A fused lobes Gene Encodes the Processing β-N-Acetylglucosaminidase in Sf9 Cells*
2008; Elsevier BV; Volume: 283; Issue: 17 Linguagem: Inglês
10.1074/jbc.m710279200
ISSN1083-351X
AutoresChristoph Geisler, Jared J. Aumiller, Donald L. Jarvis,
Tópico(s)Transgenic Plants and Applications
ResumoManα6(Manα3)Manβ4GlcNAcβ4GlcNAc-R is the core structure of the major processed protein N-glycans produced by insect cells. Ultimately, this paucimannose type structure is produced by an unusual β-N-acetylglucosaminidase, which removes the terminal N-acetylglucosamine residue from the upstream intermediate, Manα6(GlcNAcβ2Manα3)Manβ4GlcNAcβ4GlcNAc-R. Because the N-glycan processing pathways leading to the production of this intermediate are probably identical in insects and higher eukaryotes, the presence or absence of this specific, processing β-N-acetylglucosaminidase is a key factor distinguishing the processing pathways in these two different types of organisms. Recent studies have shown that the fused lobes (fdl) gene encodes the specific, processing β-N-acetylglucosaminidase of Drosophila melanogaster. However, there are conflicting reports on the identity of the gene encoding this enzyme in the lepidopteran insect, Spodoptera frugiperda. One has suggested that a gene alternatively designated SfGlcNAcase-3 or SfHex encodes this function, whereas another has suggested that this gene encodes a broad-spectrum β-N-acetylglucosaminidase that functions in glycan and chitin degradation. In this study we resolved this conflict by molecularly cloning an S. frugiperda fdl ortholog (Sf-fdl) and demonstrating that it encodes a product with the substrate specificity expected of the processing β-N-acetylglucosaminidase. Moreover, we showed that the endogenous levels of specific, processing β-N-acetylglucosaminidase activity were significantly reduced in S. frugiperda cells engineered to express a double-stranded RNA derived from the Sf-fdl gene. These results indicate that Sf-fdl encodes the specific, processing β-N-acetylglucosaminidase of S. frugiperda and validate our previous suggestion that the broad-spectrum β-N-acetylglucosaminidase encoded by the SfGlcNAcase-3/SfHex gene is more likely to be involved in N-glycan and/or chitin degradation. Manα6(Manα3)Manβ4GlcNAcβ4GlcNAc-R is the core structure of the major processed protein N-glycans produced by insect cells. Ultimately, this paucimannose type structure is produced by an unusual β-N-acetylglucosaminidase, which removes the terminal N-acetylglucosamine residue from the upstream intermediate, Manα6(GlcNAcβ2Manα3)Manβ4GlcNAcβ4GlcNAc-R. Because the N-glycan processing pathways leading to the production of this intermediate are probably identical in insects and higher eukaryotes, the presence or absence of this specific, processing β-N-acetylglucosaminidase is a key factor distinguishing the processing pathways in these two different types of organisms. Recent studies have shown that the fused lobes (fdl) gene encodes the specific, processing β-N-acetylglucosaminidase of Drosophila melanogaster. However, there are conflicting reports on the identity of the gene encoding this enzyme in the lepidopteran insect, Spodoptera frugiperda. One has suggested that a gene alternatively designated SfGlcNAcase-3 or SfHex encodes this function, whereas another has suggested that this gene encodes a broad-spectrum β-N-acetylglucosaminidase that functions in glycan and chitin degradation. In this study we resolved this conflict by molecularly cloning an S. frugiperda fdl ortholog (Sf-fdl) and demonstrating that it encodes a product with the substrate specificity expected of the processing β-N-acetylglucosaminidase. Moreover, we showed that the endogenous levels of specific, processing β-N-acetylglucosaminidase