Purification, Characterization, and Cloning of a Spodoptera frugiperda Sf9 β-N-Acetylhexosaminidase That Hydrolyzes Terminal N-Acetylglucosamine on the N-Glycan Core
2006; Elsevier BV; Volume: 281; Issue: 28 Linguagem: Inglês
10.1074/jbc.m603312200
ISSN1083-351X
AutoresNoboru Tomiya, Someet Narang, Jung Park, Badarulhisam Abdul‐Rahman, One Choi, Sundeep Singh, Jun Hiratake, Kanzo Sakata, Michael J. Betenbaugh, Karen Palter, Yuan C. Lee,
Tópico(s)Trypanosoma species research and implications
ResumoPaucimannosidic glycans are often predominant in N-glycans produced by insect cells. However, a β-N-acetylhexosaminidase responsible for the generation of paucimannosidic glycans in lepidopteran insect cells has not been identified. We report the purification of a β-N-acetylhexosaminidase from the culture medium of Spodoptera frugiperda Sf9 cells (Sfhex). The purified Sfhex protein showed 10 times higher activity for a terminal N-acetylglucosamine on the N-glycan core compared with tri-N-acetylchitotriose. Sfhex was found to be a homodimer of 110 kDa in solution, with a pH optimum of 5.5. With a biantennary N-glycan substrate, it exhibited a 5-fold preference for removal of the β(1Henrissat B. Biochem. J. 1991; 280: 309-316Crossref PubMed Scopus (2624) Google Scholar,2Henrissat B. Bairoch A. Biochem. J. 1993; 293: 781-788Crossref PubMed Scopus (1771) Google Scholar)-linked N-acetylglucosamine from the Manα(1Henrissat B. Biochem. J. 1991; 280: 309-316Crossref PubMed Scopus (2624) Google Scholar,3Henrissat B. Bairoch A. Biochem. J. 1996; 316: 695-696Crossref PubMed Scopus (1186) Google Scholar) branch compared with the Manα(1Henrissat B. Biochem. J. 1991; 280: 309-316Crossref PubMed Scopus (2624) Google Scholar,6Kubelka V. Altmann F. Kornfeld G. Marz L. Arch. Biochem. Biophys. 1994; 308: 148-157Crossref PubMed Scopus (128) Google Scholar) branch. We isolated two corresponding cDNA clones for Sfhex that encode proteins with >99% amino acid identity. A phylogenetic analysis suggested that Sfhex is an ortholog of mammalian lysosomal β-N-acetylhexosaminidases. Recombinant Sfhex expressed in Sf9 cells exhibited the same substrate specificity and pH optimum as the purified enzyme. Although a larger amount of newly synthesized Sfhex was secreted into the culture medium by Sf9 cells, a significant amount of Sfhex was also found to be intracellular. Under a confocal microscope, cellular Sfhex exhibited punctate staining throughout the cytoplasm, but did not colocalize with a Golgi marker. Because secretory glycoproteins and Sfhex are cotransported through the same secretory pathway and because Sfhex is active at the pH of the secretory compartments, this study suggests that Sfhex may play a role as a processing β-N-acetylhexosaminidase acting on N-glycans from Sf9 cells. Paucimannosidic glycans are often predominant in N-glycans produced by insect cells. However, a β-N-acetylhexosaminidase responsible for the generation of paucimannosidic glycans in lepidopteran insect cells has not been identified. We report the purification of a β-N-acetylhexosaminidase from the culture medium of Spodoptera frugiperda Sf9 cells (Sfhex). The purified Sfhex protein showed 10 times higher activity for a terminal N-acetylglucosamine on the N-glycan core compared with tri-N-acetylchitotriose. Sfhex was found to be a homodimer of 110 kDa in solution, with a pH optimum of 5.5. With a biantennary N-glycan substrate, it exhibited a 5-fold preference for removal of the β(1Henrissat B. Biochem. J. 1991; 280: 309-316Crossref PubMed Scopus (2624) Google Scholar,2Henrissat B. Bairoch A. Biochem. J. 1993; 293: 781-788Crossref PubMed Scopus (1771) Google Scholar)-linked N-acetylglucosamine from the Manα(1Henrissat B. Biochem. J. 1991; 280: 309-316Crossref PubMed Scopus (2624) Google Scholar,3Henrissat B. Bairoch A. Biochem. J. 1996; 316: 695-696Crossref PubMed Scopus (1186) Google Scholar) branch compared with the Manα(1Henrissat B. Biochem. J. 1991; 280: 309-316Crossref PubMed Scopus (2624) Google Scholar,6Kubelka V. Altmann F. Kornfeld G. Marz L. Arch. Biochem. Biophys. 