β-Fructofuranosidase Genes of the Silkworm, Bombyx mori
2008; Elsevier BV; Volume: 283; Issue: 22 Linguagem: Inglês
10.1074/jbc.m709350200
ISSN1083-351X
AutoresTakaaki Daimon, Tomohiro Taguchi, Yan Meng, Susumu Katsuma, Kazuei Mita, Toru Shimada,
Tópico(s)Invertebrate Immune Response Mechanisms
ResumoMulberry latex contains extremely high concentrations of alkaloidal sugar mimic glycosidase inhibitors, such as 1,4-dideoxy-1,4-imino-d-arabinitol (d-AB1) and 1-deoxynojirimycin (DNJ). Although these compounds do not harm the silkworm, Bombyx mori, a mulberry specialist, they are highly toxic to insects that do not normally feed on mulberry leaves. d-AB1 and DNJ are strong inhibitors of α-glucosidases (EC 3.2.1.20); however, they do not affect the activity ofβ-fructofuranosidases (EC 3.2.1.26). Althoughα-glucosidase genes are found in a wide range of organisms, β-fructofuranosidase genes have not been identified in any animals so far. In this study, we report the identification and characterization of β-fructofuranosidase genes (BmSuc1 and BmSuc2) from B. mori. The BmSuc1 gene was highly expressed in the midgut and silk gland, whereas the expression of BmSuc2 gene was not detected. BmSuc1 encodes a functional β-fructofuranosidase, whose enzymatic activity was not inhibited by DNJ or d-AB1. We also showed that BmSUC1 protein localized within the midgut goblet cell cavities. Collectively, our data clearly demonstrated that BmSuc1 serves as a sugar-digesting enzyme in the silkworm physiology. This anomalous presence of the β-fructofuranosidase gene in the B. mori genome may partly explain why the silkworm can circumvent the mulberry'ns defense system. Mulberry latex contains extremely high concentrations of alkaloidal sugar mimic glycosidase inhibitors, such as 1,4-dideoxy-1,4-imino-d-arabinitol (d-AB1) and 1-deoxynojirimycin (DNJ). Although these compounds do not harm the silkworm, Bombyx mori, a mulberry specialist, they are highly toxic to insects that do not normally feed on mulberry leaves. d-AB1 and DNJ are strong inhibitors of α-glucosidases (EC 3.2.1.20); however, they do not affect the activity ofβ-fructofuranosidases (EC 3.2.1.26). Althoughα-glucosidase genes are found in a wide range of organisms, β-fructofuranosidase genes have not been identified in any animals so far. In this study, we report the identification and characterization of β-fructofuranosidase genes (BmSuc1 and BmSuc2) from B. mori. The BmSuc1 gene was highly expressed in the midgut and silk gland, whereas the expression of BmSuc2 gene was not detected. BmSuc1 encodes a functional β-fructofuranosidase, whose enzymatic activity was not inhibited by DNJ or d-AB1. We also showed that BmSUC1 protein localized within the midgut goblet cell cavities. Collectively, our data clearly demonstrated that BmSuc1 serves as a sugar-digesting enzyme in the silkworm physiology. This anomalous presence of the β-fructofuranosidase gene in the B. mori genome may partly explain why the silkworm can circumvent the mulberry'ns defense system. Certain plants have evolved defense mechanisms against insect predation through the production of defense compounds (1Zagrobelny M. Bak S. Rasmussen A.V. Jorgensen B. Naumann C.M. Lindberg Moller B. Phytochemistry. 2004; 65: 293-306Crossref PubMed Scopus (234) Google Scholar, 2Konno K. Ono H. Nakamura M. Tateishi K. Hirayama C. Tamura Y. Hattori M. Koyama A. Kohno K. Proc. Natl. Acad. Sci. U. S. A. 2006; 103: 1337-1341Crossref PubMed Scopus (133) Google Scholar, 3Nishida R. Annu. Rev. Entomol. 