activity were significantly reduced in S. frugiperda cells engineered to express a double-stranded RNA derived from the Sf-fdl gene. These results indicate that Sf-fdl encodes the specific, processing β-N-acetylglucosaminidase of S. frugiperda and validate our previous suggestion that the broad-spectrum β-N-acetylglucosaminidase encoded by the SfGlcNAcase-3/SfHex gene is more likely to be involved in N-glycan and/or chitin degradation. Insects and other lower eukaryotes such as nematodes and plants occupy an interesting evolutionary niche in glycobiology because they produce N-glycoproteins but typically process their N-linked glycans less extensively than mammals (1Marz, L., Altmann, F., Staudacher, E., and Kubelka, V. (1995) in Glycoproteins (Montreuil, J., Vliegenthart, J. F. G., and Schachter, H., eds) pp. 543–564, Elsevier Science Publishers B. V., AmsterdamGoogle Scholar, 2Marchal I. Jarvis D.L. Cacan R. Verbert A. Biol. Chem. 2001; 382: 151-159Crossref PubMed Scopus (152) Google Scholar). This difference between lower and higher eukaryotic protein N-glycosylation pathways is biotechnologically significant because insects and plants are used to produce recombinant mammalian glycoproteins for many different biomedical research applications (3O'Reilly D.R. Miller L.K. Luckow V.A. Baculovirus Expression Vectors. W. H. Freeman and Co., New York1992Google Scholar, 4Summers M.D. Smith G.E. Texas Agricultural Experimental Station Bulletin 1555. Texas Agricultural Experimental Station, College Station, TX1987Google Scholar, 5Jarvis, D. L. (1997) in Baculoviruses (Miller, L. K., ed) pp. 389–431, Plenum Press, New YorkGoogle Scholar, 6Fischer R. Stoger E. Schillberg S. Christou P. Twyman R.M. Curr. Opin. Plant Biol. 2004; 7: 152-158Crossref PubMed Scopus (498) Google Scholar, 7Ma J.K. Drake P.M. Christou P. Nat. Rev. Genet. 2003; 4: 794-805Crossref PubMed Scopus (748) Google Scholar). Our research focuses on insects, and we believe the evolutionary status and widespread use of insect-based systems for recombinant glycoprotein production demands a much deeper understanding of their protein N-glycosylation pathways. Insect and mammalian protein N-glycosylation pathways each begin with the co-translational transfer of N-glycan precursors to nascent proteins (1Marz, L., Altmann, F., Staudacher, E., and Kubelka, V. (1995) in Glycoproteins (Montreuil, J., Vliegenthart, J. F. G., and Schachter, H., eds) pp. 543–564, Elsevier Science Publishers B. V., AmsterdamGoogle Scholar, 8Kornfeld R. Kornfeld S. Annu. Rev. Biochem. 1985; 54: 631-664Crossref PubMed Scopus (3772) Google Scholar). These precursors are subsequently trimmed and elongated by enzymes localized in the endoplasmic reticulum and Golgi apparatus of insect and mammalian cells to produce a common intermediate with the structure Manα6(GlcNAcβ2Manα3)Manβ4GlcNAcβ4GlcNAc-R. In mammalian cells, this intermediate is elongated by various glycosyltransferases to produce complex N-glycans, which often have terminal sialic acid residues. In contrast, insect cells usually fail to elongate this same intermediate and convert it instead to paucimannose N-glycans with the core structure Manα6(Manα3)Manβ4GlcNAcβ4GlcNAc-R. An unusual β-N-acetylglucosaminidase is responsible for the production of these structures (9Altmann F. Schwihla H. Staudacher E. Glossl J. Marz L. J. Biol. Chem. 1995; 270: 17344-17349Abstract Full Text Full Text PDF PubMed Scopus (193) Google Scholar). This enzyme specifically removes the terminal N-acetylglucosamine residue from the α3 branch of Manα6(GlcNAcβ2Manα3)Manβ4GlcNAcβ4GlcNAc-R, simultaneously eliminating the intermediate required for N-glycan elongation and producing the core paucimannose glycan typically found on insect cell-derived N-glycoproteins. This