1994; 308: 148-157Crossref PubMed Scopus (128) Google Scholar) branch. We isolated two corresponding cDNA clones for Sfhex that encode proteins with >99% amino acid identity. A phylogenetic analysis suggested that Sfhex is an ortholog of mammalian lysosomal β-N-acetylhexosaminidases. Recombinant Sfhex expressed in Sf9 cells exhibited the same substrate specificity and pH optimum as the purified enzyme. Although a larger amount of newly synthesized Sfhex was secreted into the culture medium by Sf9 cells, a significant amount of Sfhex was also found to be intracellular. Under a confocal microscope, cellular Sfhex exhibited punctate staining throughout the cytoplasm, but did not colocalize with a Golgi marker. Because secretory glycoproteins and Sfhex are cotransported through the same secretory pathway and because Sfhex is active at the pH of the secretory compartments, this study suggests that Sfhex may play a role as a processing β-N-acetylhexosaminidase acting on N-glycans from Sf9 cells. β-N-Acetylhexosaminidase (hexosaminidase, EC 3.2.1.52) catalyzes the hydrolysis of nonreducing terminal N-acetyl-d-hexosamine residues in N-acetyl-β-d-hexosaminides. Hexosaminidases belong to the glycosyl hydrolase (GH) 2The abbreviations used are: GH, glycosyl hydrolase; Sfhex, S. frugiperda hexosaminidase; MU-GlcNAc, 4-methylumbelliferyl 2-acetamido-2-deoxy-β-d-glucopyranoside; MU-GalNAc, 4-methylumbelliferyl 2-acetamido-2-deoxy-β-d-galactopyranoside; PA, 2-aminopyridine; HPLC, high-performance liquid chromatography; pNP-GlcNAc, p-nitrophenyl 2-acetamido-2-deoxy-β-d-glucopyranoside; PBS, phosphate-buffered saline; aa, amino acids; RACE, rapid amplification of cDNA ends; BSA, bovine serum albumin; HexB, hexosaminidase B. 3, GH20, or GH84 family (1Henrissat B. Biochem. J. 1991; 280: 309-316Crossref PubMed Scopus (2624) Google Scholar, 2Henrissat B. Bairoch A. Biochem. J. 1993; 293: 781-788Crossref PubMed Scopus (1771) Google Scholar, 3Henrissat B. Bairoch A. Biochem. J. 1996; 316: 695-696Crossref PubMed Scopus (1186) Google Scholar, 4Davies G. Henrissat B. Structure (Lond.). 1995; 3: 853-859Abstract Full Text Full Text PDF PubMed Scopus (1612) Google Scholar). Of these, family 20 hexosaminidases include mammalian lysosomal hexosaminidases, fungal exochitinases, bacterial chitobiases, and insect chitinolytic hexosaminidases. The hexosaminidase activity of insects and insect cells is of particular interest because of the role that the enzyme may play in altering the structures of N-glycans generated by these cells. The N-glycan synthesis pathway in insects differs from that in mammals in that insects and insect cells produce appreciable amounts of paucimannosidic glycans (reviewed in Ref. 5Tomiya N. Narang S. Lee Y.C. Betenbaugh M.J. Glycoconj. J. 2004; 21: 343-360Crossref PubMed Scopus (117) Google Scholar). The intracellular N-glycan processing pathway in the endoplasmic reticulum of insects has been observed to include the addition of a Glc3Man9GlcNAc2 group to the acceptor Asn residue, followed by the subsequent trimming of the initial oligosaccharide to generate Man5GlcNAc2. Insect cells also contain significant levels of N-acetylglucosaminyltransferase I, which adds a GlcNAc residue to the Manα(1Henrissat B. Biochem. J. 1991; 280: 309-316Crossref PubMed Scopus (2624) Google Scholar,3Henrissat B. Bairoch A. Biochem. J. 1996; 316: 695-696Crossref PubMed Scopus (1186) Google Scholar) branch, followed by the removal of two Man residues to produce the GlcNAcβ(1Henrissat B. Biochem. J. 1991; 280: 309-316Crossref PubMed Scopus (2624) Google Scholar,2Henrissat B. Bairoch A. Biochem. J. 1993; 293: 781-788Crossref PubMed Scopus (1771) Google Scholar)Manα(1Henrissat B. Biochem. J. 1991; 280: 309-316Crossref PubMed Scopus (2624) Google Scholar,3Henrissat B. Bairoch A. Biochem. J. 1996; 316: 695-696Crossref PubMed Scopus (1186) Google Scholar)(Man(1Henrissat B. Biochem. J. 1991; 280: 309-316Crossref PubMed Scopus (2624) Google Scholar,6Kubelka V. Altmann F. Kornfeld G. Marz L. Arch. Biochem. Biophys. 