Glycobiology vol. 7 no. 1 pp. 113-127, 1997

Isolation and characterization of a class II a- cDNA from lepidopteran insect cells

Donald LJarvis2, Dwight A.Bohlmeyer, Yung-Feng Introduction Liao1, Kristen K.Lomax, Roberta K.Merkle1, Carla 1 Weinkauf and Kelley W.Moremen Lepidopteran insect cells are used widely as hosts for foreign Department of Entomology and Center for Advanced Invertebrate Molecular gene expression by recombinant baculoviruses and, like bac- Sciences, Texas A&M University, College Station, TX 77843, USA and terial expression systems, the insect cell-baculovirus system 'Department of Biochemistry and Molecular Biology and Complex can provide high levels of foreign gene expression (Summers Carbohydrate Research Center, University of Georgia, Athens, GA 30602, and Smith, 1987; Miller, 1988; O'Reilly et al., 1992). The USA Downloaded from https://academic.oup.com/glycob/article/7/1/113/725524 by guest on 01 October 2021 insect ceU-baculovirus system also has eukaryotic protein pro- ^o whom correspondence should be addressed cessing capabilities and, therefore, is one of the best tools Lepidopteran insect cells are used routinely as hosts for currently available for foreign production. How- foreign glycoprotein expression by recombinant baculovi- ever, the nature of the N-glycosylation pathway in lepidopteran ruses, but the precise nature of their N-glycosylation path- insect cells and, particularly, in baculovirus-infected lepidop- way remains poorly defined. These cells clearly have pro- teran insect cells, remains poorly defined. cessing and mannosidases that can convert By comparison, the N-glycosylation pathway in higher eu- caryotes is well-defined (reviewed by Kornfeld and Kornfeld, precursors to Man3GlcNAc2 structures and fucosyltrans- ferases that can add fucose to the oligosaccharide core. 1985). This pathway begins with the cotranslational transfer of However, their ability to extend these structures to produce preformed GlCjMangGlcNAcj oligosaccharides from a doli- complex side chains like those found in mammalian cells chol-linked precursor to nascent polypeptide chains on the lu- remains to be determined. To begin to examine this path- minal face of the (ER), followed by way at the molecular genetic level, we isolated and charac- immediate trimming of glucose residues within the ER. Addi- terized a class II ot-mannosidase (a-mannosidase II) cDNA tional trimming of glucose residues and as many as three of the from Sf9, a lepidopteran insect cell line. In mammalian nine mannose residues can occur within the ER. As glycopro- cells, this catalyzes the committed step in the path- teins transit the Golgi complex, the last a-l,2-linked mannose way converting N-linked carbohydrates to complex forms. residues are removed by 'class F ot-mannosidases, producing a Degenerate primers against conserved regions in known Man5GlcNAc2 structure (Moremen et al., 1994). Following the class II a-mannosidase protein sequences were used to gen- addition of a single GlcNAc residue by GlcNAc I, erate an a-mannosidase H-specific PCR product from Sf9 two more mannose residues are removed by Golgi a-manno- cell DNA. Sequence information from this product was sidase II, a 'class II' mannosidase (Moremen et al, 1994). This used to isolate a partial cDNA clone, the 5' end was isolated is the committed step in the synthesis of complex type oligo- by ligation-anchored PCR, and the full length a-mannosi- saccharides, which are subsequently produced by Golgi glyco- dase II cDNA was assembled. This cDNA contained a long syltransferases that extend the trimmed structures by adding open reading frame predicted to encode an 1130 amino acid N-acetylglucosamine, galactose, fucose, and sialic acid resi- protein with 37% identity to human Golgi a-mannosidase dues (reviewed by Komfeld and Komfeld, 1985; Paulson and II and with a type II membrane topology, a feature of all Colley, 1989; Moremen et al., 1994). known Golgi processing . Southern blotting indi- Most information on the N-glycosylation pathway in lepi- cated that a-mannosidase II is a single copy gene in Sf9 dopteran insect cells has come from structural studies on for- cells. Other Lepidoptera had related a-mannosidase II eign expressed in baculovirus-infected cell lines genes, but there was variation among different genera, and or in larvae (reviewed by Jarvis and Summers, 1992; O'Reilly the Sf9 a-mannosidase II cDNA did not cross-hybridize et al., 1992; Jarvis, 1993a). These studies have demonstrated with DNA from animals outside Lepidoptera. Steady-state that lepidopteran insect cells have processing glucosidases and levels of a-mannosidase II RNA were low in uninfected Sf9 mannosidases which convert high mannose oligosaccharides to cells and even lower after baculovirus infection. The trimmed structures with as few as three mannose residues in v#ro-translated Sf9 a-mannosidase II protein had the (pauci-mannose structures). Several lines of evidence indicate expected size and was translocated and N-glycosylated by that these cells also have a fucosyltransferase that can add microsomal membranes. Expression of the Sf9 a-mannosi- fucose to the core Asn-linked GlcNAc residue (Staudacher et dase II cDNA in the baculovirus system produced large al., 1992), but it remains unclear whether insect cells have the amounts of a protein with the expected size and swainso- ability to extend these trimmed structures, as in mammalian nine-sensitive a-mannosidase II activity towards an aryl- cells. Most structural data indicate that baculovirus-expressed a-mannoside substrate. These results demonstrate that Sf9 glycoproteins (Kuroda et al., 1990; Chen and Bahl, 1991; Chen cells encode and express an a-mannosidase II with prop- etal., 1991; Wathen etal, 1991; Hogeland et al., 1992; Knep- erties similar to those of the mammalian enzyme. per et al., 1992; Grabenhorst et al., 1993; Yeh et al., 1993; Manneberg et al., 1994; Jarvis and Finn, 1995) and native Key words: class II ot-mannosidase/cDNA/lepidopteran insect insect glycoproteins (Butters et al, 1981; Hsieh and Robbins, cells/baculovirus expression system 1984; Ryan et al., 1985; Nagao et al., 1987; Williams et al., 1991) have only trimmed and fucosylated high mannose or

© Oxford University Press 113 D.LJarvis et at

pauci-mannose oligosaccharides. However, some recent stud- human a-mannosidase II (42.3% and 43.3% identity, respec- ies indicate that lepidopteran insect cell lines have GlcNAc tively) and D .discoideum and human lysosomal a-mannosi- transferase I and II activities (Altmann et al., 1993; Velardo et dase (29.4% and 26.1% identity, respectively), were identified al., 1993) and that they can produce glycoproteins with termi- by diis analysis. This suggested that the Sf9 amplimer was nal GlcNAc (Kubelka et al., 1994; Ackermann et al., 1995), derived from a gene that is related to the class II mannosidases Gal (Ogonah et al., 1996), and sialic acid (Davidson et al, and is more similar to the Golgi processing than the lysosomal 1990; Davidson and Castellino, 1991a). mannosidases. While structural biochemistry has provided much useful in- The Sf9 amplimer DNA sequence was used to design exact- formation on the N-glycosylation pathway in lepidopteran in- match primers against the putative Sf9 a-mannosidase II cod- sect cells, this approach is limited because it is indirect and ing region, and these primers were used for PCRs with total \ provides only a retrospective view of the processing pathway, DNA from an unfractionated Sf9 cDNA library, as described in which must be inferred from the structures of the end-products. Materials and methods. Electrophoretic analysis of the reaction Conclusions about the processing pathway can be complicated products revealed one major DNA fragment of about the same by degradative pathways, which might alter the product of the size as the RT-PCR product (data not shown). The same result biosynthetic pathway and lead to misinterpretations (Licari et was obtained with total A. DNA from an unfractionated Sf9

al., 1993; Jarvis and Finn, 1995). Finally, conclusions based on genomic DNA library or with the pCRII clone containing the Downloaded from https://academic.oup.com/glycob/article/7/1/113/725524 by guest on 01 October 2021 structural data from any one glycoprotein might apply only to Sf9 amplimer, but not in negative controls which lacked tem- that glycoprotein and not to the pathway in general. An alter- plate DNA or contained one specific and one nonspecific native approach that circumvents these problems is to use mo- primer (data not shown). These results indicated that the un- lecular genetics to isolate genes encoding glycoprotein pro- infected Sf9 cell cDNA library included a clone containing the cessing enzymes. This makes it possible to study these genes, putative Sf9 a-mannosidase II coding region, prompting us to their expression, and, ultimately, the properties of the enzymes proceed to isolate this cDNA by using a sibling selection and they encode. A large number of cDNAs encoding various gly- PCR screening approach (Moremen, 1989). Three positive coprotein processing enzymes have been cloned and charac- clones were isolated through sibling selection and three rounds terized in other systems (reviewed by Paulson and Colley, of plaque purification, as described in Materials and methods 1989; Lowe, 1991; Joziasse, 1992; Moremen etai, 1994; Field (data not shown). The cDNA inserts were excised as Blue- and Wainwright, 1995). However, except for the recent isola- script-based plasmids and mapped by restriction analysis. The tion of putative class I (Kerscher et al., 1995) and class II largest cDNA (about 6.5 Kb) was sequenced with universal a-mannosidase (Foster et al., 1995) cDNAs from Drosophila and gene-specific primers and compared to mammalian melanogaster, this approach has not been undertaken in insect a-mannosidase U DNA sequences. This analysis revealed ex- systems, and no cDNAs encoding carbohydrate processing en- tensive similarities but indicated that our cDNA clone lacked zymes have been isolated from Lepidoptera, the relevant hosts the 5' end of the a-mannosidase II coding region. for the baculovirus expression system. This report describes The missing 5' sequence was isolated by ligation-anchored the isolation and characterization of a cDNA encoding an PCR (Figure IB; Troutt et al., 1992). Following synthesis of a-mannosidase II with the properties of a glycoprotein pro- single stranded cDNA from Sf9 cell mRNA with random cessing enzyme from a lepidopteran insect cell line, Sf9, which hexamer primers, an oligonucleotide complementary to the T3 is the most widely used host for baculovirus-mediated foreign primer sequence was ligated onto the 3' end of the cDNA gene expression (Vaughn, 1977; Summers and Smith, 1987). product. The resulting ligation-anchored cDNA was subjected to two successive rounds of PCR with the T3 primer and nested gene-specific primers to amplify the region extending upstream Results of the Sf9 a-mannosidase coding region (Figure IB). The am- plification products were resolved on agarose gels and ana- Isolation and characterization of an a-mannosidase II cDNA lyzed by Southern blotting with the gene-specific 5' end probe from Sf9 cells shown in Figure IB. After the second round of PCR amplifi- An a-mannosidase cDNA from lepidopteran insect (Sf9) cells cation, several products were observed by ethidium bromide was isolated using a degenerate oligonucleotide PCR approach staining, but only two (175 and 750 bp) hybridized with the 5' (Moremen, 1989) based on regions of conserved amino acid end probe (data not shown). The 750 bp product was cloned sequences in two class II a-mannosidases (Moremen et al., into pCRQ and sequenced, and the results showed that it over- 1994), namely, murine Golgi a-mannosidase II (Moremen and lapped and extended the 5' end of the Sf9 a-mannosidase Robbins, 1991) and the lysosomal a-mannosidase from Dic- cDNA clone by an additional 681 bp. This new sequence in- tyostelium discoideum (Schatzle et al., 1992). The oligonucleo- formation was used to produce a 5' RT-PCR fragment, which tide primers were designed as shown in Figure 1A and used in was ligated in-frame to a downstream restriction fragment of an amplification reaction widi Sf9 cell genomic DNA, as de- the cDNA clone to produce a full-length cDNA, as shown in scribed in Materials and methods. The resulting 669 bp am- Figure 1C. plification product was identical in size to a positive control The assembled full-length open reading frame of the Sf9 RT-PCR product derived from murine liver rnRNA (data not a-mannosidase II cDNA, together with 176 bp of 5' and 179 shown). Similar RT-PCR products were obtained using mRNA bp of 3' sequence, is shown in Figure 2. The 3393 bp open isolated from either uninfected or baculovirus-infected Sf9 reading frame encodes a polypeptide of 1130 amino acids with cells (data not shown). The PCR product from Sf9 genomic a 37% identity to murine Golgi a-mannosidase II (Moremen DNA was cloned into pPCRII and sequenced, and the transla- and Robbins, 1991). The first in-frame ATG in the long open tion of the amplimer sequence was compared to a translation of reading frame is preceded by a purine at the critical -3 posi- the GenBank sequence database, as described in Material and tion, suggesting that it serves as the translation initiation site methods. Only class II mannosidases, including murine and (Kozak, 1983, 1986). Hydropathy analysis (Kyte and Doolittle,

