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Engineering N-glycosylation pathways in the baculovirus-insect cell system
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Engineering N-glycosylation pathways in the baculovirus-insect cell system Donald L Jarvis, Ziad S Kawar and Jason R Hollister The inability to produce eukaryotic glycoproteins with complex N-linked glycans is a major limitation of the baculovirus-insect cell expression system. Recent studies have demonstrated that metabolic engineering can be used to extend the glycoprotein processing capabilities of lepidopteran insect cells. This approach is being used to develop new baculovirusinsect cell expression systems that can produce more authentic recombinant glycoproteins and obtain new information on insect N-glycosylation pathways.
Addresses Department of Molecular Biology, University of Wyoming, PO Box 3944, Laramie, Wyoming 82071-3944, USA Correspondence: Donald J Jarvis; e-mail: [email protected]
Current Opinion in Biotechnology 1998, 9:528-533 http://biomednet.com/elecref/0958166900900528 :c: Current Biology Ltd ISSN 0958-1669
Abbreviations GIc
glucose
GIcNAc N-acetylglucosamine Man mannose Introduction Recombinant baculoviruses are frequently used to prodt,ce foreign proteins in lepidopteran insect cells, which usually serve as the laboratory hosts for these viruses [1,2]. One important feature of the bacuh)virus-inscct cell system is that it can produce glycosylated proteins. (;lycosylation is a comnqon covalent chemical m()dification that can influence many prntein properties, including intracelh, lar trafficking, biological function, and biochemical stabilit> Most evidence, however, suggests that the protein glycosylation pathways of lcpidopteran insect cells differ from these of higher et,karyotes [3",4]. Recent studies have described metabolic engineering apprnaches that can be used to extend the glycoprotein processing capabilities of the bacuh)virus-insect cell system. In this review, we will summarize some of the evidence leading to our current understanding of insect N-glycosylation, present a comprehensive model of insect N-g;lycosylation, and discuss current efforts to improve N-glycosylation in the baculovirus-inscct cell expression system. Protein N-glycosylation T h e protein N-glycosylation reactions of mammalian cells have been reviewed [5-7] and are summarized in Figure 1. T h e mammalian N-glycosylation pathway begins with the transfer of a preassembled oligosaccharide, Glc.~-Mang-GlcNAc z, to a nascent polypeptide. This is followed by trimming of the glucose residues to produce a 'high mannose' glycan, MangGlcNAc z. Some
N-linked glycans are processed no further and rcmain in this high mannose form. Others are processed by class I o~mannosidases, which remove four mannose residues to produce lklansGlcNAcz, and N-acetylglucosaminyltransferase I, which adds a terminal GIcNAc residue to produce GIcNAcMansGlcNAc z. Addition of the GIcNAc permits o~-mannnsidase II to remove two morc mannosc rcsiducs to produce GIcNAcMan~GIcNAcz, a trimmed intermediate that can be converted to 'complex' glycans by various glycosyltransferases, including N-acetylglucosaminyltransfcrases, fucosyltransferases, galactnsyhransferascs, N-acetylgalactosaminyhransferases, and sialvltransfcrases. N - g l y c o s y l a t i o n in insect cells Historically, two diffcrent approaches have been used to study N-glycosylation in insect cells. One was to determine the structures of N-linked glycans on glycoproteins produced by insect cells. T h e other was to assay insect cell extracts for specific glycoprotein processing activities. Recently, these biochemical approaches have been supplcmented by a molecular genetic approach. Selected biochemical studies An early survey suggested that invertebrates lacked sialic acids [81. lmtcr, it was shown that Sindbis virus glycoproreins were sialylated by mammalian but not mosquito cells, anti it was found that mosquito cell extracts lacked both sialic acid and siaMtransferase activity [9]. Structural analyses of native mosquito cell-surface glycoproteins suggested that they had only high mannose N-linked glycans [10] and activity assays indicatcd that these cells lacked N-acetylglucosaminyltransfcrascs, galactosyhransferases, and sialyltransferases [1 1]. Subseqt,ent structural studies revealed that dipteran insect cells could produce the trimannosvl core structure, Man.