1994; 308: 148-157Crossref PubMed Scopus (128) Google Scholar))-Manβ(1Henrissat B. Biochem. J. 1991; 280: 309-316Crossref PubMed Scopus (2624) Google Scholar,4Davies G. Henrissat B. Structure (Lond.). 1995; 3: 853-859Abstract Full Text Full Text PDF PubMed Scopus (1612) Google Scholar)GlcNAcβ(1Henrissat B. Biochem. J. 1991; 280: 309-316Crossref PubMed Scopus (2624) Google Scholar,4Davies G. Henrissat B. Structure (Lond.). 1995; 3: 853-859Abstract Full Text Full Text PDF PubMed Scopus (1612) Google Scholar)GlcNAc structure. Unlike mammalian cells, which subsequently modify this intermediate to yield complex, often sialylated oligosaccharides, insect cells typically hydrolyze the nonreducing terminal GlcNAc residue attached to the Manα(1Henrissat B. Biochem. J. 1991; 280: 309-316Crossref PubMed Scopus (2624) Google Scholar,3Henrissat B. Bairoch A. Biochem. J. 1996; 316: 695-696Crossref PubMed Scopus (1186) Google Scholar) branch of the N-glycan core by the action of a hexosaminidase, leading to generation of paucimannosidic structures with one or two Man attachments. Indeed, N-glycans that do not contain this GlcNAc residue are often prevalent on glycoproteins expressed in insect lines such as MB-0503 cells from Mamestra brassicae (cabbage moth), Sf21 cells from Spodoptera frugiperda (fall armyworm), and BmN cells from Bombyx mori (silk moth) (6Kubelka V. Altmann F. Kornfeld G. Marz L. Arch. Biochem. Biophys. 1994; 308: 148-157Crossref PubMed Scopus (128) Google Scholar); cells from Trichoplusia ni (cabbage looper) (7Ailor E. Takahashi N. Tsukamoto Y. Masuda K. Rahman B.A. Jarvis D.L. Lee Y.C. Betenbaugh M.J. Glycobiology. 2000; 10: 837-847Crossref PubMed Scopus (80) Google Scholar, 8Takahashi N. Tsukamoto Y. Shiosaka S. Kishi T. Hakoshima T. Arata Y. Yamaguchi Y. Kato K. Shimada I. Glycoconj. J. 1999; 16: 405-414Crossref PubMed Scopus (25) Google Scholar); and Ld652Y cells from Lymantria dispar (gypsy moth) (9Choi O. Tomiya N. Kim J.H. Slavicek J.M. Betenbaugh M.J. Lee Y.C. Glycobiology. 2003; 13: 539-548Crossref PubMed Scopus (25) Google Scholar); and in adult bodies of Drosophila melanogaster (10Fabini G. Freilinger A. Altmann F. Wilson I.B. J. Biol. Chem. 2001; 276: 28058-28067Abstract Full Text Full Text PDF PubMed Scopus (141) Google Scholar). Hexosaminidases are widely distributed in insects, including species of Lepidoptera, Coleoptera, Hemiptera, and Orthoptera (11Dziadik-Turner C. Koga D. Mai M.S. Kramer K.J. Arch. Biochem. Biophys. 1981; 212: 546-560Crossref PubMed Scopus (73) Google Scholar) and Diptera (12Filho B.P. Lemos F.J. Secundino N.F. Pascoa V. Pereira S.T. Pimenta P.F. Insect Biochem. Mol. Biol. 2002; 32: 1723-1729Crossref PubMed Scopus (85) Google Scholar). This activity was found not only in tissues, but also in the blood and molting fluid of B. mori L. (13Kimura S. Comp. Biochem. Physiol. B. 1974; 49: 345-351Crossref PubMed Scopus (28) Google Scholar) and in the secretion fluid of female accessory glands of Ceratitis capitata (mosquito) (14Marchini D. Bernini L.F. Dallai R. Insect Biochem. 