114 Lepidopteran insect a-mannosidase II cDNA

ManH-A: 5'-GGI TGG HI ATT GAT CCI TTT GGT CA-31 (sense) C C C C A A G Manll-B: 5*-GG ACG IGA HI AAA ATA ICC ICT CCA ATA-31 (antisense) T T CT G G GG G G A C

Xhol Dral B 1 157 204 2994 Downloaded from https://academic.oup.com/glycob/article/7/1/113/725524 by guest on 01 October 2021 SfManll TTTlTAA A cDNA SfManII+13+ SfManII+157- 5' end probe

3'-anchored first strand Sf31+92- cDNA First round LA-PCR product SfManU+157-

1 Second round T3 5 UTR~.ATC.ORF LA-PCR product

Xhol Sf9cDNA BgIIL..-20 601 SfMann-Al Bglll i PCR i Xhol

Bglll-Xhol pBSAK/BS Clone into pCRII SfManll cDNA Xhol-Dral Xhol Hindll-Klenow pSfMann-5' |

Bglll-Xhol | pSfManII-37] Bglll-Xhol pSfManll (full-length)

Fig. 1. Isolation of the Sf9 a-mannosidase II cDNA. The sequences of the degenerate primers designed against conserved regions in known a-mannosidase II CDNAJ are shown in (A). A diagram of the partial Sf9 a-mannosidase II cDNA clone is shown at the top of (B) and a diagram showing the ligation-anchored PCR strategy used to isolate the 5' end of the cDNA is shown underneath. (C) shows how the full-length Sf9 a-mannosidase II cDNA was assembled.

115 D.L Janis et at

GGCTTATTAACCCTCACTAAAGGGAGAGTCAGGAGCACGCTGTGGTGTTTGGCTGT -120 -1 120 HRTRVLRCRPFST R I L I, L L L FVLAFGVYCYFYN A....; P Q N Y N 40 TCTTCCCTCACTCACACCGTCAAGAGCCGAGACGAGCCAACTCCGGATCAATGCCCTG 240 KPRI SI PASMEHFKSSLTHTVKSRDEPTPDQCPALKESEA BO 360 DIDTVAIYPTFDFQPSWLRTKEFIIDKSFEDRYERIH H...R...X T 120 480 RPRLKVIVVPHSHNDPGWLKTFEQYFEWKTKNIINNIVNK 160 600 L H Q Y P W...M...I FIKTEISFLNAWWERSHPVKQKALKKLIKEGR 200 720 LEITTGGWVMPDEACTHIYALIDQFIECHHMVKTNLGVIP 240 840 K T CWSTnPFnH GATVPYLIDQ.SGLEGTIIO.RIHYAHKO.WL 280 960 AERQIEEFYMLASWATTKPSMIVHNQPFDIYSIKSTCGPH 320 1080 PSICLSFDFRKIPGEYSEYTAKHEDITEHNLHSKAKTLIE 360 1200 Downloaded from https://academic.oup.com/glycob/article/7/1/113/725524 by guest on 01 October 2021 EYDRIGSLTPHNVVLVPLGDDFRYEYSVEFDAQYVNYMKH 400 1320 FNYINAHKEIFNADVQFGTPLDYFNAMKERHQ.NIPSLKGD 440 1440 FFVYSDI FSEGKPA YWSGYYTTRP YQKILARQFEHQLRSA 460 1S60 EILFTLVSNYIRQMGRQGEFCASEKKLEKSYEQLIYARRN S20 1680 LGLFQHHDAITGTSKSSVMQDYGTKLFTSLYHCIRLQ.EAA 560 1800 LTTIMLPDQSLHSQSIIQSEVEWETYGKPPKKLQVSFIDK too 1920 KKVILFNPLAETRTEVVTVRS H...X...S NIRVYDTHKRKHVLYQ 640 2040 IMPSITIQDNGKSIVSDTTFDIMFVATIPPLTSISYKLQ.E 680 2160 H T H...X...S. HHCVIFCNNCEQYQKSNVFQIKKMMPGDIQLENAV 720 2280 LKLLVNRNTGFLRQVYRKDIRKRTVVDVQFGAYQSAQRHS 760 2400 GAYLFMPHYDSPEKNVLHPYTNQNNMQDDNIIIVSGPIST 800 2520 EITTMYLPFLVHTIRIYNVPDPVLSRAILLETDVDFEAPP 640 2640 KNRETELFMRLQTDIQNGDIPEFYTDQNGFQYQKRVKVNK 880 2760 LGIEANYYPITTHACLQ.DEETRLTLLTNHAQGAAAYEPGR 920 2880 LEVMLDRRTLYDDFRGIGEGVVDNKPTTFQNMILIESMPG 960 3000 VTRAKRDTSEPCFKFVNERRFGPGQKESPYQVPSQTADYL 1000 3120 SRMFNYPVNVYLVDTSEVGEIEVKPYQSFLQSFPPGIHLV 1040 3240 TLRTITDDVLELFPS H...R...S.. YMVLHRPGYSCAVGEKPVAKSP 1080 3360 KFSSKTRFNGLNIQ H...1...X.. AVSLTGLKSLRPLTGLSDIHLNA 1120 TAAAGACTG- 3480 MEVKTYKIRF 1130 3572

Fig. 2. Nucleotide sequence of the Sf9 a-mannosidase II cDNA and predicted amino acid sequence. The nucleotide sequence of the Sf9 a-mannosidasc II cDNA is shown with the predicted amino acid sequence encoded by the single long open reading frame underneath. Both nucleotides and amino acids are numbered from the beginning of the open reading frame and nucleotides upstream of the putative translational initiation site are assigned negative numbers. The conserved regions used to isolate the original PCR product are underlined, the putative transmembrane domain is double underlined, and the potential N-glycosylation sites are marked by dotted underlines.

1982) revealed a potential transmembrane domain between typified by the mammalian Golgi glycoprotein processing en- amino acids 14 and 34 (double-underlined in Figure 2) and zyme, a-mannosidase II, and a recently cloned homolog, predicted a type II transmembrane topology similar to all other a-mannosidase II* (Misago et al., 1996; Figure 3B). The re- cloned Golgi processing and gions of lowest sequence similarity are localized to the NH2 (Paulson and Colley, 1989; Lowe, 1991; Moremen et al., terminal 124 amino acids of the Sf9 a-mannosidase polypep- 1994). In addition, the putative Sf9 a-mannosidase protein tide, which encode the putative cytoplasmic tail, transmem- would have seven potential N-glycosylation sites (dotted un- brane domain, and 'stem region' (Figure 3A). These regions derlines in Figure 2). were previously shown to be unessential for the catalytic ac- The protein encoded by the Sf9 a-mannosidase cDNA has tivity of mammalian a-mannosidase II (Moremen et al., 1991), extensive amino acid sequence similarity to a subclass of the indicating that there would be little selective pressure to main- class II mannosidases (Moremen et al., 1994). This subclass is tain their primary sequences during evolution. Surprisingly, the 116 Lepidopteran insect ot-mannosidase II cDNA

Y«ast_Vac_Man MSSEDII YDaOF»E>VQGI YHH^RQBlDTOGDTHDLWLPItFYDKKRHSLDHDHVKVWWYQVgFERGSBPV EPDK[3PSWKS II ERDKBGELEFHE QPPHPawSTTWFK Rat_Br_Man HAAAgFL^lWRTTFg.gVEKflv SP^Y FTDCNLRGRLFGDgcF VTLgsFLTPEgLP Y EKAVaQNFSP Di cty_Lys_Man House_Manli Huian.Ksoii Drotoph Hanii Sf9_Manii Yeaat_Vac_Ma I DPJtTLI PVTAFBGGERT ..TSDGKHP Rat_Er_Man T . .KBGEKTS Dicty_Lyi_Ma LHAADPRgLTL

Human_Haniix Droooph Minii QIKPHTBH IE Sf9 Manii FmHDRSSVRKCRBBLOREYrDSFLBSnVY rjAQQjfKWLLE Rat_Er_Man AQQLEHVKN Di cty_Ly8_Man CHAQPETYPAAEAB AJBFF Hunan_Lys_Nan Mouae^Kanii Kunan^Mani i Huwan^Haniix Drosoph_Mani i Sf9^Manii

Yeast_Vac_Man 347 gHHgFFN[JVLI PKI Rat_Er_Man 308 QYgGLYAQ.LQEFA Downloaded from https://academic.oup.com/glycob/article/7/1/113/725524 by guest on 01 October 2021

KQN TNN..RDKG QFTQVALEYAT

Human_Kaniix 343 Drosopfa.Hanii 32 1 ALF Sf9_Maoii 300 SKIV

Dicty_Lys_Man 325 Y Hu»an_Lys_Man 330 YS

DGMABIEER YIRQMGROGEPGASEFFI.[3K Y«8Bt_Vac_Man 62 8 VLPS SCIE Rit.ET^Nao 578 VVfj j SClR EPGP[3GLRHYQHTALE Di cty_Lye^Man 4 22 Mouse_Manii 570 Hunas_Manii 57l Human_Maniix 57l S36 Sf9_Manii 529

Yea«t_Vac_Ma Rat_Er Han

Hunan^Manii Drosoph Man

Human.Lys^Man 6 15 R Hunan.Kinii 790 Human^Mani ix 796 Dro«oph_Mani i 772 Sf9 Mani i 74 7

as M P GBT R ABB DT S E

PAgLSLgHPflLPHFTTTOO^ •• Zj' + ' •%. GGJJA^ VLSG .