2002; 47: 57-92Crossref PubMed Scopus (433) Google Scholar). This is reflected by the limited number of insects capable of feeding on such plants. In particular, plant latex often contains a wide variety of toxic compounds, such as alkaloids and proteases, which play an important role in a plant'ns defense mechanism against insect herbivory (2Konno K. Ono H. Nakamura M. Tateishi K. Hirayama C. Tamura Y. Hattori M. Koyama A. Kohno K. Proc. Natl. Acad. Sci. U. S. A. 2006; 103: 1337-1341Crossref PubMed Scopus (133) Google Scholar, 4Dussorurd D.E. Stamp N.E. Casey T.M. Caterpillars: Ecological and Evolutionary Constraints on Foraging. Chapman & Hall, New York1993: 92-131Google Scholar, 5Dussourd D.E. Eisner T. Science. 1987; 237: 898-901Crossref PubMed Scopus (222) Google Scholar, 6Konno K. Hirayama C. Nakamura M. Tateishi K. Tamura Y. Hattori M. Kohno K. Plant J. 2004; 37: 370-378Crossref PubMed Scopus (251) Google Scholar).Mulberry latex contains extremely high concentrations of alkaloidal sugar mimic glycosidase inhibitors, such as 1,4-dideoxy-1,4-imino-d-arabinitol (d-AB1), 2The abbreviations used are: d-AB1, 1,4-dideoxy-1,4-imino-d-arabinitol; DNJ, 1-deoxynojirimycin; DAPI, 4′,6-diamidino-2-phenylindole dihydrochloride; MSG, middle silk gland; sALP, soluble alkaline phosphatase; mALP, membrane-bound alkaline phosphatase; CBP, carotenoid-binding protein; DIG, digoxigenin; EST, expressed sequence tag; RT, reverse transcription. 2The abbreviations used are: d-AB1, 1,4-dideoxy-1,4-imino-d-arabinitol; DNJ, 1-deoxynojirimycin; DAPI, 4′,6-diamidino-2-phenylindole dihydrochloride; MSG, middle silk gland; sALP, soluble alkaline phosphatase; mALP, membrane-bound alkaline phosphatase; CBP, carotenoid-binding protein; DIG, digoxigenin; EST, expressed sequence tag; RT, reverse transcription. 1-deoxynojirimycin (DNJ), and 1,4-dideoxy-1,4-imino-d-ribitol (2Konno K. Ono H. Nakamura M. Tateishi K. Hirayama C. Tamura Y. Hattori M. Koyama A. Kohno K. Proc. Natl. Acad. Sci. U. S. A. 2006; 103: 1337-1341Crossref PubMed Scopus (133) Google Scholar, 7Asano N. Yamashita T. Yasuda K. Ikeda K. Kizu H. Kameda Y. Kato A. Nash R.J. Lee H.S. Ryu K.S. J. Agric. Food Chem. 2001; 49: 4208-4213Crossref PubMed Scopus (317) Google Scholar). These sugar mimic alkaloids are not toxic to larvae of the silkworm Bombyx mori (family Bombycidae), which feed only on mulberry leaves and have been reared on them for thousands of years (2Konno K. Ono H. Nakamura M. Tateishi K. Hirayama C. Tamura Y. Hattori M. Koyama A. Kohno K. Proc. Natl. Acad. Sci. U. S. A. 2006; 103: 1337-1341Crossref PubMed Scopus (133) Google Scholar). However, these compounds are highly toxic to other caterpillars, such as the eri-silkworm Samia cynthia ricini (family Saturniidae) and cabbage moth Mamestra brassicae (family Noctuidae), for which mulberry trees are not the host plant in natural conditions (2Konno K. Ono H. Nakamura M. Tateishi K. Hirayama C. Tamura Y. Hattori M. Koyama A. Kohno K. Proc. Natl. Acad. Sci. U. S. A. 2006; 103: 1337-1341Crossref PubMed Scopus (133) Google Scholar). This indicates that the silkworm has evolved an unknown mechanism to circumvent the toxic effects of such sugar mimic alkaloids, thus enabling it to feed and grow well on mulberry leaves (2Konno K. Ono H. Nakamura M. Tateishi K. Hirayama C. Tamura Y. Hattori M. Koyama A. Kohno K. Proc. Natl. Acad. Sci. U. S. A. 2006; 103: 1337-1341Crossref PubMed Scopus (133) Google Scholar, 8Hirayama C. Konno K. Wasano N. Nakamura M. Insect. Biochem. Mol. Biol. 2007; 37: 1348-1358Crossref PubMed Scopus (80) Google Scholar).Sucrases are digestive enzymes that hydrolyze α-glucosyl (α-glucosidase, EC 3.2.1.20) or β-fructosyl residue (β-fructofuranosidase, EC 3.2.1.26) of the substrate. d-AB1 and DNJ are strong inhibitors of α-glucosidases; however, they do not exhibit inhibitory activity against β-fructofuranosidases (7Asano N. Yamashita T. Yasuda K. Ikeda K. Kizu H. Kameda Y. Kato A. Nash R.J. Lee H.S. Ryu K.S. J. Agric. Food Chem. 2001; 49: 4208-4213Crossref PubMed Scopus (317) Google Scholar). Although α-glucosidases are found in many types of organisms, including bacteria, fungi, plants, and animals, it has been generally assumed that β-fructofuranosidases do not exist in animals (9Krasikov V.V. Karelov D.V. Firsov L.M. Biochemistry (Mosc.). 