1989; 19: 549-555Crossref Scopus (17) Google Scholar). Several studies have reported hexosaminidase activity in culture media as well as in cell extracts of lepidopteran insect cell lines such as S. frugiperda Sf9, T. ni TN-368, Malacosoma disstria MD108, and B. mori cells (15Licari P.J. Jarvis D.L. Bailey J.E. Biotechnol. Prog. 1993; 9: 146-152Crossref PubMed Scopus (57) Google Scholar); T. ni Tn-5B1-4 cells (16van Die I. van Tetering A. Bakker H. van den Eijnden D.H. Joziasse D.H. Glycobiology. 1996; 6: 157-164Crossref PubMed Scopus (80) Google Scholar); and a dipteran insect cell line, D. melanogaster Kc cells (17Sommer U. Spindler K.D. Arch. Insect Biochem. Physiol. 1991; 17: 3-13Crossref PubMed Scopus (13) Google Scholar, 18Sommer U. Spindler K.D. Arch. Insect Biochem. Physiol. 1991; 18: 45-53Crossref PubMed Scopus (7) Google Scholar). The culture medium of Culex quinquefasciatus (southern house mosquito) cells was also found to contain hexosaminidase activity (19Dziadik-Turner C. Koga D. Kramer K.J. Insect Biochem. 1981; 11: 215-219Crossref Scopus (11) Google Scholar). A few studies have reported the purification of a hexosaminidase from the hemolymph (20Kimura S. Biochim. Biophys. Acta. 1976; 446: 399-406Crossref PubMed Scopus (32) Google Scholar) and larval integument tissue (21Nagamatsu Y. Yanagisawa I. Kimoto M. Okamoto E. Koga D. Biosci. Biotechnol. Biochem. 1995; 59: 219-225Crossref PubMed Scopus (50) Google Scholar) of B. mori; the larval or pupal molting fluid, hemolymph, and integument tissue of Manduca sexta (tobacco hornworm) (11Dziadik-Turner C. Koga D. Mai M.S. Kramer K.J. Arch. Biochem. Biophys. 1981; 212: 546-560Crossref PubMed Scopus (73) Google Scholar, 22Koga D. Mai M.S. Kramer K.J. Comp. Biochem. Physiol. B. 1983; 74: 515-520Crossref Scopus (29) Google Scholar); the secretion fluid of female accessory glands of C. capitata (14Marchini D. Bernini L.F. Dallai R. Insect Biochem. 1989; 19: 549-555Crossref Scopus (17) Google Scholar); the culture medium and cell extract of D. melanogaster Kc cells (17Sommer U. Spindler K.D. Arch. Insect Biochem. Physiol. 1991; 17: 3-13Crossref PubMed Scopus (13) Google Scholar); and the culture medium of C. quinquefasciatus (19Dziadik-Turner C. Koga D. Kramer K.J. Insect Biochem. 1981; 11: 215-219Crossref Scopus (11) Google Scholar). Two closely related genes that encode insect hexosaminidases were cloned from B. mori (21Nagamatsu Y. Yanagisawa I. Kimoto M. Okamoto E. Koga D. Biosci. Biotechnol. Biochem. 1995; 59: 219-225Crossref PubMed Scopus (50) Google Scholar) and M. sexta (23Zen K.C. Choi H.K. Krishnamachary N. Muthukrishnan S. Kramer K.J. Insect Biochem. Mol. Biol. 1996; 26: 435-444Crossref PubMed Scopus (71) Google Scholar). The former is known to be an exochitinase. However, because these studies used synthetic substrates and not N-glycan substrates in the enzyme assay, it is not known whether or not previously purified or cloned insect hexosaminidases can hydrolyze the terminal GlcNAc residue linked to the N-glycan core. In addition to the above-mentioned studies, a microsomal membrane-associated hexosaminidase activity in lepidopteran insect cells that can catalyze such a reaction has been reported (24Altmann F. Schwihla H. Staudacher E. Glossl J. Marz L. J. Biol. Chem. 1995; 270: 17344-17349Abstract Full Text Full Text PDF PubMed Scopus (194) Google Scholar), and the importance of a cellular hexosaminidase activity in N-glycan processing in insect cells was supported by the finding of an inverse relationship between the level of cellular hexosaminidase activity and the level of GlcNAc-containing N-glycans in glycoproteins expressed in S. frugiperda Sf9 cells and Estigmene acrea cells (25Wagner R. Geyer H. Geyer R. Klenk H.-D. J. Virol. 