. . .gG^GGDHPHAREgLCV£V KPVHERRFGPjQKgS jj. 33 Y LVDTS EVGE I E VK . . . gY0@F LOSFi3p'; :

Fig. 3. Continued on next page

117 D.LJarvis et al

• Yeast Vacuolar a-Mannosidase suggesting that these cells have single copy, closely related (16.4%) a-mannosidase II genes. Pstl digests of DNA from these four cell lines produced identical multiple banding patterns (there - Rat ER a-Mannosidase were only four bands because two of the six expected Pstl (19%) fragments were too small to be retained by the gel) with similar hybridization intensities, supporting the idea that they all have • Dictyostelium Lysosomal a-Mannosidase closely related a-mannosidase II genes. By contrast, HindlH (26.4%) and Pstl digests of genomic DNA from High 5 and Ea cells produced different banding patterns and weaker hybridization - Human Lysosomal a-Mannosidase signals, suggesting that the a-mannosidase II genes in these (263%) cell lines differ from the a-mannosidase II genes in the former cell lines. This conclusion was supported by the Southern blot- — Murine Golgi a-Mannosidase II (35.6%) ting results obtained after digesting genomic DNA from Sf9, High 5, and Ea cells with six different enzymes (Figure 4C), — Human Golgi a-Mannosidase II which revealed significant differences in the restriction maps Downloaded from https://academic.oup.com/glycob/article/7/1/113/725524 by guest on 01 October 2021 (37.0%) of the a-mannosidase II genes in these cell Lines. The Sf9 probe failed to hybridize with DNA from COS cells (Figure 4A,B), -Human a-Mannosidase II indicating that the Sf9 a-mannosidase II gene is less closely (31.9%) related to the a-mannosidase II genes in these mammalian cells than it is to the a-mannosidase II genes in the various lepidop- - Drosophila a-Mannosidase teran insect cell lines. When the blots shown in Figure 4 were (31.8%) K stripped and rehybridized under lower stringency, the hybrid- ization signals obtained with DNA from the various lepidop- • Sf9 a-Mannosidase teran insect cells were approximately equal, but we still de- tected no hybridization with COS cell DNA (data not shown). Fig. 3. Comparison of the protein encoded by the Sf9 a-mannosidase cDNA The nearly full-length Sf9 a-mannosidase II probe also was with the protein sequences of other class II a-mannosidases. (A) An optimized multiple sequence alignment was generated using the Pileup and used for low stringency hybridizations on Southern blots of Boxshade subroutines, as described in Materials and methods. Protein HindBI (Figure 5A) and Pstl (Figure 5B) digests of genomic sequences included in the alignment are the yeast vacuolar mannosidase DNA from various insect larvae and Xenopus laevis. The re- (Yoshinisa and AnraVu, 1989; accession no. M29146), rat ER sults showed that the probe hybridized with DNA from two a-mannosidase (Bischoff et al., 1990; accession no. M57547), Dictyostelium discoideum lysosomal a-mannosidase (Schatzle et al., 1992; accession no. different lepidopteran insects, Heliothis virescens (tobacco M82822), human lysosomal a-mannosidase (Nebes and Schmidt, 1994; liao budworm) and Helicoverpa zea (corn earworm), but the hy- and Moremen, unpublished observations; accession no. UO5572), mouse bridization signal was weaker and the digestion patterns were a-mannosidase II (Moremen and Robbins, 1991; accession no. X61172), different, when compared to the Sf9 cell controls. The Sf9 human a-mannosidase II (Misago et al., 1996; accession no. U31520), human a-mannosidase IP (Misago et al., 1996; accession no. D55649), a-mannosidase II probe failed to detectably hybridize to any Drosophila a-mannosidase II (Foster et al., 1995; accession DO. X77652), nonlepidopteran insect DNA, including beetle (Coleoptera), and Sf9 a-mannosidase IL Sequences shown with white text on a black locust (Orthoptera), cockroach (Blattaria), and fruitfly (Dip- background are identical in a least two of the aligned proteins. Dots indicate tera), or to frog (Xenopus) DNA. These results supported the gaps introduced to optimize the sequence alignment. (B) A dendogram of idea that the Sf9 a-mannosidase II gene is more closely related the aligned polypeptide sequences in (A) generated with the Pileup subroutine as described in Materials and methods. The numbers in to the a-mannosidase II genes of other lepidopteran insects parentheses represent the percent identity of the indicated sequence to the than to these same genes in animals outside of the order Lepi- Sf9 a-mannosidase II polypeptide. doptera. Transcription of the Sf9 a-mannosidase II gene Initially, we used Northern blotting to try to examine transcrip- predicted Sf9 a-mannosidase protein is more similar to the tion of the Sf9 a-mannosidase II gene in uninfected and bacu- mammalian a-mannosidase H proteins than to the putative lovirus-infected Sf9 cells, but this approach failed even using a-mannosidase II protein encoded by a cDNA recently isolated 20 (xg of poly A+ RNA (data not shown), despite being able to from Drosophila (Foster et al., 1995). obtain specific RT-PCR products from these RNAs. Therefore, we turned to the use of a more sensitive technique, ribonucle- Southern blotting analysis of the Sf9 a-mannosidase II gene ase protection (Lee and Costlow, 1987), with a 431 bp anti- sense riboprobe consisting of 19 bp of vector sequence fol- Southern blotting analyses were done to examine the structure lowed by 412 bp of sequence from the middle of the Sf9 of the Sf9 a-mannosidase II gene and its relationship to a-man- a-mannosidase II cDNA (positions 966—1384 in Figure 2). The nosidase II genes from other insects and higher eucaryotes. results showed that RNA from mock-infected Sf9 cells pro- Genomic DNA from several different lepidopteran insect cell tected a 412 bp fragment of the probe (Figure 6), indicating lines or from a mammalian cell line were digested with HindTR that the a-mannosidase II gene is expressed in these cells. The (Figure 4A), which does not cut the Sf9 a-mannosidase II steady state levels of a-mannosidase II—specific RNA must be cDNA, or with PstI (Figure 4B), which cuts this cDNA five very low, as we were unable to detect any protection in assays times, and Southern blots were probed with a nearly full-length with 20 \ig of total RNA (data not shown) and large amounts Sf9 a-mannosidase II fragment (Xhol-Dral in Figure IB). The (20 p,g) of poly A+ RNA produced only a relatively weak Hindm digests of DNA from Sf9, Sf21, Bm, and Md cells signal. The signals observed with poly A+ RNA from infected produced single bands with similar hybridization intensities, cells were even weaker than the signal obtained from unin-

118 Lepidopteran insect a-mannosidase II cDNA

J S B 23.1. 23.1. 9.4- 9.4- 6.6" 6.6- 4.4- 4.4- 2.3- 2.3- 2.0" Downloaded from https://academic.oup.com/glycob/article/7/1/113/725524 by guest on 01 October 2021

EcoRI HinDIlI Pstl Sspl Styl Xbal

23.1 -*•

9A-+- 6.6-*- 4.4-*-

2.3 _• 2.0 -•>

Fig. 4. Southern blotting analysis of a-mannosidase II genes in various lepidopteran insect cell lines. Genomic DNAs from Sf9, Sf21, High Five, Ea, Bm, Md, or CXDS cells were digested with HindJR (A) or Pstl (B). The digests were transferred to nylon filters, and the filters were probed under high stringency conditions with a nearly full-length Sf9 a-mannosidase II probe, as described in Materials and methods. (C) shows analogous Southern blots on genomic DNAs from Sf9, Ea, or High Five cells after digestion with various restriction endonucleases, as indicated above the figure. The positions of //mdHI-digested X DNA standards are marked by their sizes (in kb) on the left-hand side of each panel.

fected cells and little, if any, protection was observed with II cDNA (Moremen and Robbins, 1991), and might be ex- RNA from cells infected for 48 h (Figure 6). Ethidium bromide plained by premature translational termination or degradation staining of the poly A+ RNA samples used for this experiment of the full-length product When translations were done in the indicated that equal amounts had been loaded (data not shown), presence of canine pancreatic microsomal membranes, a new suggesting that steady state levels of Sf9 a-mannosidase II product was observed that was larger than the largest product RNA are reduced by baculovirus infection. translated in the absence of microsomes. Unlike the smaller products, this one was not degraded by subsequent trypsin treatment but was converted to a slightly smaller form. This In vitro translation and processing of the SJ9 a-mannosidase result indicated that the majority of the protein was oriented II protein towards the lumen of the microsomes, where it was protected To obtain further evidence that the Sf9 a-mannosidase II open from trypsin as a result of cotranslational translocation during reading frame encodes a protein, a plasmid construct contain- synthesis. In support of this conclusion, this product was com- ing the Sf9 a-mannosidase II cDNA was Linearized, tran- pletely degraded when trypsin treatments were performed in scribed in vitro, and the resulting RNA was used for in vitro the presence of a nonionic detergent to solubilize the mem- translation experiments, as described in Materials and meth- branes. The larger size of the translation product prior to pro- ods. The results showed that a protein of about the expected teolysis also suggested that one or more of the potential N- size was produced when a rabbit reticulocyte lysate was primed glycosylation sites are utilized during synthesis. Together, with Sf9 a-mannosidase II RNA (Figure 7). We also observed these results indicate that the Sf9 a-mannosidase II cDNA several additional lower molecular weight proteins, which had encodes a protein with the characteristics of a type II mem- been seen previously in translations of murine a-mannosidase brane glycoprotein.

119 D.L Jarvis et al

HinDm PstI en en C 2 2 u en w E O. g* ' Q. i i •= 2 I Sihs 5 a« s.2 - i » 1 f 1 i § £ o S O J- i2 -- a> C- B 3J U O « Q X CO 23.1 9.4 6.6 i

4.4 Downloaded from https://academic.oup.com/glycob/article/7/1/113/725524 by guest on 01 October 2021

t Fig. 5. Relationship among a-mannosidase II genes in various insects. Southern blotting analyses were done on HindXR (A) or Pstl (B) digests of genomic DNA from insects belonging to the Orders Lepidoptera (H.virescens and Hjea), Coleoptera (T.molitor), Orthoptera (S.gregana), Blattaria (B.discoidalis), or Diptera (D.melanogaster), or from Xenopus laevis. Analogous digests on genomic DNA from Sf9 cells were used as positive controls. Hybridizations were done under low stringency conditions with a nearly full-length Sf9 a-mannosidase II probe, as described in Materials and methods. The positions of X DNA standards are indicated by their sizes (in kb) on the left-hand side of (A).