same enzyme is also responsible for the production of core paucimannose N-glycans in nematodes (10Zhang W. Cao P. Chen S. Spence A.M. Zhu S. Staudacher E. Schachter H. Biochem. J. 2003; 372: 53-64Crossref PubMed Google Scholar, 11Gutternigg M. Kretschmer-Lubich D. Paschinger K. Rendic D. Hader J. Geier P. Ranftl R. Jantsch V. Lochnit G. Wilson I.B. J. Biol. Chem. 2007; 282: 27825-27840Abstract Full Text Full Text PDF PubMed Scopus (76) Google Scholar). Thus, the presence of a processing β-N-acetylglucosaminidase is a key difference between the protein N-glycosylation pathways of lower and higher eukaryotes. In a seminal study on this topic, Altmann et al. (9Altmann F. Schwihla H. Staudacher E. Glossl J. Marz L. J. Biol. Chem. 1995; 270: 17344-17349Abstract Full Text Full Text PDF PubMed Scopus (193) Google Scholar) demonstrated that IPLB-Sf21AE, a cell line derived from the lepidopteran insect Spodoptera frugiperda (12Vaughn J.L. Goodwin R.H. Thompkins G.J. McCawley P. In Vitro. 1977; 13: 213-217Crossref PubMed Scopus (999) Google Scholar), has a membrane-associated β-N-acetylglucosaminidase activity that can specifically cleave the terminal N-acetylglucosamine residue from the α3 branch of a biantennary N-glycan in vitro. Subsequently, it was shown that cell lines derived from Estigmene acrea, another lepidopteran insect, produce hybrid and complex N-glycans containing terminal N-acetylglucosamine or galactose residues because they lack this intracellular β-N-acetylglucosaminidase activity (13Wagner R. Geyer H. Geyer R. Klenk H.D. J. Virol. 1996; 70: 4103-4109Crossref PubMed Google Scholar). Together, these studies strongly supported the idea that the N-glycosylation pathway of at least some insect cells includes a processing β-N-acetylglucosaminidase, as described above. However, unequivocal proof of this concept awaited the isolation of an insect gene encoding this enzyme together with evidence that the gene product has the exquisite substrate specificity of the N-glycan processing enzyme. The first proof of this kind was provided by a more recent study from the Altmann group (14Leonard R. Rendic D. Rabouille C. Wilson I.B. Preat T. Altmann F. J. Biol. Chem. 2006; 281: 4867-4875Abstract Full Text Full Text PDF PubMed Scopus (136) Google Scholar) in which they demonstrated that the Drosophila melanogaster fused lobes (Dm-fdl) 2The abbreviations used are: Dm, D. melanogaster; AcMNPV, A. californica nucleopolyhedrovirus; FDL, fused lobes; GnGn, GlcNAcβ2Manα6 (GlcNAcβ2Manα3)Manβ4GlcNAcβ4GlcNAc-PA; GnM, GlcNAcβ2Manα6 (Manα3)Manβ4GlcNAcβ4GlcNAc-PA; MGn, Manα6(GlcNAcβ2Manα3) Manβ4GlcNAcβ4GlcNAc-PA; MM, Manα6(Manα3)Manβ4GlcNAcβ4GlcNAc-PA; PA, pyridylamine; Sf, S. frugiperda; RACE, rapid amplification of cDNA ends; GST, glutathione S-transferase; HPLC, high performance liquid chromatography; RNAi, RNA-mediated interference. 2The abbreviations used are: Dm, D. melanogaster; AcMNPV, A. californica nucleopolyhedrovirus; FDL, fused lobes; GnGn, GlcNAcβ2Manα6 (GlcNAcβ2Manα3)Manβ4GlcNAcβ4GlcNAc-PA; GnM, GlcNAcβ2Manα6 (Manα3)Manβ4GlcNAcβ4GlcNAc-PA; MGn, Manα6(GlcNAcβ2Manα3) Manβ4GlcNAcβ4GlcNAc-PA; MM, Manα6(Manα3)Manβ4GlcNAcβ4GlcNAc-PA; PA, pyridylamine; Sf, S. frugiperda; RACE, rapid amplification of cDNA ends; GST, glutathione S-transferase; HPLC, high performance liquid chromatography; RNAi, RNA-mediated interference. gene encodes the specific, processing β-N-acetylglucosaminidase in this organism. Importantly, this study demonstrated that the Dm-fdl gene product has several features distinguishing it from degradative hexosaminidases and chitinases, which also have β-N-acetylglucosaminidase activities. These features include its specificity for the terminal N-acetylglucosamine residue linked to the α3 branch of N-glycan