1996; 70: 4103-4109Crossref PubMed Google Scholar). In this study, we describe the purification of a hexosaminidase from the culture broth of S. frugiperda Sf9 cells (Sfhex). This enzyme is a homodimer of 110 kDa with maximum activity at pH 5.5 and was found to preferentially remove terminal GlcNAc residues from the Manα(1Henrissat B. Biochem. J. 1991; 280: 309-316Crossref PubMed Scopus (2624) Google Scholar,3Henrissat B. Bairoch A. Biochem. J. 1996; 316: 695-696Crossref PubMed Scopus (1186) Google Scholar) branch of the N-glycan core. The N-terminal sequence of the purified enzyme was subsequently used to isolate two corresponding cDNA clones from S. frugiperda mRNA. When introduced into insect cells, the Sfhex cDNA resulted in enhanced hexosaminidase activity in the medium. On the basis of our characterization of Sfhex, we believe that this hexosaminidase from lepidopteran insect cells is capable of removing the terminal GlcNAc residue linked to the N-glycan core and therefore of generating paucimannosidic N-glycans. The identification of this type of hexosaminidase not only contributes to a better understanding of N-glycan processing in insect cells, but will be important in engineering insect cells capable of generating complex N-glycans that can be used for baculovirus expression of heterologous mammalian proteins (26Luckow V.A. Summers M.D. Virology. 1988; 167: 56-71Crossref PubMed Scopus (148) Google Scholar, 27Luckow V.L. Prokop A. Bajpai R.K. Ho C.S. Recombinant DNA Technology and Applications. McGraw-Hill Book Co., New York1991: 97-152Google Scholar). Materials—The following materials were obtained from the sources indicated: β-galactosidase (jack bean), β-N-acetylglucosaminidase (jack bean), α-l-fucosidase (bovine kidney), 2-aminopyridine, borane-dimethylamine complex, human apotransferrin, human IgG, bovine pancreatic ribonuclease B, 4-methylumbelliferyl 2-acetamido-2-deoxy-β-d-glucopyranoside (MU-GlcNAc), 4-methylumbelliferyl 2-acetamido-2-deoxy-β-d-galactopyranoside (MU-GalNAc), MU-GlcNAc-6SO4, and Nonidet P-40 (Sigma); tri-N-acetylchitotriose (Seikagaku America, East Falmouth, MA); 2-acetamido-1,2-dideoxynojirimycin (Toronto Research Chemicals Inc., North York, Ontario, Canada); Sephacryl S-200 HR (Amersham Biosciences); Shim-pack CLC-ODS column (6 × 150 mm; Shimadzu Scientific Instruments, Columbia, MD); Amide-80 column (2 × 250 mm; Tosoh Biosep LLC, Montgomeryville, PA); Sf-900 II SFM medium (Invitrogen); and peptide N-glycosidase F (ProZyme, San Leandro, CA). Oligosaccharides—The structures of oligosaccharides used in this work are shown in Table 1. The oligosaccharides were derivatized with 2-aminopyridine (PA) by the method of Kondo et al. (28Kondo A. Suzuki J. Kuraya N. Hase S. Kato I. Ikenaka T. Agric. Biol. Chem. 1990; 54: 2169-2170Crossref PubMed Scopus (195) Google Scholar). GnGn-PA was prepared from a PA derivative of a desialylated biantennary oligosaccharide prepared from human transferrin (Sigma). Briefly, sialic acids were removed by treating the PA-derivatized sialylated biantennary oligosaccharide in 20 mm HCl for 1 h at 80°C, and then terminal Gal residues were removed with jack bean β-galactosidase. MGn-PA and GnM-PA were prepared from PA derivatives of two positionally isomeric monogalactosylated biantennary oligosaccharides from human IgG (29Takahashi N. Ishii I. Ishihara H. Mori M. Tejima S. Jefferis R. Endo S. Arata Y. Biochemistry. 1987; 26: 1137-1144Crossref PubMed Scopus (157) Google Scholar) by sequential digestion with jack bean β-N-acetylglucosaminidase, β-galactosidase, and bovine kidney α-l-fucosidase. M3-PA and M5-PA were prepared from quail ovomucoid (30Hase S. Sugimoto T. Takemoto H. Ikenaka T. Schmid K. J. Biochem. (Tokyo). 