2001; 66: 267-281Crossref PubMed Scopus (47) Google Scholar). However, several reports have suggested that β-fructofuranosidases are present anomalously in certain species of insects (10Santos C.D. Terra W.R. Insect Biochem. 1986; 16: 819-824Crossref Scopus (36) Google Scholar, 11Sumida M. Yuan X.L. Matsubara F. Comp. Biochem. Physiol. B Biochem. Mol. Biol. 1994; 107: 273-284Crossref Scopus (12) Google Scholar, 12Sumida M. Yuan X.L. Matsubara F. Comp. Biochem. Physiol. A Biochem. Mol. Biol. 1994; 108: 255-264Google Scholar, 13Carneiro C.N. Isejima E.M. Samuels R.I. Silva C.P. J. Insect Physiol. 2004; 50: 1093-1101Crossref PubMed Scopus (21) Google Scholar), including the silkworm. In the silkworm, sucrases in larval midgut tissue showed a kinetic property that is characteristic of β-fructofuranosidase (11Sumida M. Yuan X.L. Matsubara F. Comp. Biochem. Physiol. B Biochem. Mol. Biol. 1994; 107: 273-284Crossref Scopus (12) Google Scholar, 12Sumida M. Yuan X.L. Matsubara F. Comp. Biochem. Physiol. A Biochem. Mol. Biol. 1994; 108: 255-264Google Scholar), and more importantly, silkworm sucrases were shown to be less affected by sugar mimic alkaloids than those of other organisms (7Asano N. Yamashita T. Yasuda K. Ikeda K. Kizu H. Kameda Y. Kato A. Nash R.J. Lee H.S. Ryu K.S. J. Agric. Food Chem. 2001; 49: 4208-4213Crossref PubMed Scopus (317) Google Scholar, 8Hirayama C. Konno K. Wasano N. Nakamura M. Insect. Biochem. Mol. Biol. 2007; 37: 1348-1358Crossref PubMed Scopus (80) Google Scholar).Although there has been no direct experimental evidence that the β-fructofuranosidase gene is encoded in the genome of animals, these observations lead us to hypothesize that the β-fructofuranosidase gene is encoded in the silkworm genome and could play an important role in the silkworm'ns ability to feed on mulberry leaves. By using β-fructofuranosidases as digestive enzymes, the silkworm could possibly avoid the toxic effects of d-AB1 and DNJ that are present in extremely high concentrations in the mulberry latex. As the first step toward understanding the mechanism by which the silkworm circumvents the mulberry'ns defense system, we have cloned and functionally characterized the β-fructofuranosidase genes from the silkworm, B. mori.We successfully isolated two β-fructofuranosidase genes from the silkworm. This is the first report of isolation of β-fructofuranosidase genes from an animal species. One of them (BmSuc1) appears to encode a functional enzyme, whereas the expression of the other (BmSuc2) was not detected. We then expressed the recombinant BmSUC1 protein and investigated its enzymatic properties. We further examined the expression and localization of BmSUC1 protein. Our data clearly indicated that the functional β-fructofuranosidase gene is actually encoded in the silkworm genome, and the product serves as a digestive enzyme in the silkworm.EXPERIMENTAL PROCEDURESInsects and Cell Line—The B. mori strains p50T and N4 were reared on an artificial diet in a conditioned insect rearing room (25 °C, 12-h light/12-h dark photoperiod) (14Daimon T. Hamada K. Mita K. Okano K. Suzuki M.G. Kobayashi M. Shimada T. Insect Biochem. Mol. Biol. 2003; 33: 749-759Crossref PubMed Scopus (77) Google Scholar). The Spodoptera frugiperda cell line (Sf9) was maintained in TC-100 