substrates and its inability to degrade chito-oligosaccharides. Furthermore, it was shown that flies with no functional fdl gene produced a higher proportion of N-glycans with terminal N-acetylglucosamine residues linked to the α3 branch as compared with wild type. These findings together with the finding that the D. melanogaster hexosaminidase genes (hexo1 and hexo-2) encode enzymes that can cleave chito-oligosaccharides, but not N-glycans, strongly suggested that Dm-FDL is the β-N-acetylglucosaminidase responsible for N-glycan processing in this fly. The properties of Dm-FDL also were consistent with the idea that it is the fly ortholog of the lepidopteran insect N-glycan processing enzyme first detected by Altmann et al. (9Altmann F. Schwihla H. Staudacher E. Glossl J. Marz L. J. Biol. Chem. 1995; 270: 17344-17349Abstract Full Text Full Text PDF PubMed Scopus (193) Google Scholar) in microsomal membranes from IPLB-Sf21AE cells. Subsequently, two laboratory groups independently reported molecular cloning of genes encoding β-N-acetylglucosaminidases from Sf9 cells, which are a clonal derivative of the IPLB-Sf21AE cell line (15Tomiya N. Narang S. Park J. Abdul-Rahman B. Choi O. Singh S. Hiratake J. Sakata K. Betenbaugh M.J. Palter K.B. Lee Y.C. J. Biol. Chem. 2006; 281: 19545-19560Abstract Full Text Full Text PDF PubMed Scopus (47) Google Scholar, 16Aumiller J.J. Hollister J. Jarvis D.L. Protein Expression Purif. 2006; 47: 571-590Crossref PubMed Scopus (32) Google Scholar). Our group described the isolation of three β-N-acetylglucosaminidase genes from Sf9 cells, which were designated SfGlcNAcase-1,-2,-3 (16Aumiller J.J. Hollister J. Jarvis D.L. Protein Expression Purif. 2006; 47: 571-590Crossref PubMed Scopus (32) Google Scholar). SfGlcNAcase-1 was clearly distinct from the other two, which were nearly identical to each other and likely allelic variants of the same gene. Further analysis of the SfGlcNAcase-1 and SfGlcNAcase-3 gene products showed that they had high sequence homology to known hexosaminidases and that each also had β-N-acetylglucosaminidase activity when assayed against relevant substrates. However, neither had the tight α3 branch specificity of the processing enzyme activity originally described by Altmann et al. (9Altmann F. Schwihla H. Staudacher E. Glossl J. Marz L. J. Biol. Chem. 1995; 270: 17344-17349Abstract Full Text Full Text PDF PubMed Scopus (193) Google Scholar). In fact, each could remove the terminal N-acetylglucosamine residues from either the α3 or the α6 branch of various N-glycan substrates, and each also could release N-acetylglucosamine monomers from a chito-oligosaccharide substrate. Accordingly, we concluded that none of these S. frugiperda genes encoded the N-glycan processing enzyme but more likely encoded broad-spectrum β-N-acetylglucosaminidases involved in N-glycan and chitin degradation. In a similar study, Tomiya et al. (15Tomiya N. Narang S. Park J. Abdul-Rahman B. Choi O. Singh S. Hiratake J. Sakata K. Betenbaugh M.J. Palter K.B. Lee Y.C. J. Biol. Chem. 2006; 281: 19545-19560Abstract Full Text Full Text PDF PubMed Scopus (47) Google Scholar) also molecularly cloned two allelic variants of an Sf9 cell β-N-acetylglucosaminidase gene, which they termed Sfhex. Further analysis of the Sfhex gene product, which is identical to the gene product we designated SfGlcNAcase-3, confirmed that the SfGlcNAcase-3/SfHex gene product lacks the α3 branch specificity of the processing enzyme activity originally described by Altmann et al. (9Altmann F. Schwihla H. Staudacher E. Glossl J. Marz L. J. Biol. Chem. 1995; 270: 17344-17349Abstract Full Text Full Text PDF PubMed Scopus (193) Google Scholar). However, because this enzyme had a 2–5-fold higher preference for the terminal