Expression of the Sf9 a-mannosidase II protein in the tion, the overexpressed protein had low specific activity. baculovirus system However, this was not unexpected, as previous studies have In vivo evidence that the Sf9 a-mannosidase II cDNA encodes shown that production of enzymatically active secretory path- a protein was obtained by using a recombinant baculovirus to way proteins in the baculovirus system is inefficient due to express the cDNA under the control of the strong polyhedrin adverse effects of virus infection on host cell secretory path- promoter in infected Sf9 cells. SDS-PAGE analysis of total way function (Jarvis and Summers, 1989; Jarvis et al., 1996). protein lysates of Sf9 cells infected for 48 or 72 h with this Finally, the a-mannosidase activity detected in the recombi- recombinant virus revealed large amounts of a new protein of nant baculovirus-infected cell extracts was sensitive to swain- about the expected size, which was not detected in mock- or sonine, a known inhibitor of class U a-mannosidases (More- wild-type virus-infected lysates at any time after infection (Fig- men et al., 1994). ure 8). This result, together with the kinetics of appearance and accumulation of this protein during recombinant baculovirus infection, suggested that this protein is the product of the Sf9 Discussion a-mannosidase II cDNA. Evidence that the protein encoded by Baculoviruses are used routinely as vectors for high-level for- the Sf9 a-mannosidase II cDNA is actually an a-mannosidase eign gene expression and, due to the eukaryotic protein pro- II was obtained by biochemical activity assays with p-nitro- cessing capabilities of their hosts, the baculovirus-insect cell phenyl-a-D-mannopyranoside as the substrate (Figure 9). Ex- system is well-suited for foreign glycoprotein production tracts from Sf9 cells infected with the recombinant baculovirus (Summers and Smith, 1987; O'Reilly et al., 1992). However, clearly contained higher levels of pNP-a-mannosidase activity the protein glycosylation pathways of lepidopteran insect cells than extracts from wild-type virus-infected controls. The levels are poorly defined, and it remains difficult to predict the struc- of a-mannosidase activity increased with increasing time of tures of carbohydrate side-chains on foreign glycoproteins pro- infection with the recombinant, but not the wild type virus. The duced in this system. Most of what we know about the N- putative SfManll protein accumulated in the recombinant vi- glycosylation pathway in lepidopteran insect cells is derived rus-infected cells to levels that could be detected by SDS- retrospectively from biochemical studies designed to elucidate PAGE and Coomassie blue staining of total cell lysates pre- the structures of N-linked oligosaccharides from glycoproteins pared at 48 hr postinfection. At this very late time after infec- synthesized in these cells. The overall conclusion from most of

120 Lepidopteran insect a-mannosidase II cDNA

these structures are merely transient intermediates necessary for fucosylation (Altmann et al., 1993; Kubellca et al., 1994; Altmann et al, 1995) and subsequent removal of the terminal N-acetylglucosamine by an exoglycosidase could produce the fucosylated ManjGlcNAcj structures seen in many biochemi- 872- cal studies. In support of this model, it has been shown that certain plant storage glycoproteins are processed via a GlcNAc extension followed by a cleavage (Vitale and Chrispeels, 1984) and N-acetylglucosaminidase activity has 603- been detected in Sf9 and other lepidopteran insect cell lines (Licari et al, 1993; Altmann et al, 1995; Wagner etal, 1996). Alternatively, if the requisite glycosyltransferases are available in insect cells, GlcNAcMan3GlcNAc2 could be elongated to produce complex-type structures analogous to those found in

431- mammalian cells. This latter possibility is supported by bio- Downloaded from https://academic.oup.com/glycob/article/7/1/113/725524 by guest on 01 October 2021 chemical data showing that baculovirus-infected insect cells 412- can produce human 7-interferon with N-linked side-chains containing outer chain N-acetylglucosamine and terminal ga- lactose (Ogonah et al, 1996) and human plasminogen with fully complex side chains containing outer chain N- acetylglucosamine, galactose, and sialic acid (Davidson et al, 1990; Davidson and Castellino, 1991a). It has been proposed that synthesis of complex side chains could require baculovi- 310- rus-induced alterations in the cellular N-glycosylation machin- ery, as the proportion of complex side chains on plasminogen is higher later in infection and certain processing activities are Fig. 6. Steady-state levels of Sf9 a-mannosidase II RNA in uninfected and induced by baculovirus infection (Davidson and Castellino, baculovirus-infected Sf9 cells. RNase protection assays were done on 20 jig 1991b; Davidson et al, 1991; Velardo et al., 1993; Ren et al, of poly A+ RNA from mock-infected Sf9 cells (Mock SF9) or Sf9 cells that had been infected for 12 (12 hpi Sf9), 24 (24 hpi Sf9), or 48 h (48 hpi Sf9) 1995). However, this idea is not supported by the results of a with wild-type baculovirus. The full-length Sf9 a-mannosidasc II riboprobe study which showed that there was no difference in the total (431 bp) is shown in the lanes marked in vitro. The 412 bp protected glycopeptide profiles of uninfected and infected Sf9 cells fragment is shown in a positive control in which the RNase protection assay (Kretzschmar et al, 1994). Perhaps only select glycoproteins, was done with 1 |ig of total RNA from Sf9 cells infected with a recombinant virus that overexpresses the Sf a-mannosidase II cDNA (Rec typified by human 7-interferon and plasminogen, can be con- BEV). The corresponding negative control with 1 jig of total RNA from verted to complex end-products due to special properties that wild-type vims-infected Sf9 cells is shown in the lane marked WT BV. The make them unusually good substrates for processing or unusu- positions of//aelll-digested 0X174 size standards, the full-length riboprobe ally poor substrates for degradative pathways in these cells (431), and the protected Sf9 a-mannosidase II fragment (412) are marked (Licari et al, 1993; Jarvis and Finn, 1995). (in kb) on the left-hand side of the figure. Thus, our current understanding of the N-glycosylation path- way in lepidopteran insect cells seems to reflect both the value and the limitations of using a structural biochemical approach these data is that the N-glycosylation pathway in lepidopteran to investigate this pathway. Whereas the biochemical approach insect cells, and probably all insect cells, differs from the well- has provided a great deal of valuable information on glycopro- characterized pathway in mammalian cells. Specifically, insect tein biosynthesis and processing in these cells, it has been cells appear to be able to N-glycosylate newly synthesized difficult to use structural data from any one model glycoprotein proteins and convert their side-chains to trimmed and fuco- to infer a generalized view of the processing pathway. The sylated structures, but most biochemical data suggest that they biochemical approach also is complicated by the potential post- cannot further process these side-chains to produce structures synthetic effects of exoglycosidases on the structures of gly- with outer chain N-acetylglucosamine, galactose, and sialic coprotein end-products produced by these cells (Licari et al, acid (Butters et al, 1981; Hsieh and Robbins, 1984; Ryan et 1993) and the uncertain effects of viral infection on cellular al, 1985; Nagao et al, 1987; Kuroda et al., 1990; Chen and protein processing pathways (Jarvis and Summers, 1989; Dav- Bahl, 1991; Chen etal., 1991; Wathen etal., 1991; Williams et idson and Castellino, 1991b; Davidson etal., 1991; Velardo et al., 1991; Hogeland et al., 1992; Knepper et al., 1992; Gra- al, 1993; Kretzschmar et al, 1994; Ren et al, 1995; Jarvis et benhorst et al., 1993; Yeh et al., 1993; Manneberg et al., 1994; al, 1996). Overall, any attempt to use structural data from Jarvis and Finn; 1995). biochemical studies to elucidate the N-glycosylation pathway It must be recognized, however, that this model of the insect in the baculovirus—insect cell system must consider both bio- cell N-glycosylation pathway is equivocal. Lepidopteran insect synthetic and degradative pathways, the relative quality of in- cells have N-acetylglucosaminyltransferase I and II activities dividual model glycoproteins as substrates for each, and the (Altmann et al, 1993; Velardo et al., 1993), indicating that effects of viral infection. This is a formidable task and, for this they can elongate trimmed high mannose structures, and it has reason, we have turned to the use of molecular genetics to been shown that N-linked side-chains with terminal N- begin to explore the N-glycosylation pathway in this system. acetylglucosamine (GlcNAcMan3GlcNAc2) can be produced By using degenerate primers designed against conserved re- by these cells (Kubelka el al., 1994; Ackermann et al., 1995; gions in known a-mannosidase II protein sequences, we suc- Ogonah et al., 1996; Wagner et al., 1996). It is possible that cessfully isolated the first lepidopteran insect cell cDNA en-