medium containing 10% fetal bovine serum as described previously (15Daimon T. Katsuma S. Iwanaga M. Kang W. Shimada T. Insect Biochem. Mol. Biol. 2005; 35: 1112-1123Crossref PubMed Scopus (52) Google Scholar).Cloning of β-Fructofuranosidase Genes—Expressed sequence tags (ESTs) (16Mita K. Morimyo M. Okano K. Koike Y. Nohata J. Kawasaki H. Kadono-Okuda K. Yamamoto K. Suzuki M.G. Shimada T. Goldsmith M.R. Maeda S. Proc. Natl. Acad. Sci. U. S. A. 2003; 100: 14121-14126Crossref PubMed Scopus (230) Google Scholar) and genome sequences (17Mita K. Kasahara M. Sasaki S. Nagayasu Y. Yamada T. Kanamori H. Namiki N. Kitagawa M. Yamashita H. Yasukochi Y. Kadono-Okuda K. Yamamoto K. Ajimura M. Ravikumar G. Shimomura M. Nagamura Y. Shin I.T. Abe H. Shimada T. Morishita S. Sasaki T. DNA Res. 2004; 11: 27-35Crossref PubMed Scopus (555) Google Scholar, 18Xia Q. Zhou Z. Lu C. Cheng D. Dai F. Li B. Zhao P. Zha X. Cheng T. Chai C. Pan G. Xu J. Liu C. Lin Y. Qian J. Hou Y. Wu Z. Li G. Pan M. Li C. Shen Y. Lan X. Yuan L. Li T. Xu H. Yang G. Wan Y. Zhu Y. Yu M. Shen W. Wu D. Xiang Z. Yu J. Wang J. Li R. Shi J. Li H. Su J. Wang X. Zhang Z. Wu Q. Li J. Zhang Q. Wei N. Sun H. Dong L. Liu D. Zhao S. Zhao X. Meng Q. Lan F. Huang X. Li Y. Fang L. Li D. Sun Y. Yang Z. Huang Y. Xi Y. Qi Q. He D. Huang H. Zhang X. Wang Z. Li W. Cao Y. Yu Y. Yu H. Ye J. Chen H. Zhou Y. Liu B. Ji H. Li S. Ni P. Zhang J. Zhang Y. Zheng H. Mao B. Wang W. Ye C. Wong G.K. Yang H. Science. 2004; 306: 1937-1940Crossref PubMed Scopus (932) Google Scholar) showing homology to β-fructofuranosidase genes were investigated using the BLAST program. By sequencing and assembling them, we obtained two nonredundant genes (BmSuc1 and BmSuc2) that putatively encode for full-length β-fructofuranosidases.Genomic DNA Blot and RNA Blot Analysis—Genomic DNA blot and RNA blot experiments were performed as described previously (14Daimon T. Hamada K. Mita K. Okano K. Suzuki M.G. Kobayashi M. Shimada T. Insect Biochem. Mol. Biol. 2003; 33: 749-759Crossref PubMed Scopus (77) Google Scholar). Briefly, 5 μg of genomic DNA fully digested with restriction enzymes or total RNA was electrophoresed and transferred onto a nylon membrane. Labeling, hybridization, and signal detection were performed using the digoxigenin (DIG) labeling and detection system (Roche Applied Science) following the supplier'ns instructions. DNA probes were amplified by PCR using the EST clone mg-0575 (for BmSuc1) (16Mita K. Morimyo M. Okano K. Koike Y. Nohata J. Kawasaki H. Kadono-Okuda K. Yamamoto K. Suzuki M.G. Shimada T. Goldsmith M.R. Maeda S. Proc. Natl. Acad. Sci. U. S. A. 2003; 100: 14121-14126Crossref PubMed Scopus (230) Google Scholar) or genomic DNA (for BmSuc2) as templates. Primers used for PCR are listed in Table 1.TABLE 1List of primersPrimerSequence (5′ to 3′)PurposeBmSuc1S-FAATCCAGTCCTCTCCTACGTGCProbe synthesis for BmSuc1BmSuc1S-RTCCGGTCTGATACGTGTTCTTGBmSuc2S-FACGTGCAACTGTGACTCTCCTGProbe synthesis for BmSuc2BmSuc2S-RCTGATGCCTCCTGTTAGGGAAGpBacSUC1-FCCTAGGCCTAGTTATGTTCGCCTGGAGCACACCGGTGConstruction of His-tagged BmSUC1pBacSUC1-RCCTAGGCCTTCAATGGTGATGGTGATGATGACCAGCGGGTACACTTCTTCTCAATCGBmSuc1RT-FGGGCTGGAATTCTTTACGACCRT-PCR of BmSuc1BmSuc1RT-RGCTGGATGAATGACCCTAACG Open table in a new tab Phylogenetic Analysis—Sequence homologies were analyzed by the BLAST program for public DNA/protein data bases. The amino acid sequences were aligned with the ClustalX program (19Thompson J.D. Gibson T.J. Plewniak F. Jeanmougin F. Higgins D.G. Nucleic Acids Res. 1997; 25: 4876-4882Crossref PubMed Scopus (35195) Google Scholar), and phylogenetic trees were constructed by neighbor-joining methods using the MEGA3 program (20Kumar S. Tamura K. Nei M. Brief Bioinform. 