1986; 99: 1725-1733Crossref PubMed Scopus (40) Google Scholar) and bovine pancreatic ribonuclease B, respectively. M5Gn-PA was synthesized from M5-PA by the action of N-acetylglucosaminyltransferase I (a kind gift from Dr. H. Schachter). (GlcNAc)3-PA was prepared from tri-N-acetylchitotriose. All PA-derivatized oligosaccharides were successively purified using normal-phase (Amide-80) and reversed-phase (Shim-pack CLC-ODS) HPLC columns before use.TABLE 1Structures of oligosaccharides Open table in a new tab Enzyme Assay—When 4-methylumbelliferyl glycosides were used as substrates, the substrates (10 nmol) were incubated in a 96-well plate with an appropriately diluted enzyme in 100 μl of 50 mm sodium citrate/phosphate buffer (pH 5.5) at 37 °C for 30 min. The reaction was quenched by the addition of 200 μl of 0.4 m glycine/NaOH buffer (pH 10.5), and released 4-methylum-belliferone was measured by fluorescence (excitation = 355 nm and emission = 460 nm) using a VICTOR Model 1420 multilabel counter (Wallac, Gaithersburg, MD) in a fluorometric mode. When p-nitrophenyl 2-acetamido-2-deoxy-β-d-glucopyranoside (pNP-GlcNAc) was used as the substrate, the substrate was incubated in a 96-well plate with an appropriately diluted enzyme in 100 μl of 30 mm sodium citrate/phosphate buffer (pH 5.5) at 37 °C for 30 min. The reaction was terminated as describe above, and released p-nitrophenol was measured at 415 nm using a Benchmark microplate reader (Bio-Rad). When PA-derivatized oligosaccharides were used as substrates, the reaction mixture contained substrates (500 pmol in 5 μl), an appropriately diluted enzyme solution (10 μl), and 0.1 m sodium citrate/phosphate buffer (pH 5.5; 10 μl). The mixture was incubated at 37 °C for predetermined periods of time. The reaction was terminated by heating the sample for 5 min in boiling water and centrifuged to obtain a clear supernatant. Substrates and products were analyzed using a normal-phase HPLC column (Amide-80). Enzyme activity was determined by the decrease of a substrate or the production of a product upon reaction. When GnGn-PA was used to determine the branch specificity of hexosaminidase, the enzyme reaction mixture was first subjected to normal-phase HPLC to separate the substrate, mono-N-acetylglucosaminylated M3-PA products, and the M3-PA product. A fraction containing mono-N-acetylglucosaminylated M3-PA products was isolated and then subjected to reversed-phase HPLC to separate MGn-PA and GnM-PA. When human asialoor aglactotransferrin was used as the substrate, the protein (24 μg) was incubated with an appropriately diluted enzyme solution (1 μl) in 20 mm sodium citrate/phosphate buffer (pH 5.5) at 37 °C for 5.5 h. After the reaction, 6-O-methylgalactose was added to the sample as an internal standard, and the sample was heated for 5 min in boiling water and centrifuged to obtain a clear supernatant. Released GlcNAc was measured by high-performance anion-exchange chromatography using a CarboPac PA20 column (3 × 150 mm; Dionex Corp., Sunnyvale, CA) and 10 mm sodium hydroxide as an eluent. Sugars were detected by pulsed amperometry. One unit of hexosaminidase is defined as the amount of enzyme required to catalyze the release of 1 μmol of terminal GlcNAc residue from substrates in 1 min at 37 °C. HPLC—All HPLC separations were performed using an LC-10Ai HPLC system (Shimadzu Scientific Instruments). Substrate PA-derivatized oligosaccharides and their degradation products were routinely analyzed by normal-phase HPLC using an Amide-80 column (2 × 250 mm). A sample (4 μl) was injected onto an Amide-80 column pre-equilibrated with Solvent A (80:20 (v/v) acetonitrile and 10 mm ammonium formate (pH 7)), and PA-derivatized oligosaccharides were eluted at a flow rate of 0.2 ml/min using a gradient consisting of Solvent A and Solvent B (50:50 (v/v) acetonitrile and 10 mm ammonium formate (pH 7)) by increasing the proportion of Solvent B (0% (t = 0 min), 50% (t = 20 min), and 100% (t = 70 min)). For reversed-phase HPLC, a sample (10 μl) was injected onto a Shim-pack CLC-ODS column (6 × 150 mm) pre-equilibrated with Solvent C (10 mm