121 D.LJarvis et al

RNA- MM- Trypsin- NP40

97 Downloaded from https://academic.oup.com/glycob/article/7/1/113/725524 by guest on 01 October 2021

68

43

Fig. 7. In vitro translation and processing of the Sf9 a-mannosidase II protein. A plasmid containing the full-length Sf9 a-mannosidase II cDNA was linearized, transcribed in vitro, and the resulting RNA was translated in a rabbit reticulocyte lysate in the absence (MM-) or presence (MM+) of canine pancreatic microsomal membranes, as described in Materials and methods. Control translations were done with no exogenous RNA (RNA-) in the absence or presence of microsomal membranes. After translation, newly synthesized proteins were treated with water (trypsin —) or trypsin (trypsin +) in the absence (NP40 -) or presence (NP40 +) of nonionic detergent, as described in Materials and methods. The final reaction products were acetone precipitated, redissolved in a protein sample buffer, and analyzed by SDS-PAGE and fluorography, as described in Materials and methods. The positions of molecular weight standards are indicated by their sizes (in kDa) on the left-hand side of the figure. coding a glycoprotein processing enzyme. We have classified this cDNA as an a-mannosidase II because of the strategy used for its isolation and because computer analysis showed that it encodes a protein which is most similar to this subgroup of the class II a-mannosidase family (Moremen et al., 1994). Hy- 24 48 72 dropathy analysis predicted that the Sf9 a-mannosidase II pro- MW R MWR MW R tein would have type II membrane topology, as predicted for 200- all other cloned Golgi processing hydrolases and glycosyltrans- ferases (Paulson and Colley, 1989; Lowe, 1991; Moremen et al., 1994). A protein of the expected size was produced when the Sf9 a-mannosidase II coding region was transcribed and 97- translated in vitro. This protein was translocated and N- 68- glycosylated when the lysate was supplemented with micro- somal membranes and protease protection experiments sug- gested that it had type II membrane topology. A protein of the 43- expected size also was produced when Sf9 cells were infected with recombinant baculoviruses containing the Sf9 a-manno- sidase II coding region, and finally, biochemical assays with 29- •Polyhedrin aryl-a-mannoside showed that this protein had swainsonine- sensitive a-mannosidase activity. Thus, the Sf9 a-mannosidase 18.4- II cDNA clearly encodes a protein that has the hallmark char- acteristics of a mammalian Golgi a-mannosidase EL Fig. 8. Overexpression of the Sf9 a-mannosidase II protein in The results of Southern blotting analyses suggested that Sf9 baculovirus-infected Sf9 cells. Sf9 cells were mock-infected (M) or infected a-mannosidase EL is a single copy gene in Sf9 cells and that with wild-type baculovirus (W) or a recombinant virus (R) designed to Sf21, Bm, and Md cells have very closely related genes. High express the Sf9 a-mannosidase n cDNA under the control of the polyhedrin Five and Ea cells also have related a-mannosidase II genes, but promoter. Total cellular proteins were extracted at 24, 48, or 72 h postinfection, equal aliquots were analyzed by SDS-PAGE, and the gel was the restriction maps of these genes differed substantially from stained with Coomassie brilliant blue. The positions of molecular weight the Sf9 a-mannosidase II gene. This Ending is extremely in- standards are indicated by their sizes (in kDa) on the left-hand side of the teresting in view of data which indicate that High Five cells figure.

122 Lepldopteran insect a-mannosidase II cDNA

cDNA in hand, we can begin to test this idea by manipulating expression of the a-mannosidase II cDNA and determining -SfManll how glycoprotein processing is influenced. This cDNA also will facilitate further studies on the biochemical activities and cell biology of the insect a-mannosidase II gene product, pro- viding a more direct and unequivocal view of the N- glycosylation pathway in the baculovirus-insect cell system.

Materials and methods Cells and cell culture Sf9 cells are derived from the EPLB-Sf21-AE cell line, which was originally isolated from Spodoptera frugiperda (fall armyworm) ovaries (Vaughn et al., 1977). Sf9 cells were maintained as a suspension culture at densities between 0.3 and 3.0 x 106 cells per ml in TNM-FH medium (Summers and Smith,

1987) supplemented with 10% (v/v) heat-inactivated fetal bovine serum (Ha- Downloaded from https://academic.oup.com/glycob/article/7/1/113/725524 by guest on 01 October 2021 zelton Research Products, Lenexa, KS), 1.25 (JLg/ml amphotericin B (Sigma Chemical Co., St. Louis, MO), 25 u.g/ml gentamicin (Sigma), and 0.1% (w/v) pluronic F68 (BASF Wynandotte, Parsippany, NJ; Murhammer and Goochee, 1988). The other lepidopteran insect cell lines used in this study were main- tained as adherent or semiadherent cultures at 28°C at densities between 0.1 and 1.0 x 10* cells per ml in the same medium. These additional cell lines were IPLB-Sf21-AE (Sf21), described above; BTI-Tn-5B1^ (High Five; Wickham et al.. 1992), derived from Trichoplusia ni (cabbage looper) eggs; MdlOS (Md; Hink and Hall, 1989), derived from Malacosoma disstria (forest tent caterpil- lar) hemocytes; Bm5 (Bm; Grace, 1967), derived from Bombyx mori (silk- moth) ovaries; and BTI-EaA (Ea; Granados and Naughton, 1975), derived from Estigmene acrea (saltmarsh caterpillar). An SV40-transformed monkey kidney cell line, COS-1 (Gluzman, 1981), was maintained as an adherent culture at 37°C in a humidified environment of 5% CO2 in Dulbecco's minimal 0.15 1.5 Cof)c#nlfstion of SwHnsonlfiv ( essential medium (GIBCO-BRL Life Sciences, Grand Island, NY) supple- mented with 10% (v/v) fetal bovine serum, 0.3% (v/v) sodium bicarbonate, and 25 u,g/ml of gentamicin sulfate. Fig. 9. Enzymatic activity of the Sf9 a-mannosidase II protein m baculovirus-infected Sf9 cells. Sf9 cells were infected with wild-type baculovirus or a recombinant designed to express the Sf9 a-mannosidase II cDNA under the control of the polyhedrin promoter. Cell extracts were Isolation of an SJ9 a-mannosidase II cDNA clone prepared at 24, 36, or 48 h postinfection and analyzed by SDS-PAGE with Genomic DNA was isolated from Sf9 cells by a standard method (Sambrook Coomassie brilliant blue staining (A). The lanes marked (W) contain the et al., 1989) and used for PCR (Saiki et al., 1985) with degenerate oligo- wild-type extracts and those marked (R) contain the recombinant extracts. nucleotide primers designed against conserved regions in rodent (Moremen, The sizes of the molecular weight standards (lane MW) are indicated in 1989; Moremen and Robbins, 1991) and Dictyosteliwn discoideum (Schatzle el kDa on the left hand side of the figure and the position of the putative Sf9 al., 1992) class II a-mannosidase cDNAs. The sequences of these degenerate a-mannosidase II product is marked on the right. a-Mannosidase II activity primers are shown in Figure 1A. PCRs were done in a total volume of 25 u.1 assays were performed on the same extracts with the p-nitrophenyl containing 10 mM Tris-HCl (pH 8.3), 50 mM KC1, 2.5 mM MgCl2, 0.01% a-mannoside substrate (B); open bars, wild type; cross-hatched bars, (w/v) gelatin, 0.2 mM dNTPs, 1 JLM primers, 1 u,g of genomic DNA, and 2-5 recombinant). Activity assays also were performed in the presence of U of Taq polymerase (Perkin-Elmer, Norwalk, CT). After 40 cycles of dena- various concentrations of swainsonine (C); dotted lines, wild-type; solid turation (45 s at 92°O, annealing (45 s at 45°C), and extension (3 min at 72°C) lines; recombinant; open squares, 24 h postinfection; crosses, 36" h in a Perkin-Elmer thermal cycler, a final extension was done for 5 min at 72°C postinfection; closed circles, 48 h postinfection). and the amplification products were analyzed on 1% agarose gels. A specific amplification product was recovered and cloned into a plasmid vector designed to facilitate direct cloning of PCR products (pCRU Invitrogen, San Diego, might process N-linked oligosaccharides more efficiently CA), and its sequence was determined by the chain termination method (Davis et ai, 1993) and that Ea cells can process N-linked (Sanger et al., 1977). Subsequently, this sequence was used to design exact- match primers against the putative Sf9 a-mannosidase II gene, and these oligosaccharides more extensively (Ackermann et ai, 1995; primers were used to screen subpopulations of an Sf9 cDNA library in XZAPII Ogonah et ai, 1996) than Sf9 cells. One might speculate that (Short et al., 1988; Stratagene, La Jolla, CA) by sibling selection and PCR, as differences in the processing capabilities of these cells reflect described previously (Moremen, 1989). Briefly, the library was split into 43 differences in the biochemical activities of their individual pools of 50,000 clones, each pool was amplified in E.coli, and total X DNA a-mannosidase II gene products. We are currently isolating was prepared from 2 x 10* progeny using a commercial anti-lambdaphage immunosorbent (Lambdasorb), according to the manufacturer's instructions High Five and Ea a-mannosidase II cDNAs in order to explore (Promega Corp., Madison, WI). These DNAs were used as templates for PCRs this possibility. It also was interesting to find that the steady with the exact-match primers to determine which pools included an Sf9 state levels of Sf9 a-mannosidase II RNA were extremely low a-mannosidase n clone. One positive pool was split into eight subpools of in uninfected and baculovirus-infected Sf9 cells. This supports 10,000 clones, each subpool was reamplified in E.coli, total X DNA was isolated, and the PCR screening process was repeated. Finally, one positive our previous speculation (Jarvis and Finn, 1995) that limited subpool, which theoretically included an Sf9 a-mannosidase II clone at a availability of key processing enzymes, such as a-mannosi- frequency of at least 1 in 10,000, was screened by plaque hybridization (Ben- dases and N-acetylglucosaminyltransferases, might impair the ton and Davis, 1977; Sambrook, 1989). The hybridization probe, which was ability of baculovirus-infected cells to efficiently convert re- the original Sf9 a-mannosidase n PCR amplimer that had been cloned into pCRTI, was excised with EcoRI, gel-purified twice, and uniformly labeled by linked carbohydrates to complex forms unless the glycoprotein the random primer method (Feinberg and Vogelstein, 1983). Positive plaques of interest presents an unusually good substrate for these en- from high-density plates were taken through two additional rounds of low zymes. This could explain why complex side chains have been density plaque hybridization for further purification and screening. The cDNA found on only a handful of glycoproteins produced in the bacu- inserts in two XZAPII clones that remained positive through all three rounds of screening were excised as Bluescript-based plasmid subclones by coinfection lovirus-insect cell system. With the Sf9 a-mannosidase II