N-acetylglucosamine residue on the α3 branch of an N-glycan substrate, Tomiya et al. (15Tomiya N. Narang S. Park J. Abdul-Rahman B. Choi O. Singh S. Hiratake J. Sakata K. Betenbaugh M.J. Palter K.B. Lee Y.C. J. Biol. Chem. 2006; 281: 19545-19560Abstract Full Text Full Text PDF PubMed Scopus (47) Google Scholar) concluded that the SfGlcNAcase-3/SfHex gene encodes the processing β-N-acetylglucosaminidase of Sf9 cells. In the present report we provide data that resolve the discrepancy in the conclusions drawn from these two previous reports. Briefly, we molecularly cloned a new β-N-acetylglucosaminidase cDNA from Sf9 cells, which turned out to be the S. frugiperda ortholog of the Dm-fdl gene. This gene, which we have designated Sf-fdl, encodes a membrane-associated product that specifically cleaves the terminal N-acetylglucosamine residue from the α3 branch of N-glycan substrates, which has virtually no activity against chito-oligosaccharide substrates and which has precisely the same pH profile as the activity originally identified by Altmann et al. (9Altmann F. Schwihla H. Staudacher E. Glossl J. Marz L. J. Biol. Chem. 1995; 270: 17344-17349Abstract Full Text Full Text PDF PubMed Scopus (193) Google Scholar) in IPLB-Sf21AE cell microsomes. Furthermore, Sf9 cells engineered to express an Sf-fdl-specific double-stranded RNA had significantly lower levels of specific, processing β-N-acetylglucosaminidase activity. These results indicate that the specific, processing β-N-acetylglucosaminidase activity originally detected by Altmann et al. (9Altmann F. Schwihla H. Staudacher E. Glossl J. Marz L. J. Biol. Chem. 1995; 270: 17344-17349Abstract Full Text Full Text PDF PubMed Scopus (193) Google Scholar) is encoded by the Sf-fdl gene. The definitive identification of this new gene and the demonstration that it can be used to suppress a key, endogenous N-glycan processing activity sets the stage for an effort to create a transformed Sf9 cell line lacking this activity, which would be an improved host for recombinant glycoprotein production by baculovirus expression vectors. Cells and Cell Culture—Sf9 cells, which are a subclone of the IPLB-Sf21-AE cell line derived from S. frugiperda ovaries (12Vaughn J.L. Goodwin R.H. Thompkins G.J. McCawley P. In Vitro. 1977; 13: 213-217Crossref PubMed Scopus (999) Google Scholar), were routinely maintained as shake flask cultures in either TNM-FH medium containing 10% fetal bovine serum (HyClone, Logan, UT) or ESF 921 serum-free medium (Expression Systems, Woodland, CA) as described previously (16Aumiller J.J. Hollister J. Jarvis D.L. Protein Expression Purif. 2006; 47: 571-590Crossref PubMed Scopus (32) Google Scholar). Molecular Cloning of an fdl Gene Homolog from Sf9 Cells—The Apis mellifera, Bombyx mori, and Tribolium castaneum genomic databases were searched using tBLASTn (17Altschul S.F. Madden T.L. Schaffer A.A. Zhang J. Zhang Z. Miller W. Lipman D.J. Nucleic Acids Res. 1997; 25: 3389-3402Crossref PubMed Scopus (59762) Google Scholar) with the derived amino acid sequence of Dm-FDL isoform C (accession number NM_165909) as the query. Exons encoding fragments of putative processing β-N-acetylglucosaminidases were joined in silico using an online splice site prediction algorithm available through the NetGene2 Server (18Brunak S. Engelbrecht J. Knudsen S. J. Mol. Biol. 1991; 220: 49-65Crossref PubMed Scopus (628) Google Scholar), and the predicted amino acid sequences were aligned using ClustalX version 1.83 (19Thompson J.D. Gibson T.J. Plewniak F. Jeanmougin F. Higgins D.G. Nucleic Acids Res. 1997; 25: 4876-4882Crossref PubMed Scopus (35414) Google Scholar) with the default settings. Highly conserved amino acid sequences were used to design degenerate oligonucleotide primers (SPDEG and ASPDEG; Fig. 