes a protein of 195 amino acids. The calculated molecular mass of the translated unmodified protein was 22.95 kDa, which is close to the mass of HP19 detected in HGLFB, the tissue that synthesizes the protein (see Fig. 6e). The polypeptide comprises 12.3% basic (9 Arg, 1 His, and 14 Lys) and 13.3% acidic residues (10 Asp and 16 Glu) but no Cys residue. The estimated isoelectric point (pI) is 5.36. Comparison of the C. cephalonica HP19 (CcHP19) cDNA with the sequences in the GenBank™ showed 67% identity with Choristoneura fumiferana GST (CfGST) (38Feng Q.L. Davey G.K. Pang A.S.D. Primavera M. Ladd T.R. Zheng S.C. Sohi S.S. Retankaran A. Palli S.R. Insect Biochem. Mol. Biol. 1999; 29: 779-793Crossref PubMed Scopus (48) Google Scholar). Similarities of HP19 cDNA with other invertebrate GST were found to be less than 38%. The comparison of the amino acid sequences of CcHP19 with the four best matching invertebrate GSTs is shown in Fig. 4. Although the CcHP19 cDNA sequence revealed 67% identity with CfGST, affinity-purified GST from Corcyra had no enhancing effect on the 20E-dependent ACP activity when compared with purified HP19 or recombinant HP19 (Fig 5a). Furthermore, the hemolymph as well as the purified HP19 had negligible GST activity (Fig. 5b).Fig. 4Alignment of the deduced amino acid sequence of C. cephalonica HP19 (CcHP19) with GST sequences of other insects (BLAST search).CfGST, C. fumiferana (AF128867); BgGST, Blattella germanica (U92412); MsGST, Manduca sexta (L32092); MdGST, Musca domestica (U02616). The identical amino acid positions are shaded, and gaps are indicated by dashes. CcHP19 showed 67% identity with CfGST, 35% with BgGST, 32% with MsGST, and 31% with MdGST.View Large Image Figure ViewerDownload (PPT)Fig. 5Effects of HP19 on the ACP activity in fat bodies compared with GST.a, ability of affinity-purified cytosolic Corcyra GST on 20E-dependent fat body ACP activity. Note that the presence of purified or cloned HP19 (CcHP19) mediated the 20E-stimulated enzyme activity, whereas the presence of GST did not show any effect. b, GST activity in different larval tissues and in purified HP19. Note that both hemolymph as well as purified HP19 have negligible GST activity. PNP, p-nitrophenol.View Large Image Figure ViewerDownload (PPT) Tissue-specific Appearance of HP19 —Co-culturing of different larval tissues with fat body demonstrates that the HGLFB is the only HP19-synthesizing tissue. A stimulation of the ACP activity by 20E was only observed when it was co-cultured with HGLFB (Fig. 6a). The hemolymph used in all experiments was cell-free and, therefore, cannot be the site of HP19 synthesis. The tissue specificity of HP19 biosynthesis was further confirmed by immunohistochemical staining of different tissue sections using HP19 antibody. Again,
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