123 DXJarvis et al

with M13R4O8 helper phage, as described previously (Short et al., 1988). The done overnight at 68°C in the same buffer with a twice gel-punfied, random resulting plasmids were isolated by standard alkaline lysis extraction and CsCl- primer-labeled (Feinberg and Vogelstein, 1983), Xhol-Dral fragment of the EtBr gradient centrifugalion procedures (Sambrook et al., 1989) and used as Sf9 a-mannosidase D cDNA clone (Figure IB). After hybridization, the filters templates to sequence the cDNA inserts with universal and gene-specific prim- were washed for 15 min at room temperature with 2 x SSC plus 0.1% SDS, ers (Sanger et al., 1977). then with 0.5 x SSC plus 0.1% SDS, then with 0.1 x SSC plus 0.1% SDS, and finally, for 30 min with 0.1 x SSC plus 1% SDS that had been prewarmed to 50°C. For low stringency hybridizations, the hybridization temperature was Ligation-anchored PCR and assembly of a full-length Sf9 a-mannosidase II reduced to 55°C and the last wash was done at room temperature. After cDNA washing, the filters were sealed in plastic bags and exposed to Kodak (Roch- ester, NY) X-OMAT AR film with Fisher (Pittsburgh, PA) intensifying screens The 5' end of the Sf9 a-mannosidase II cDNA was isolated by using ligation- for various times at -85°C. anchored PCR (Troutt et al., 1992) as outlined in Figure IB. Total RNA was prepared from a log phase culture of uninfected Sf9 cells by the method of Chirgwin and coworkers (1979) and used to prepare poly A+ RNA by oligo-dT Isolation and analysis of RNA cellulose column chromatography (Aviv and Leder, 1972). One microgram of the poly A+ RNA was used for first-strand cDNA synthesis with random Sf9 cells were grown in 500 ml spinner flasks (Bellco Glass Co., NJ) to a hexamer primers and an RNase H-minus form of MoMuLV reverse transcrip- density of 1 x 10* cells per ml and either mock-infected or infected with tase (Superscript H Life Technologies, Gaithersburg, MD). After reverse tran- wild-type baculovirus (Autographa californica multicapsid nuclear polyhedro- scription, the RNA was digested with RNase H and the reaction mixture was sis virus) at a multiplicity of five plaque-forming units per cell. After adsorp- Downloaded from https://academic.oup.com/glycob/article/7/1/113/725524 by guest on 01 October 2021 diluted and desalted by ultrafiltration in a Microcon 100 filter (Amicon, Bev- tion for 1 h at 28°C, the cells were separated from the inoculum by low speed erly, MA). The single-stranded cDNA was recovered and a 5'-phosphorylated, centrifugation, gently resuspended in 500 ml of TN-MFH medium supple- 3'-blocked primer complementary to the T3 primer (5'- mented with 10% serum, antibiotics, and pluronic F68, and returned to the TCCCTTTAGTGAGGGTTAATTT-NH2-3') was ligated to its 3' end with T4 spinner flask. Total RNA either was extracted immediately from the mock- RNA (New England Biolabs, Beverly, MA). The resulting anchored infected cells or was extracted 12, 24, or 48 h later from the infected cells and first-strand cDNA product was used as the template for a PCR with T3 as the used to isolate poly A+ RNA, as described above. Twenty microgram samples upstream primer and an Sf9 a-mannosidase II-specific oligonucleotide of each mRNA preparation were analyzed by RNase protection assays (Lee (SD1+92- in Figure IB) as the downstream primer, under the conditions and Costlow, 1987) using a commercial kit according to the manufacturer's described by Apte and Siebert (1993). The amplification product was extracted instructions (Ambion, Austin TX). The riboprobe for these assays was syn- with phenol-chloroform and a fraction was used as the template for a second- thesized in vitro with T7 RNA polymerase (Melton et al., 1984) and [a-32P]- ary PCR under the same conditions as the primary PCR with T3 as the up- GTP (800 Ci/mmol; DuPont NEN, Boston, MA) using a commercial kit (Am- stream primer and a different Sf9 a-mannosidase II-specific oligonucleotide bion) according to the manufacturer's instructions. The DNA template for the (SfManII+157- in Figure IB) as the downstream primer. The secondary prod- transcription reactions was pSfManHABst, a derivative of pSfManll (Figure ucts were extracted with phenol-chloroform, gel-purified on a 1% agarose gel, 1C), linearized at the Pmh site. Transcription of this template produced a 431 and a band of interest identified by Southern blotting was recovered and cloned bp antisense RNA consisting of 19 bp from the vector followed by 412 bp into pCRII. Clones containing the 5' end of the putative Sf9 a-mannosidase II beginning at the BstXl site and ending at the Pmh site of the Sf9 a-manno- cDNA were identified by colony hybridization as described previously (Sam- sidase II cDNA (positions 1384 and 966, respectively, in Figure 2). Protected brook et al., 1989). The hybridization probe used for both Southern blots and fragments were analyzed on 5% acrylamide, 7 M urea gels, as described colony lifts was a 144 bp PCR product from the 5' end of the Sf9 a-manno- previously (Jarvis, 1993b), and the gels were dried and exposed to Kodak sidase II partial cDNA clone (produced with primers SfManII+13+ and Sf- X-OMAT AR film with intensifying screens for various times at -85°C. ManH+157- in Figure IB), which had been gel-purified twice and uniformly labeled by the random primer method (Feinberg and Vogelstein, 1983). Plas- mid DNA was isolated from positive clones, purified, and the inserts were In vitro translation and processing of the Sf9 a-mannosidase II cDNA. sequenced by the chain termination method (Sanger et al., 1977). For these experiments, the full-length Sf9 a-mannosidase II open reading The resulting sequence information was used to design exact-match primers frame was subcloned downstream of an SP6 promoter in the plasmid for amplification of the 5' end of the putative a-mannosidase II cDNA from pGem7Zf+ (Promega) to produce pGemSfManH RNA was synthesized in the original Sf9 cell cDNA preparation, as diagrammed in Figure 1C. The 5' vitro from Jffcal-linearized pGemSfMardl as described above, except SP6 RNA primer (BglU...—20 in Figure 1C) was designed to incorporate a unique BglU polymerase was used instead of T7 RNA polymerase and the [a-32P]-GTP was site at position -20, with respect to the putative translational initiation site, and replaced by nonradioactive rGTP. A portion of the in vitro transcribed RNA the 3' primer (SfManH-Al in Figure 1C) was located downstream of a unique was used for in vitro translation reactions in a rabbit reticulocyte lysate (Pro- Xhol site. This amplification product was cloned into pCRII to produce pSf- mega) in the presence or absence of canine pancreatic microsomal membranes ManTI-5', several independent transformants were sequenced, and the BglH- (Blobel and Dobberstein, 1975; Promega), as described previously (Moremen Xhol fragment from a representative clone was excised and gel-purified. In and Robbins, 1991). Subsequently, translation reactions were treated with parallel, the Xhol-Dral fragment of the partial a-mannosidase n cDNA clone water, 100 u.g/ml trypsin, or trypsin plus 0.1% (v/v) Nonidet-P40 (Zilberstein from the Sf9 library was subcloned into a modified form of pBSKS+ (pBSAK/ et al., 1980), as described previously (Moremen and Robbins, 1991). The BS in Figure 1Q to produce pSfManII-3'. Finally, the full-length Sf9 a-man- reaction products were acetone-precipitated, and the precipitates were dried, nosidase II cDNA was assembled by inserting the BglU-Xhol fragment of rcdissolved in protein disruption buffer (50 mM Tris-HCl, pH 6.8; 4% (w/v) pSfManII-5' into Bg/11-.X/ioI-digested pSfManII-3' to produce pSfManll, as SDS; 4% (v/v) B-mercaptoethanol), and heated at 65°C for 10 min. Total shown in Figure 1C. The complete sequence of the Sf9 a-raannosidase D solubilized proteins were analyzed by SDS-PAGE using the discontinuous cDNA (Figure 2) was assembled and analyzed using version 8.1 of the Uni- buffer system of Laemmli (1970) and the gels were fluorographed with Au- versity of Wisconsin Genetics Computer Group software package (Program tofluor (National Diagnostics, Atlanta, GA), dried, and exposed to Kodak Manual for the Wisconsin Package, Version 8.1, Genetics Computer Group, X-OMAT AR film at -85°C. Madison, WI).

Baculovirus-mediated expression of the Sf9 a-mannosidase II cDNA in Isolation and analysis of genomic DNA insect cells A standard method (Sambrook et al., 1989) was used to isolate genomic DNA A standard method was used to isolate a recombinant baculovirus containing from Sf9, Sf21, High Five, Md, Ea, Bm, or COS cells. Genomic DNA from the Sf9 a-mannosidase II cDNA (Summers and Smith, 1987; O'Reilly et al., various insect larvae or Xenopus laevis was kindly provided by Dr. Jay Brad- 1992). The intact Sf9 a-mannosidase II open reading frame was excised from field, Texas A&M University, Department of Entomology. Twenty micro- pSfManll and subcloned into the baculovirus transfer vector, pVL1392 (Webb grams of each DNA was digested with various restriction endonucleases and and Summers, 1990). The resulting plasmid, in which the Sf9 a-mannosidase the digests were resolved on a 1% agarose gel. The DNA was transferred to a II cDNA was positioned downstream of the strong polyhedrin promoter, was positively charged nylon filter (Zetaprobe; Bio-Rad Laboratories, Hercules, mixed with wild type viral DNA and the mixture was used to cotransfect Sf9 CA) under alkaline conditions (Southern, 1975; Reed and Mann, 1985), and cells by a modified calcium phosphate precipitation method (Summers and the filter was prehybridized for at least 1 h at 68°C in a buffer containing 1.5 Smith, 1987). Viral progeny were harvested 5 days after transfection and x SSPE (15 mM NaPO_, pH 7.0; 270 mM NaCl, and 15 mM EDTA), 1% (w/v) resolved by plaque assay in Sf9 cells, as described previously (Summers and sodium dodecyl sulfate (SDS), 0.5% (w/v) nonfat dry milk, and 150 u.g/ml of Smith, 1987). Recombinants were identified by their occlusion-negative sheared salmon sperm DNA (Sigma). High stringency hybridizations were plaque phenotypes and taken through two additional rounds of plaque-