ammonium formate (pH 4.3) containing 0.1% 1-butanol). PA-derivatized oligosaccharides were eluted by increasing the concentration of 1-butanol from 0.1 to 0.175% over 30 min at a flow rate of 1.0 ml/min. PA-derivatized oligosaccharides were monitored by fluorescence (excitation = 300 nm and emission = 360 nm for normal-phase HPLC and excitation = 315 nm and emission = 380 nm for reversed-phase HPLC). Preparation of GlcNAc-amidine Affinity Adsorbent—GlcNAc-amidine-immobilized Toyopearl 650M was prepared as described previously (31Hiratake J. Sakata K. Methods Enzymol. 2003; 363: 421-444Crossref PubMed Scopus (6) Google Scholar, 32Inoue K. Hiratake J. Mizutani M. Takada M. Yamamoto M. Sakata K. Carbohydr. Res. 2003; 338: 1477-1490Crossref PubMed Scopus (18) Google Scholar) with a minor modification. TSK-GEL Toyopearl Carboxyl-650M (wet volume of 50 ml) modified with maleimido groups via a spacer (4,7,10-trioxatridecane-1,13-diamine) was treated with 2-acetamido-2-deoxy-β-d-glucopyranosylamine (5.5 g, 25 mmol) and 2-iminothiolane HCl (3.4 g, 25 mmol) in dry pyridine (100 ml) (33Kato M. Uno T. Hiratake J. Sakata K. Bioorg. Med. Chem. 2005; 13: 1563-1571Crossref PubMed Scopus (14) Google Scholar). The mixture was shaken at room temperature for 24 h and filtered. The resin was washed with ethanol and water successively and suspended in 0.1 m sodium citrate buffer (pH 6) until used. Cell Culture—Serum-free adapted Sf9 cells (Invitrogen) were routinely grown in serum-free Sf-900 II SFM medium in shaker flasks at 140 rpm and 27 °C. The cells were passaged every 4 days at a seeding density of 0.8 × 106 cells/ml. Cell Extract and Supernatant Preparation—The suspension culture was typically harvested at 96 h post-seeding, and a clarified cell culture supernatant and a cell pellet were obtained by centrifugation at 350 × g for 10 min. The cell pellet was washed with chilled phosphate-buffered saline (PBS) (Invitrogen). Cells were lysed by resuspending the cell pellet in chilled PBS containing 0.5% Nonidet P-40, followed by two cycles of sonication with a Tekmar sonic disruptor for 30 s at a 50% duty cycle and a power setting of 5. The cell debris was removed by centrifugation, and the clear extract was used for analysis. Purification of Hexosaminidase—At 96 h post-seeding, the suspension culture (1.8 liters) was centrifuged at 350 × g for 10 min, and the cell-free supernatant was collected. Purification was carried out at 4 °C. To the clarified cell culture supernatant were added sodium chloride and sodium citrate to final concentrations of 1 m and 10 mm, respectively, and the pH was adjusted to 6.0 with a dilute sodium hydroxide solution. The sample was loaded at a flow rate of 5 ml/min onto the GlcNAc-amidine affinity column (2.5 × 6 cm, 30 ml) pre-equilibrated with 10 mm sodium citrate buffer (pH 6.0) containing 1 m sodium chloride. After washing the column with 3 bed volumes of the same buffer, bound protein was eluted with 10 mm sodium citrate/phosphate buffer (pH 7.0) containing 1 m d-Gl-cNAc at a flow rate of 5 ml/min by pumping the elution buffer in the reverse direction, and 5-ml fractions were collected. Hexosaminidase activity in each fraction was measured with MU-GlcNAc as the substrate, and the protein concentration was determined by the method of Bradford (34Bradford M.M. Anal. Biochem. 