1 and Table S1), which were then used for PCRs (20Innis, M. A., and Gelfand, D. H. (1990) in PCR Protocols: A Guide to Methods and Applications (Innis, M. A., Gelfand, D. H., Sninsky, J. J., and White, T. J., eds) Academic Press, Inc., San Diego, CAGoogle Scholar) with Sf9 genomic DNA or cDNA as the templates. The methods used to isolate genomic DNA, total RNA, and cDNA from Sf9 cells and to degenerate PCR conditions are given in the supplemental data. The degenerate PCRs yielded specific amplification products of about the expected size (420 bp), which were recovered, purified, and directly sequenced using the degenerate PCR primers specified above. The resulting nucleotide sequences were assembled using ContigExpress (Vector NTI Advance 10.3.0; Invitrogen). These data were used to design gene-specific primers for primary and nested 5′- and 3′-RACE reactions, which are described in the supplemental data. These reactions yielded the full-length, putative Sf-fdl gene sequence shown in supplemental Fig. S1. Subsequently, the Sf-fdl open reading frame was amplified from Sf9 cell genomic DNA and cDNA, and accuracy of the amplification products was checked by sequencing, as described in the supplemental data. Isolation of Recombinant Baculoviruses Encoding Native or GST-tagged Soluble Domains of β-N-Acetylglucosaminidases—Baculovirus transfer plasmids encoding the relevant full-length, untagged N-acetylglucosaminidases or their GST-tagged soluble domains were produced by using PCR to amplify the appropriate nucleotide sequences, as described in the supplemental data, and error-free clones were identified by sequencing. The transfer plasmids encoding the native proteins were used to produce recombinant baculoviruses by the BaculoDirect™ method (Invitrogen) according to the manufacturer's protocol, whereas those encoding the GST-tagged soluble domains were used to produce viruses by a standard allelic transplacement method (3O'Reilly D.R. Miller L.K. Luckow V.A. Baculovirus Expression Vectors. W. H. Freeman and Co., New York1992Google Scholar, 4Summers M.D. Smith G.E. Texas Agricultural Experimental Station Bulletin 1555. Texas Agricultural Experimental Station, College Station, TX1987Google Scholar) with Bsu36I-digested BacPAK6 viral DNA (21Kitts P.A. Possee R.D. Biotechniques. 1993; 14: 810-817PubMed Google Scholar) as the target for homologous recombination. Each recombinant baculovirus vector was plaque-purified, amplified in Sf9 cells, and titered by plaque assay on Sf9 cells, as described previously (4Summers M.D. Smith G.E. Texas Agricultural Experimental Station Bulletin 1555. Texas Agricultural Experimental Station, College Station, TX1987Google Scholar). The recombinant viruses encoding the native β-N-acetylglucosaminidases were designated AcSfGlcNAcase-3 (16Aumiller J.J. Hollister J. Jarvis D.L. Protein Expression Purif. 2006; 47: 571-590Crossref PubMed Scopus (32) Google Scholar), AcDm-FDL, and AcSf-FDL, whereas those encoding the N-terminal GST-tagged ectodomains of these enzymes were designated AcGSTSfGlcNAcase-3, AcGSTDm-FDL, and AcGSTSf-FDL, respectively. The parental virus used to produce these viruses, which also served as a negative control for some of the experiments included in this study, was Autographa californica nucleopolyhedrovirus (AcMNPV). Expression of Recombinant Proteins in Insect Cells—Sf9 cells were seeded into 100 ml of ESF 921 medium in 250-ml DeLong flasks (Corning Glass Works, Corning, NY) and allowed to grow to a density of about 1.5–2.0 × 106 cells/ml at 28 °C and 125 rpm in a model 4580 rotary platform shaker-incubator (Forma Scientific, Inc., Marietta, OH). The cells were then infected with the appropriate baculovirus at a multiplicity of infection of about 1 plaque-forming unit/cell and incubated for another 72 h under the same conditions. Isolation of Purified Microsomal Fractions—The isolation of microsomal fractions from baculovirus-infected Sf9 cells has been described previously (16Aumiller J.J. Hollister J. Jarvis D.L. Protein Expression Purif. 