124 Lepidopteran insect a-mannosidase II cDNA

purification. Vims stocks were prepared and titered by plaque assay in Sf9 and expression of cDNA encoding a rat liver endoplasmic reuculum alpha- cells and stored frozen in the dark at -85°C (Jarvis and Garcia, 1994). The mannosidase. J. Biol. Chem., 165, 17110-17117. procedures used for baculovims infections and analysis of recombinant protein Blobel.G. and DobbersteinjJ. (1975) Transfer of proteins across membranes, biosynthesis have been described previously (Jarvis and Summers, 1989; Jarvis n. Reconsn'tution of functional rough microsomes from heterologous com- et al., 1991). Briefly, Sf9 cells were seeded into 6-well plates (Coming Glass ponents. J. Cell Biol., 67, 852-862. Works, Coming, NY) at a density of 1 x 10* cells per well, mock-infected or Butters.TX)., Hughes,R.C. and Vischer.P. (1981) Steps in the biosynthesis of infected at a multiplicity of about 5 plaque-forming units per cell, and incu- mosquito cell membrane glycoproteins and the effects of tunicamycin. Bio- bated at 28°C until 24, 36, 48, or 72 h postinfection. At these time points, the chim. Biophys. Acta, 640, 672-686. cells were gently squirted off the plastic into the medium, pelleted, 0.5 ml of Chen.W. and Bahl.OJ. (1991) Recombinant carbohydrate variant of human protein disruption buffer was added, and the cell pellets were triturated through choriogonadotropin P-subunit (hCGfi) descarboxyl terminus (115-145): ex- a 1 ml syringe equipped with a 22 ga needle. The sheared lysates were boiled pression and characterization of carboxy-terminal deletion mutant of hCGB for 3 min, total solubilized proteins were resolved by SDS-PAGE, as described in the baculovirus system. J. Biol. Chem., 266, 6246-6251. above, and the gels were stained with Coomassie brilliant blue, destained, and Chen.W., Shen,Q.-X. and BaW,O.P. (1991) Carbohydrate variant of the re- photographed. combinant P-subunit of human choriogonadotropin expressed in baculovirus expression system. J. Biol. Chem., 266, 4081-4087. a-Mannosidase II activity assays ChirgwinJ.M., PrzybylaA-E., MacDonaldJU. and Rutter.WJ. (1979) Isola- tion of biologically active ribonucleic acid from sources enriched in ribo- Sf9 cells were infected with wild-type or recombinant baculoviruses as de- nuclease. Biochemistry, 18, 5294-5299. Downloaded from https://academic.oup.com/glycob/article/7/1/113/725524 by guest on 01 October 2021 scribed above and harvested by centrifugation at various times after infection, DavidsonJDJ. and CasteUinoJ'J. (1991a) Asparagine-linked ohgosaccharide and pellets containing 1x10* cells were resuspended in 100 u.1 of assay buffer processing in lepidopteran insect cells. Temporal dependence of the nature (0.1 M MES, pH 6.3; 0.5% Triton- X-100). The cell suspensions were assayed of the oligosaccharides assembled on asparagine-289 of recombinant human for a-mannosidase activity by mixing 25 p-I of the cell extracts with 25 u.1 of plasminogen produced in baculovirus vector infected Spodoptera frugiperda 10 mM p-nitrophenyl a-D-mannopyranoside in the presence or absence of (TPLB-SF-21AE) cells. Biochemistry, 30, 6167-6174. various concentrations of swainsonine. The reaction mixtures were incubated Davidson,DJ. and CastellinoJU. (1991b) Structures of the asparagine-289- in a microtiter plate for 1 hr at 37°C with gentle agitation, and then quenched linked oligosaccharides assembled on recombinant human plasminogen ex- by the addition of 200 u,l of stop solution (133 mM , 67 mM NaCl, 83 pressed in a Mamestra brassicae cell line (1ZU-MBO503). Biochemistry, 30, mM Na CO ). Absorbance was measured at 410 nm on a plate reader (Dy- 2 3 6689-6696. natech model MR 5000), corrected for light scattering at 570 nm, and the corrected absorbance values were converted to nmol p-nitrophenol using a DavidsonJDJ., FraserJvlJ. and Castellino.FJ. (1990) Oligosaccharide process- standard curve produced on the same plate reader. ing in the expression of human plasminogen cDNA by lepidopteran insect {Spodoptera frugiperda) cells. Biochemistry, 29, 5584-5590. DavidsonJJJ., Bretthauer,R.K. and Castellino.FJ. (1991) a-mannosidase Computer methods catalyzed trimming of high mannose glycans in noninfected and baculovi- rus-infected Spodoptera frugiperda cells (IPLB-SF-21AE). A possible con- The cDNA sequence was assembled using the sequence assembly package of tributing regulatory mechanism for assembly of complex-type oligosaccha- Staden (1987). The sequence of the amplimer translation was compared to the rides in infected cells. Biochemistry, 30, 9811-9815. six frame translation of the GenBank nonredundant DNA sequence database Davis.T.R., ShuleT,MX., Granados,R.R. and WcxxLH.A. (1993) Comparison (version 91) using the TFASTA subroutine of the University of Wisconsin of oligosaccharide processing among various insect cell lines expressing a Genetics Computer Group (GCG) software package (Program Manual for the secreted glycoprotein. In Vitro Cell. Dev. Biol., 29A, 842-846. Wisconsin Package, Version 8.1, Genetics Computer Group, Madison, WT). Feinberg^A.P. and Vogelstein3. (1983) A technique for radiolabeling DNA The pairwise sequence comparisons were performed using the Bestfit subrou- restriction endonuclease fragments to high specific activity. Anal. Biochem , tine and multiple sequence alignments and dendrograms were prepared using the Pileup and Boxshade subroutines of the GCG software package. 132, 6-13. Field,M.C. and WainwrightXJ. (1995) Molecular cloning of eukaryotic gly- coprotein and glycosyltransferases: a survey. Glycobiology, 5, 463-472. Acknowledgments FosterJ.M., Yudkir^B-, LockyerAE. and Roberts.D.B. (1995) Cloning and sequence analysis of GrnTI, a Drosophila melanogaster homologue of the We thank Dr. Max Summers for providing the various insect cell lines, Dr. cDNA encoding murine Golgi alpha-mannosidase II. Gene, 154, 183-186. Linda Guarino for providing the Sf9 cell cDNA library, and Dr. Jay Bradfield Gluzman.Y. (1981) SV40-transformed simian cells support the replication of for providing the larval DNAs. This work was supported by grants from the early SV40 mutants. Cell, 23, 175-182. NIH (GM49734 to D.LJ. and GM47533 and RR05351 to K.W.M.). Grabenhorst^E., HofeT.B., Nimtzjvl., Jager.V. and Conradt,H.S. (1993) Bio- synthesis and secretion of human interleukin 2 glycoprotein variants from baculovirus-infected Sf21 cells. Characterization of polypeptides and post- References translational modifications. Ear. J. Biochem., 215, 189-197. Grace.T.D.C. (1967) Establishment of a line of cells from the silkworm Bom- Ackermann.M., Nimtz^M., GrabenhorsUE., ConradtJI.S. and Jager.V. (1995) byx mori. Nature, 216, 613. Pilot-scale production of glycoproteins with recombinant baculoviruses: Granados,R.R. and Naughtonjd. (1975) Development of Amsacta moorei en- Evaluation of different host cell lines with respect to productivity, protein tomopoxvirus in ovarian and haemocyte cultures from Estigmene acrea integrity, and glycosylation. Baculovirus and Insect Cell Gene Expr. Conf. larvae. Intervirology, 5, 62-68. Abstr., ST13. Hink.W.F. and Hall,R.L. (1989) Recently established invertebrate cell lines. In Altmann.R, Komfeld,G., Dalik.T., Staudacher,E. and GlosslJ. (1993) Process- MitsuhashiJ. (ed), Invertebrate Cell System Applications, Vol U. CRC ing of asparagine-linked oligosaccharides in insect cells. N- Press, Boca Raton, pp. 269—293 acetylglucosaminyltransferase I and n activities in cultured lepidopteran Hogeland.K.E.Jr., ArbogasUJ. and Deinzer.M.L. (1992) Liquid secondary ion cells. Glycobiology, 3, 619-625. mass spectrometry analysis of permethylated, n-hexylamine derivatized oli- Altmann.R, Schwihia,H., Staudacher,E., GlossLJ. and MarzX- (1995) Insect gosaccharides. Application to baculovirus-expressed mouse interleukin-3. /. cells contain an unusual, membrane-bound P-N-acetylglucosaminidase Am. Soc. Mass Spectrom., 3, 345-352. probably involved in the processing of protein N-glycans. /. Biol. Chan., HsiehJ". and Robbins,P.W. (1984) Regulation of asparagine-linked oligosac- 270, 17344-17349. charide processing. Oligosaccharide processing in Aedes albopictus mos- Apte,A.N. and SiebertJ'.D. (1993) Anchor-ligated cDNA libraries: a technique quito cells. /. Biol. Chem., 259, 2375-2382. for generating a cDNA library for the immediate cloning of the 5' ends of JarvisJ5.L. (1993a) Continuous foreign gene expression in stably-transformed mRNAs. Biotechniqucs, 15, 890-893. insect cells, In Goosen,M.F.A, Daugulis,A. and Faulkner,P. (eds), Insect Aviv.H. and Leder,P. (1972) Purification of biologically active globin mes- Cell Culture Engineering. Marcel DekkeT, New York, pp. 193-217. senger RNA by chromatography on oligothymidylic acid-cellulose. Proc. JarvisJ3.L. (1993b) Effects of baculovirus infection on IE 1-mediated foreign Nat. Acad. Sci. USA, 69, 1408-1412. gene expression in stably-transformed insect cells. /. Virol., 67, 2583-2591. Benton.W.D. and Davis,R.W. (1977) Screening lambda gt recombinant clones Jarvis.D.L. and Finn^E.E. (1995) Biochemical analysis of the N-glycosylation by hybridization to single plaques in situ. Science, 196, 180-182. pathway in baculovirus-infected lepidopteran insect cells. Virology, 212, BischoffJ., Moremenjf. and Lodish.H.F. (1990) Isolation, characterization, 500-511. 125 D.L Jarvis et al

JarvisJD.L. and Garcia^A- (1994) Long-term stability of baculovimses stored asparagine-linked oligosaccharide processing pathway. Glycobiology, 4, under various conditions. BioTechniques, 16, 508—513. 113-125. JarvisJD.L. and Summers,M.D. (1989) Glycosylation and secretion of human MurhammeTjD.W. and Goochee.C.F. (1988) Scaleup of insect cell cultures: tissue plasminogen activator in recombinant baculovirus-infected insect protective effects of pluronic F68. Bio/Technology, 6, 1411-1418. cells. Mol. Cell. Biol., 9, 214-223. Nagao,E., TakahashiJ"}. and ChinoJ-L (1987) Asparagine-liriked oligosaccha- Jarvis,D.L. and Summers^M.D. (1992) Baculovirus expression vectors. In rides of locust lipophorin. Insect Biochem., 17, 531-538. Isaacson,R.E. (ed), Recombinant DNA Vaccines: Rationale and Strategies. Nebes.V.L. and SchmidtJvl.C. (1994) Human lysosomal alpha-mannosidase: Marcel Dekker, New York, pp. 265-291. isolation and nucleotide sequence of the full-length cDNA. Biochem. Bio- JarvisJD.L., Bohlmeyer,D.A. and Garcia^A. (1991) Requirements for nuclear phys. Res. Commun., 200, 239-245. localization and supramolecular assembly of a baculovirus polyhedrin pro- Ogonah.O.W., FreedmanJR.B., Jenkins,N., PateLK. and Rooney.B. (1996) Iso- tein. Virology, 185, 795-810. lation and characterization of an insect cell line able to perform complex JarvisJD.L., Weinkauf.C. and GuarinoJL.A. (1996) Immediate early baculovi- N-linked glycosylation on recombinant proteins. Bio/Technology, 14, 197— rus vectors for foreign gene expression in transformed or infected insect 202. cells. Protein Expression and Purification, 8, 191-203. O'ReillyJD.R., MiUeT,L.K. and Luckow.V.A. (1992) Baculovirus Expression JoziasseJD.H. (1992) Mammalian glycosyltransferases: genomic organization Vectors. W. H. Freeman, New York. and protein structure. Glycobiology, 2, 271-277. PaulsonJ.C. and Colley.KJ. (1989) Glycosyltransferases. Structure, localiza- Kerscher.S., Albert.S., Wucherpfennig.D., Heisenberg,M. and Schneuwly.S. tion, and control of cell type-specific glycosylation. J. Biol. Chem., 264,