1976; 72: 248-254Crossref PubMed Scopus (216377) Google Scholar) using bovine serum albumin as a standard. SDS-PAGE—The purified Sf hex protein (0.5 μg) was analyzed by SDS-PAGE (10% acrylamide gel) under reducing conditions, and proteins were visualized by staining with Coomassie Brilliant Blue R-250. Gel Filtration Chromatography—The purified Sfhex protein (30 μg in 0.5 ml) was applied to a Sephacryl S-200 HR column (1.6 × 60 cm) pre-equilibrated with 10 mm sodium citrate/phosphate buffer (pH 6.5) containing 0.15 m sodium chloride. After loading a sample, protein was eluted with the same buffer at a flow rate of 25 ml/h, and 1-ml fractions were collected. Hexosaminidase activity in each fraction was measured with MU-GlcNAc as the substrate as described above. The column was calibrated with bovine thyroglobulin, human IgG, bovine serum albumin, chicken ovalbumin, bovine β-lactoglobulin, bovine ribonuclease A, and uridine. Deglycosylation—N-Glycans attached to Sfhex was released by digesting the purified Sfhex protein with peptide N-glycosidase F. The reaction was performed according to the manufacturer's protocol. Sequencing of N-terminal Amino Acids—N-terminal sequence analysis of the protein sample was carried out by Edman degradation using an Applied Biosystems Procise Model 494A protein sequencer. Isolation of a cDNA Clone Encoding Sfhex and DNA Sequencing—Aligning the amino acid sequences of hexosaminidases from human, mouse, D. melanogaster, B. mori, M. sexta, and T. ni showed a highly conserved internal peptide (H(L/M)GG-DEV, amino acids (aa) 318-324 of the human hexosaminidase α-chain) that forms part of the catalytic domain. This segment is common to all of these hexosaminidases and many other hexosaminidases in the GH20 family. Degenerate primers derived from both the N-terminal (LSIVNPGPQYPPTKGSIWPRP) and internal sequences were used for reverse transcription-PCR of Sf9 RNA to amplify a cDNA corresponding to a portion of the Sfhex gene. We reasoned that, although the downstream primer is common to all known hexosaminidase proteins, the upstream primer is specific for the Sfhex gene, and therefore, only the cDNA corresponding to the Sfhex gene would be amplified. The forward primer SF1′ (5′-CACTAAGCTTAAYCCNGGNCCNCARTAYCC) contained a HindIII site (shown in italics) and sequence corresponding to aa 23-29 (Supplemental Fig. S3). The reverse primer SF5′ (5-AGTGAAGCTTACYTCRTCNCCNCCNADRTG) contained a HindIII site (shown in italics) and sequence corresponding to aa 333-339. Total RNA prepared by the TRIzol method (Invitrogen) from Sf9 cells treated with amplification-grade DNase I (Invitrogen) was used as the template. Reverse transcription-PCR was performed using the 3′-SMART RACE kit (Clontech) to perform first-strand cDNA synthesis with 0.6 μg of template RNA. Subsequently, 2.5 μl (of the 110-μl reverse transcription reaction) was introduced into a 100-μl PCR at the following cycle settings: 94 °C for 5 min; 40 cycles at 94 °C for 1 min, 55 °C for 1.5 min, and 72 °C for 2 min; 72 °C for 10 min; and hold at 4 °C. PCR reagents were purchased from Applied Biosystems, and PCR was performed using AmpliTaq Gold in 3.5 mm MgCl2 with an Applied Biosystems GeneAmp 2400 thermal cycler. The 948-bp product was subcloned into pUC18 and sequenced on both strands using BigDye terminators (PerkinElmer Life Sciences) by the Nucleic Acid/Protein Core Research Facility of the Children's Hospital of Philadelphia. The DNA sequence of the partial cDNA fragment matched the seven N-terminal amino acids used for designing the upstream degenerate primer, as well as the next 10 amino acids of the determined N-terminal sequence of Sfhex, confirming that the desired cDNA had been isolated. The full-length cDNA for the Sfhex gene was obtained by performing both 5′- and 3′-RACE using the SMART RACE cDNA amplification and BD Advantage 2 PCR kits (Clontech). For 3′-RACE, first-strand cDNA synthesis was performed as described above, and 2.5 μl (of 110 μl) was introduced into a 50-μl PCR using the gene-specific upstream primer SF6 (5′-GCTCCTGGGGCGTTGCGTATCCAA, aa 280-288) and the universal primer UPM as the downstream primer at the following cycle settings: 94 °C for 5
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