2006; 47: 571-590Crossref PubMed Scopus (32) Google Scholar). Microsomes were solubilized in β-N-acetylglucosaminidase assay buffer (100 mm citrate phosphate buffer, pH 6.0) containing 0.5% (v/v) Triton-X-100, total protein concentrations were determined using a commercial bicinchoninic acid assay (Pierce), and samples containing equal amounts of total protein were assayed for β-N-acetylglucosaminidase activity as described below. Glutathione Affinity Chromatography—The GST-tagged ectodomains of the various β-N-acetylglucosaminidases examined in this study were purified from the extracellular fraction of Sf9 cells infected with AcGSTSfGlcNAcase-3, AcGSTDm-FDL, or AcGSTSf-FDL as described in the supplemental data. The purity of the final affinity-purified protein preparations was assessed by SDS-PAGE with Coomassie Blue staining (supplemental Fig. S3A), and GST-tagged protein content was measured by SDS-PAGE and immunoblotting with a GST-specific antiserum (supplemental Fig. S3B). β-N-Acetylglucosaminidase Activity Assays—Enzyme activity assays were performed using either solubilized microsomal fractions or affinity-purified recombinant proteins isolated from baculovirus-infected Sf9 cells. For the microsomal membrane assays, microsomes were prepared and extracted as described above, and samples containing equal amounts of total protein were assayed in a total volume of 100 μl containing 20 pmol of various pyridylamine (PA)-tagged glycan substrates, which were generously provided by Drs. Friedrich Altmann and Iain Wilson of the Glycobiology group in the Department of Chemistry, University of Natural Resources and Applied Life Sciences, Vienna. The enzymatic activity of the affinity-purified recombinant proteins was assayed under identical conditions, except the amounts of purified protein used for these assays were equalized by immunoblotting rather than by total protein assays. The substrates used in this study included GlcNAcβ2Manα6 (GlcNAcβ2Manα3)Manβ4GlcNAcβ4GlcNAc-PA (GnGn), GlcNAcβ2Manα6(Manα3)Manβ4GlcNAcβ4GlcNAc-PA (GnM), and Manα6(GlcNAcβ2Manα3)Manβ4GlcNAcβ4GlcNAc-PA (MGn). After being incubated for various times at 37 °C, each reaction was diluted to 150 μl with water, and the products were analyzed by reverse phase high performance liquid chromatography as described previously (22Altmann F. Kornfeld G. Dalik T. Staudacher E. Glossl J. Glycobiology. 1993; 3: 619-625Crossref PubMed Scopus (136) Google Scholar). GnGn, GnM, MGn, and also Manα6(Manα3)Manβ4GlcNAcβ4GlcNAc-PA (MM) were used as standards for the chromatographic analyses. RNA Interference—In general, the RNA interference approach used in this study involved transforming Sf9 cells with an immediate early expression plasmid encoding an inverted repeat derived from a portion of the Sf-fdl coding sequence, with the inverted repeat separated by a D. melanogaster white gene intron, as originally described by Lee and Carthew (23Lee Y.S. Carthew R.W. Methods. 2003; 30: 322-329Crossref PubMed Scopus (307) Google Scholar). A detailed description of the construction of this expression plasmid is given in the supplemental data. Ultimately, a sequence-verified Sf-fdl inverted repeat and white gene intron cassette was inserted into pIE1HR3 (24Jarvis D.L. Weinkauf C. Guarino L.A. Protein Expression Purif. 