(1995) Molecular and genetic analysis of the Drosophila mas-1 (mannosi- 17615-17618. Downloaded from https://academic.oup.com/glycob/article/7/1/113/725524 by guest on 01 October 2021 dase-1) gene which encodes a glycoprotein processing alpha 1,2- ReedJC.C. and MannJD.A. (1985) Rapid transfer of DNA from agarose gels to mannosidase. Dev. Biol, 168, 613-626. nylon membranes. Nucleic Acids Res., 13, 7207-7221. Knepper.T.P., ArbogastJ}., SchreursJ. and Deinzer,M.L. (1992) Determina- RenJ., Bretthauer,R. and CasteUino,FJ. (1995) Purification and properties of tion of the glycosylation patterns, disulfide linkages, and protein heteroge- a golgi-derived (a-l,2)-mannosidase-I from baculovirus-infected lepidop- neities of baculovirus-expressed mouse intcrleukin-3 by mass spectrometry. teran insect cells (IPLB-Sf21AE) with preferential activity towards man- Biochemistry, 31, 11651-11659. noses-N-acetylglucosamine. Biochemistry, 34, 2489-2495. KomfeldJR. and Komfeld,S. (1985) Assembly of asparagine-linked oligosac- RyanJi.O., AndersonJD.R., Grimes.WJ. and LawJ.H. (1985) Arylphorin from charides. Annu. Rev. Biochem., 54, 631—664. Manduca sexta: carbohydrate structure and immunological studies. Arch. Kozakjvl. (1983) Comparison of initiation of protein synthesis in procaryotes, Biochem. Biophys., 243, 115-124. eucaryotes, and organelles. Microbiol. Rev., 47, 1-45. SaikiJt.K., Scharf.S., FaloonaJ'., MullisJC.B., Horn.G.T., Eriich.H.A. and Kozakjvl. (1986) The scanning model for translation: an update. J. Cell Biol., Amheim,N. (1985) Enzymatic amplification of beta-globin genomic se- 108, 229-241. quences and restriction site analysis for diagnosis of sickle cell anemia. Kretzschmar.E., Geyerji. and Klenk,H.-D. (1994) Baculovirus infection does Science, 230, 1350-1354. not alter N-glycosylation in Spodoptera frugiperda cells. Biol Chem. SambrookJ., FritschJi.F. and Maniatis.T. (1989) Molecular Cloning: A Labo- Hoppe-Seyler, 375, 323-327. ratory Manual, 2nd ed. Cold Spring Harbor Laboratory, Cold Spring Har- Kubelka,V., AltmannJ1., K.omfeld,G. and MarzJ,. (1994) Structures of the bor, NY. N-linked oligosaccharides of the membrane glycoproteins from three lepi- SangerJ7., Nicklen.S. and Coulson,AJt. (1977) DNA sequencing with chain- dopteran cell lines (Sf-21, IZD-Mb-O5O3, Bm-N). Arch. Bioch. Biophys., terminating inhibitors. Proc. Natl Acad. Sci. USA, 74, 5463-5467. 308, 148-157. SchatzleJ., Bush J. and Cardelli J. (1992) Molecular cloning and characteriza- KurodaJC., Geyer,H., GeyerJ*., DoerfleT.W. and Klenkji.-D. (1990) The oli- tion of the structural gene coding for the developmentally regulated lyso- gosaccharides of influenza virus hemagglutinin expressed in insect cells by somal enzyme, a-mannosidase, inDictyostelium discoideum. J. Biol. Chem., a baculovirus vector. Virology, 174, 418—429. 267, 400CM007. KyteJ. and Doolittle,R.F. (1982) A simple method for displaying the hydro- ShortJ.M., FemandczJ.M., SorgeJ.A. and Huse.W.M. (1988) X ZAP: a bac- pathic character of a protein. J. Mol. Biol, 157, 105-132. teriophage X expression vector with in vivo excision properties. Nucleic Laemmli.U.K. (1970) Cleavage of structural proteins during the assembly of Acids Res., 16, 7583-7600. the head of bacteriophage T4. Nature, 227, 680-685. Southem,E.M. (1975) Detection of specific sequences among DNA fragments LeeJJ. and Costlow,N.A. (1987) A molecular titration assay to measure tran- separated by gel electrophoresis. J. Mol. Biol, 98, 503-517. script prevalence levels. Methods Eniymol., 152, 633-648. Stadenjt. (1987) Computer handling of DNA sequencing project. In Bish- LicariJ"., JarvUJD.L. and Bailey J.E. (1993) Insect cell hosts for baculovirus op.MJ. and Rawlings,CJ.(eds), Nucleic Acid and Protein Sequence Analy- expression vectors contain endogenous exoglycosidase activity. Biotech. sis, A Practical Approach. IRL Press, Eynsham, England, pp. 173-217. Prog., 9, 146-152. Staudacherji, Kubelka,V. and MarzJ,. (1992) Distinct N-glycan fucosylation LoweJ.B. (1991) Molecular cloning, expression, and uses of mammalian gly- potentials of three lepidopteran cell lines. Eur. J. Biochem., 207, 987-993. cosyltransferases. Sem. Cell Biol, 2, 289-307. SummersJvl.D. and Smith.G.E. (1987) A manual of methods for baculovirus Manneberg.M., Friedlein,A., Kurth.H., Lahm,H.-W. and Fountoulakis.M. vectors and insect cell culture procedures. Tex. Agric. Exp. Stn. Bull, 1555. (1994) Structural analysis and localization of the carbohydrate moieties of a Troutt,A.B., McHeyzer-Williams.M.G., Pulendran.B. and Nossal.GJ.V. soluble human interferon G receptor produced in baculovirus-infected insect (1992) Ligation-anchored PCR: a simple amplification technique with cells. Protein Science, 3, 30-38. single-sided specificity. Proc. Natl. Acad. Sci. USA, 89, 9823-9825. MeltonJD.A., Krieg J>., RebagliatiJvI.R., Maniatis.T., Zinh.K. and Green,M.R. VaughnJ.L., GoodwinJt.H., Thompltins.GJ. and McCawleyJ5. (1977) The (1984) Efficient in vitro synthesis of biologically active RNA and RNA establishment of two insect cell lines from the insect Spodoptera frugiperda hybridization probes from plasmids containing a bacteriophage SP6 pro- (Lepidoptera:Noctuidae). In Vitro, 13, 213-217. moter. Nucleic Acids Res., 12, 7035-7055. Velardo.M.A., BretthauerJ*.K-, Boutaud^A., ReinholoVB., Reinhold,V.N. and Miller,L.K. (1988) Baculoviruses as gene expression vectors. Annu. Rev. Mi- CasteUinoJ^J. (1993) The presence of UDP-N-acetylglucosamine:a-3-r> crobiol, 42, 177-199. mannoside fi-1,2-N-acetylglucosaminyl transferase I activity in Spodoptera MisagoJVL, Liao,Y.-F., Kudo.S., Eto,S., Mattei,M.-G., MoremenJC.W. and frugiperda cells (IPLB-SF-21AE) and its enhancement as a result of bacu- FukudaJvI.N. (1995) Molecular cloning and expression of cDNAs encoding lovirus infection. J. Biol. Chem., 268, 17902-17907. human alpha-mannosidase II and a previously unrecognized alpha- Vitale^A. and ChrispeelsJvlJ. (1984) Transient N-acetylglucosamine in the mannosidase IT isozyme. Proc. Natl. Acad. Sci. USA, 92, 11766-11770. biosynthesis of phytohemagglutinin: attachment in the Golgi apparatus and MoremenJC.W. (1989) Isolation of a rat liver Golgi mannosidase II clone by removal in protein bodies. /. Cell Biol, 99, 133-140. mixed oligonucleotide-primed amplification of cDNA. Proc. Natl Acad. Wagner.R., Geyer.H., Geyer.R. and Klenk,H.-D. (1996) N-Acetyl-0- Sci. USA, 86, 5276-5280. glucosaminidase accounts for differences in glycosylation of influenza virus MoremenJC.W. and RobbinsJ'.W. (1991) Isolation, characterization, and ex- hemagglutinin expressed in insect cells from a baculovirus vector. J. Virol, pression of cDNAs encoding murine a-mannosidase II, a Golgi enzyme that 70, 4103^109. controls conversion of high mannose to complex N-glycans. /. Cell Biol, WathenJvl.W., AeedJ'.A. and Elhammer^A.P. (1991) Characterization of oli- 115, 1521-1534. gosaccharide structures on a chimeric respiratory syncytial virus protein Moremen.K.W., Touster.O. and Robbins,P.W. (1991) Novel purification of the expressed in insect cell line Sf9. Biochemistry, 30, 2863-2868. catalytic domain of Golgi a-mannosidase IL Characterization and compari- Webb,N.R. and Summers,M.D. (1990) Expression of proteins using recombi- son with the intact enzyme. /. Biol. Chem., 266, 16876-16885. nant baculoviruses. Technique, 2, 173-188. Moremen.K.W., TrimbleJt.G. and HerscovicsA (1994) Glycosidases of the Wickham.TJ., Davis.T., Granadosjt.R., Shuler^4i. and WoodJI.A. (1992) 126 Lepidopteran insect a-mannosidase II cDNA

Screening of insect cell lines for the production of recombinant proteins and infectious virus in the baculovirus expression system. Biotech. Prog., 8, 391-396. Williams.PJ., Wormala\M.R., DwekJtA., Rademacher.TW., Parker.G.F. and Roberts.D.B. (1991) Characterization of oligosaccharides from Drosophila melanogaster glycoproteins. Biochim. Biophys. Acta, 1075, 146-153. YehJ.-C., SealsJ.R., Murphy.C.I., van Halbeek.H. and Cummijigs.R.D. (1993) Site-specific N-glycosylation and oligosaccharide structures of re- combinant HTV-1 gpl20 derived from a baculovirus expression system. Biochemistry, 32, 11087-11099. YoshihisaJ". and Anraku.Y. (1989) Nucleotide sequence of AMS1, the struc- ture gene of vacuolar alpha-manDOsidase of Saccharomyces cerevisiae. Bio- chem Biophys. Res. Commun., 163, 908-915. ZilbersteinA, Snider.M.D., PorteT,M. and Lodish^i.F. (1980) Mutants of ve- sicular stomatitis virus blocked at different stages in maturation of the viral glycoprotein. Cell, 21, 417-427.

Received on June 20, 1996; revised on July 25, 1996; accepted on July 25, 1996 Downloaded from https://academic.oup.com/glycob/article/7/1/113/725524 by guest on 01 October 2021

127