2004; 5: 150-163Crossref PubMed Scopus (10630) Google Scholar) as previously described (15Daimon T. Katsuma S. Iwanaga M. Kang W. Shimada T. Insect Biochem. Mol. Biol. 2005; 35: 1112-1123Crossref PubMed Scopus (52) Google Scholar).Expression and Purification of BmSUC1—The recombinant Autographa californica nucleopolyhedrovirus (AcMNPV) was constructed using a Bac-to-Bac system (Invitrogen). The coding region of BmSuc1 with a His tag sequence at the C terminus was PCR-amplified with primers pBacSUC1-F and pBacSUC1-R (Table 1), using the EST clone mg-0757 as a template. The PCR product was cloned into the cloning site (StuI) of the pFastBac1 vector (Invitrogen). Virus propagation and infection were performed as described previously (15Daimon T. Katsuma S. Iwanaga M. Kang W. Shimada T. Insect Biochem. Mol. Biol. 2005; 35: 1112-1123Crossref PubMed Scopus (52) Google Scholar). Monolayers of Sf9 cells cultured in a 150-mm dish were infected with the virus at a multiplicity of infection of 5. After 72 h, the culture medium was centrifuged at 20,000 × g for 5 min, and the supernatant was collected. Protein purification was performed using nickel chromatography. The medium was then concentrated from 300 to 20 ml using Centriprep YM-30 (Amicon), dialyzed against a binding buffer (20 mm sodium phosphate, pH 7.4, 500 mm NaCl, and 10 mm imidazole) overnight at 4 °C, and loaded onto a HiTrap column (Amersham Biosciences). After washing with 5 ml of a wash buffer (20 mm sodium phosphate, pH 7.4, 500 mm NaCl, and 40 mm imidazole), elution was performed using an elution buffer (20 mm sodium phosphate, pH 7.4, 500 mm NaCl, and 200 mm imidazole). The eluate was dialyzed against a 20 mm sodium phosphate buffer (pH 7.4) and stored at 4 °C or -80 °C until use.Enzyme Assay—Enzyme activity was assayed by two methods using glucose as a standard substrate. Glucose released in the reaction was detected by the glucose oxidase-peroxidase method, and the reducing sugars were determined by the Somogyi-Nelson method. A 100-μl reaction containing 1 μg of purified protein, 100 mm substrate, and 10 mm sodium phosphate buffer (pH 7.0), was incubated for 1–20 min at 30 °C. After incubation, the reaction was stopped by boiling for 5 min. To measure the glucose liberated, a glucose test kit (Wako) was used as indicated by the supplier'ns protocol. The reducing sugars that were liberated during the reaction were estimated by the Somogyi-Nelson method. To establish the effect of pH on the sucrose hydrolytic activity, a 100-μl reaction containing 1 μg of purified protein, 100 mm sucrose, and 20 mm Britton-Robinson'ns wide range buffer (pH 4.0–11.0) was incubated for 1–20 min at 30 °C. The glucose liberated was measured using a glucose test kit (Wako). To see the effect of DNJ, an inhibitor of α-glucosidase, the sucrose hydrolytic activity was determined by a procedure identical to that described above except for the addition of DNJ (Sigma) to the reaction. For comparison, α-glucosidase from a bacterium Bacillus stearothermophilus (Sigma) was also assayed.RT-PCR Analysis—Total RNA from the tissues was extracted using TRIZOL reagent (Invitrogen). cDNAs were obtained using the Superscript II reverse transcriptase (Invitrogen), following the manufacturer'ns instructions. PCR conditions were as follows: 94 °C for 2 min followed by 30 cycles of 94 °C for 30 s; 55 °C for 30 s; and 72 °C for 1 min. Primers used for PCR are listed in Table 1. PCR products were analyzed on a 1.0% agarose gel.SDS-PAGE and Immunoblot Analysis—SDS-PAGE and immunoblot were performed as described previously (15Daimon T. Katsuma S. Iwanaga M. Kang W. Shimada T. Insect Biochem. Mol. Biol. 2005; 35: 1112-1123Crossref PubMed Scopus (52) Google Scholar). An antiserum against recombinant BmSUC1 was raised in rabbits (Operon Biotechnology). Protein samples were extracted from B. mori tissues as follows. Frozen tissues were homogenized in a 10 mm phosphate buffer (pH 7.0) containing the Complete protein inhibitor mixture (Roche Applied Science) using liquid nitrogen and centrifuged at 20,000 × g for 10 min at 4 °C, and the supernatants were collected. The primary antibody was the penta-His antibody (Qiagen) at a dilution of 1:2,000 or the anti-BmSUC1 antiserum at a dilution of 1:4,000. Signals were detected with the Immobilon Western Kit (Millipore) using the LAS1000 Plus imaging system (Fuji Film).Immunohistochemistry—Immunohistochemistry was carried out as described previously (15Daimon T. Katsuma S. Iwanaga M. Kang W. Shimada T. Insect Biochem. Mol. Biol. 2005; 35: 1112-1123Crossref PubMed Scopus (52) Google Scholar). Sections of the middle part of the midgut were obtained from 3-day-old fifth instar of strain p50T. Sections of silk glands were obtained from the same developmental stage of strain N4, since this strain expresses carotenoid-binding protein (CBP) (21Tsuchida K. Jouni Z.E. Gardetto J. Kobayashi Y. Tabunoki H. Azuma M. Sugiyama H. Takada N. Maekawa H. Banno Y. Fujii H. Iwano H. Wells M.A. J. Insect Physiol. 2004; 50: 363-372Crossref PubMed Scopus (24) Google Scholar), which was used for control experiments. The primary antibody was anti-BmSUC1 at a dilution of 1:200, and the secondary antibody was an AlexaFluor488- or AlexaFluor546-labeled goat anti-rabbit IgG F(ab)2 fragment (Invitrogen) at a dilution of 1:200. The slides were counterstained with a 4′,6-diamidino-2-phenylindole dihydrochloride solution (DAPI; Wako) or AlexaFluor594-conjugated phalloidin (Molecular Probes, Inc., Eugene, OR). The fluorescence was observed under a fluorescence microscope (Olympus BX51) and photographed. For control experiments, antisera against soluble and membrane-bound alkaline phosphatase of B. mori (sALP and mALP) (22Azuma M. Eguchi M. J. Exp. Zool. 1989; 251: 108-112Crossref Scopus (29) Google Scholar), and CBP (21Tsuchida K. Jouni Z.E. Gardetto J. Kobayashi Y. Tabunoki H. Azuma M. Sugiyama H. Takada N. Maekawa H. Banno Y. Fujii H. Iwano H. Wells M.A. J. Insect Physiol. 2004; 50: 363-372Crossref PubMed Scopus (24) Google Scholar) were also used at a dilution of 1:200, following the procedure described above.RESULTSCloning of β-Fructofuranosidase Genes from B. mori—We performed homology searches against the whole genome data of B. mori and found two genes that show significant homology to bacterial β-fructofuranosidase (BmSuc1 and BmSuc2) (Fig. 1). BmSuc1 was found also in the silkworm EST data base (e.g. clone mg-0575) (16Mita K. Morimyo M. Okano K. Koike Y. Nohata J. Kawasaki H. Kadono-Okuda K. Yamamoto K. Suzuki M.G. Shimada T. Goldsmith M.R. Maeda S. Proc. Natl. Acad. Sci. U. S. A. 2003; 100: 14121-14126Crossref PubMed Scopus (230) Google Scholar), whereas BmSuc2 was not.The BmSuc1 gene contained an open reading frame of 1464 bp encoding 488 amino acid residues with a predicted molecular mass of 55.9 kDa. The BmSuc2 gene contained an open reading frame of 1518 bp encoding 506 amino acid residues with a predicted molecular mass of 58.0 kDa. There was no intron in the coding region of either of the genes.These genes were predicted to encode proteins that exhibited high homology to bacterial β-fructofuranosidases (Fig. 1). BmSUC1 and BmSUC2 showed 45 and 39% identity (in amino acids) to Bacillus licheniformis β-fructofuranosidase (GenBank™ accession number AAU25612), respectively. The active site of invertase (Asp-23 in yeast invertase) (23Reddy A. Maley F. J. Biol. Chem. 1996; 271: 13953-13957Abstract Full Text Full Text PDF PubMed Scopus (128) Google Scholar) was conserved in BmSUC1 (Asp-63) but not in BmSUC2 (His-54) (Fig. 1), suggesting that BmSUC2 might not possess β-fructofuranosidase activity. The N-terminal sequence of both predicted polypeptides consisted of a putative signal peptide that contained hydrophobic residues. Signal P prediction (24Emanuelsson O. Brunak S. von Heijne G. Nielsen H. Nat. Protoc. 