1996; 8: 191-203Crossref PubMed Scopus (126) Google Scholar) to produce a plasmid designated pIE1HR3SfFdlRNAi. This plasmid was then used along with pIE1Neo to co-transfect Sf9 cells using a modified calcium phosphate method, as described previously (4Summers M.D. Smith G.E. Texas Agricultural Experimental Station Bulletin 1555. Texas Agricultural Experimental Station, College Station, TX1987Google Scholar), and neomycin-resistant clones were isolated by limiting dilution, as described previously (25Harrison R.L. Jarvis D.L. Methods Mol. Biol. 2007; 388: 299-316Crossref PubMed Google Scholar). The levels of specific, processing β-N-acetylglucosaminidase activity in the parental and transformed cells were compared by HPLC analysis of the products obtained by reacting microsomal membrane preparations with GnGn, as described above. Isolation and Characterization of an fdl Gene Homolog from Sf9 Cells—Our effort to isolate an fdl gene homolog from Sf9 cells was informed and facilitated by the availability of genome sequence data from several insect species and also by our previous efforts to isolate the gene encoding the processing β-N-acetylglucosaminidase activity from this cell line. tBLASTn analyses of the A. mellifera, B. mori, and T. castaneum genomes with the predicted amino acid sequence of Dm-FDL as the query yielded putative exons from each species encoding peptides related to Dm-fdl (data not shown). We subsequently used a splice site prediction algorithm to join the relevant exons and identify open reading frames encoding at least partial, putative β-N-acetylglucosaminidases from each insect species. Importantly, a ClustalW alignment revealed that these open reading frames encoded amino acid sequences that were conserved in the Dm-fdl gene product but not in the SfGlcNAcase-1 or SfGlcNAcase-3 gene products identified in our previous study (Fig. 1; Ref. 16Aumiller J.J. Hollister J. Jarvis D.L. Protein Expression Purif. 2006; 47: 571-590Crossref PubMed Scopus (32) Google Scholar). These conserved sequences (boxed in Fig. 1) were used to design degenerate oligonucleotides for high fidelity PCRs with Sf9 cDNA or genomic DNA as the templates. These PCRs yielded an amplification product of about the expected size (420 bp) that appeared to be specific because it was not observed in control reactions in which either one of the degenerate oligonucleotides was excluded (data not shown). This product was directly sequenced, and the derived translation product was found to be highly similar to the corresponding fragments of the known D. melanogaster and putative A. mellifera, B. mori, and T. castaneum FDL proteins. Accordingly, we used the nucleotide sequence of this amplification product to design gene-specific primers for 5′- and 3′-RACE reactions. The 5′-RACE reactions yielded a specific 1.4-kilobase amplification product that overlapped with the sequence of the original degenerate PCR product, extended it by 1180 bp in the 5′ direction, and included a potential translational initiation site (data not shown). The 3′-RACE reactions yielded a specific 1.0-kilobase amplification product that also overlapped with the sequence of the original degenerate PCR product, extended it by 734 bp in the 3′ direction, and encoded a translational termination site. A contiguous nucleotide sequence of 2319 bp was assembled by joining the sequences of the degenerate amplimer, the 5′-RACE product, and the 3′-RACE product. The accuracy of this sequence was confirmed by PCR with gene-specific primers using both Sf9 cDNA and genomic DNA as the templates followed by direct sequencing of the products as described under "Experimental Procedures." In Silico Analysis of the Sf9 Cell fdl Gene Homolog—The full-length Sf-fdl nucleotide sequence and derived amino acid sequence of the Sf-FDL polypeptide are shown in supplemental Fig. S1. The nucleotide sequence includes a single long ope
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