2007; 2: 953-971Crossref PubMed Scopus (2601) Google Scholar) positioned the putative cleavage site between residues 21 and 22 for BmSUC1 and between residues 18 and 19 for BmSUC2 (Fig. 1).Genomic DNA Blot and Phylogenetic Analysis—To eliminate the possibility that β-fructofuranosidase genes were obtained because of the contamination of the microbial DNA or RNA, genomic DNA blot analysis was performed (data not shown). Genomic DNA of the B. mori p50T strain was digested with selected restriction enzymes, blotted onto a nylon membrane, and then probed with DIG-labeled PCR products of either BmSuc1 or BmSuc2. A single band was detected in each case, and the molecular size of the signal was different between them, suggesting that both BmSuc1 and BmSuc2 are single copy genes located on the silkworm genome (data not shown).To investigate the evolutionary relationship between the BmSuc1, BmSuc2, and other β-fructofuranosidase genes, phylogenetic analysis was performed (Fig. 2). We searched proteins that show homology to BmSUC1 and BmSUC2 from public data bases with the BLAST program. The BLAST search retrieved many bacterial, fungal, and plant sucrases with high sequence homology. Their amino acid sequences were aligned with the ClustalX program (19Thompson J.D. Gibson T.J. Plewniak F. Jeanmougin F. Higgins D.G. Nucleic Acids Res. 1997; 25: 4876-4882Crossref PubMed Scopus (35195) Google Scholar), and a phylogenetic tree was constructed with the neighbor-joining method (Fig. 2). BmSUC1 and BmSUC2 were shown to form a monophyletic clade, suggesting that they may have been derived from a gene duplication event in an ancestral insect. Interestingly, these two genes were shown to belong to bacterial lineages. This may suggest a horizontal gene transfer from bacteria to an ancestral insect, although the phylogeny showed a deep division between bacterial and silkworm β-fructofuranosidases.FIGURE 2Phylogenetic analysis of β-fructofuranosidases. Amino acid sequences of β-fructofuranosidases were aligned by the ClustalX program, and the phylogenetic tree was constructed by the neighbor-joining method using the MEGA3 program package (20Kumar S. Tamura K. Nei M. Brief Bioinform. 2004; 5: 150-163Crossref PubMed Scopus (10630) Google Scholar). Bootstrap values after 1,000 replications are shown. GenBank™ or SwissProt accession numbers of sequences are shown in parentheses.View Large Image Figure ViewerDownload Hi-res image Download (PPT)Expression and Purification of Recombinant BmSUC1 Protein—To characterize the enzymatic property, recombinant His-tagged BmSUC1 protein was expressed using a baculovirus expression system (Fig. 3). Since the expression of BmSuc2 was not detected in our RT-PCR analysis, only BmSuc1 was analyzed further.FIGURE 3Expression and purification of the His-tagged BmSUC1 protein. Recombinant BmSUC1 protein with the His tag at the C terminus was expressed using a baculovirus expression system. BmSUC1 was purified from the medium of virus-infected cells by nickel chromatography. Protein samples were analyzed by SDS-PAGE and stained with CBB (a), and the same samples were analyzed by immunoblot with the anti-His antibody (b). The molecular masses of the protein standards are shown on the left, and the estimated molecular mass of BmSUC1 is shown on the right. The following are the protein samples used. Mock, the medium from mock-infected cells; WT, the medium from parental AcMNPV-infected cells; BmSUC1-His, the medium from cells infected with the recombinant AcMNPV that expressed BmSUC1 protein; purified, the purified BmSUC1 protein.View Large Image Figure ViewerDownload Hi-res image Download (PPT)The BmSUC1 protein of ∼56 kDa, which was consistent with the predicted molecular mass, was purified by nickel-chelating chromatography from the culture medium of virus-infected cells (Fig. 3a). The presence of the His tag sequence at the C terminus was confirmed by immunoblot analysis (Fig. 3b). The purified protein was used for further examination of the enzymatic properties.Enzyme Assay of BmSUC1—The substrate specificity of BmSUC1 was assayed using several sugar substrates (sucrose, isomaltose, maltose, raffinose, or stachyose) (Fig. 4). BmSUC1 was active on sucrose, raffinose, and stachyose, which possess the nonreducing β-d-fructofuranoside residues (Fig. 4b). However, BmSUC1 was not active on isomaltose or maltose, which lacks the β-fructofuranoside residue (Fig. 4a). This result clearly indicates that the BmSuc1 gene encodes a β-fructofuranosidase (EC 3.2.1.26) that hydrolyzed the β-fructosyl residue of the substrate. The pH profile of BmSUC1 was determined using sucrose as the substrate (Fig. 4c). The assay was performed in a pH range of 4.0–11.0, and the condition of pH 7.0 was shown to be optimal.FIGURE 4Enzymatic properties of recombinant BmSUC1 protein. BmSUC1 protein was incubated with selected substrates (sucrose, isomaltose, maltose, raffinose, or stachose), and the substrate specificity of BmSUC1 was measured. a, the liberated glucose was measured by the glucose oxidase method; b, the reducing sugars released were estimated by the Somogyi-Nelson method. Bars, mean ± S.D. (n = 3). c, pH-activity relationship of the recombinant BmSUC1, determined using sucrose as a substrate. The points indicate the mean ± S.D. (n = 3). d, inhibitory assay of DNJ on the recombinant BmSUC1 (filled circle) and the α-glucosidase (open triangle) from a bacterium (Sigma), using sucrose as a substrate. The same unit (nmol of glucose produced/min) of the BmSUC1 and bacterial α-glucosidase was used for the analysis. The points indicate the mean ± S.D. (n = 3).View Large Image Figure ViewerDownload Hi-res image Download (PPT)Next, we analyzed the inhibitory activities of DNJ on BmSUC1 activity (Fig. 4d). For comparison, α-glucosidase from bacterium Bacillus stearothermophilus was also used in the assay. As expected, DNJ strongly inhibited the α-glucosidase with an IC50 value of 10 μm. In contrast, BmSUC1 activity was not inhibited by DNJ (Fig. 4d), even at a final concentration of 1200 μm (IC50 > 1200 μm; data not shown).Expression Profiles of BmSuc1 and BmSuc2 Genes—The expression profile of BmSuc1 mRNA was examined by RT-PCR (Fig. 5a). On the 3rd day of the fifth instar larvae, BmSuc1 mRNA was expressed in midgut and, interestingly, also in the silk gland. Signals were detected throughout the midgut (i.e. anterior, middle, and posterior parts of the midgut) but were not detected in the foregut and hindgut of B. mori. In the silk glands, signals were detected in the anterior silk gland and anterior and middle part of the middle silk gland (MSG) but were not detected in the posterior part of MSG and in the posterior silk gland. Signals were not detected in other tissues, such as testis, fat bodies, epidermis, trachea, or malphigian tubules.FIGURE 5Expression profiles of BmSuc1 mRNA.a, RT-PCR analysis of BmSuc1 expression. Total RNA from the 3rd day of
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