~GlcNAc z, but not complex N-linked glycans [12]. Together, these observations led to the general conclusion that insect cells can produce N-linked glycoproteins with high mannose glycans and trim them to produce trimannosvl core structures, but they lack the glycosyhransferases needed to convcrt the trimmed glycans to complex structures. This conclusion was derived mainly from studies of dipteran insect cells, but structural studies of N-linked glycans from native lepidopteran insect cell glycoproteins suggested that these cells had the same capabilities [13-16]. Further information on protein N-glycosylation in lepidopteran insect cells came with the developnaent of baculovims expression vectors, which led to widespread use of these cells for recombinant glycoprotein production. One of the first direct analyses of the N-linked glycans from a recombinant glycoprotein produced by the baeulovirt,s-insect cell system demonstrated that they had high mannose or
Engineering N-glycosylation pathways in the baculovirus-insect cell system Jarvis, Kawar and Hollister
529
Figure 1
.u , C)-~
~ A S N
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=Fucose
O = Galactose O = G.atNAc.,
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N-aeetylglucosaminyltransferaseI ASN H cz-mannosidaseII ~7 Fucosyltransferases
, N-acetylglucosaminidase
[ ~
~ A S N ~ ~
, N-acetylglucosaminyl transferase II ~k~
~
~ A S N
Trimannosyl core
NialAy/tetaYIngs faleaCts°eSY It ransferas ~ / / /
~
Current Opinion in Biotechnology
A
S
N
ASN
~
~
Giall~l~trO::lf~raans::rases
A
S
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Complex N-linked glycans
A comprehensive working model of insect N-glycosylation pathways. The model proposes that there are multiple branches for the processing of Nlinked glycans on newly synthesized insect glycoproteins. Depending upon the levels of competing processing enzyme activities in a specific insect cell type under a specific set of conditions, high mannose, trimannosyl core, or complex N-linked glycans may be produced. We also propose that the net result of the N-glycosylation pathway is strongly influenced by the structural characteristics of the glycoprotein being processed. This model stems from observations described in many different studies from many different labs, including the representative studies cited in this review, as well as additional studies cited in references [3 °] and [4]. GalNAc, N-acetylgalactosamine; RER, rough endoplasmic reticulum.
od,6-fucosylated trimannosyl core structures [17]. Subsequent structural analyses of N-linked glycans from other recombinant glycoproteins produced by this system provided analogous results [18-20,Z1",22-27], The inability to produce complex N-linked glycans suggested that bacuh)virus-infected lepidopteran insect cells lacked glycosyltransferases. If they lacked all glycosyltransferases, however, including N-acetylglucosaminyltransferase I, these cells would be unable to convert MansGlcNAc 2 to GlcNAcMansGlcNAc 2, which is required for o~-mannosidase II to complete the trimming reactions [5,6]. Thus, insect cells would need a different way to produce trimannosyl core structures. Ultimately, N-acetylglucosaminyl-
transferase 1 activity was found in lepidopteran insect cells [28,29] and this indicated that mannose trimming could proceed by the conventional pathway. However, this still failed to explain how terminal GIcNAc was removed from GIcNAcMan,~GIcNAc2 to produce the trimannosyl core. A possible explanation arose from the finding that insect cells have high levels of N-acetylglucosaminidase activity, which could remove terminal GlcNAc in a degradative fashion [30]. A more elegant explanation arose with the demonstration that some lepidopteran insect cells have a membranebound N-acetylglucosaminidase that might fimction as a processing enzyme that converts GIcNAcMan3GIcNAc z to Man.~GlcNAc z [31].
530 Expressionvectorsand deliverysystems
Molecular genetic studies More recentl'~, molecular genetics has been used to study insect cell N-glycosylation pathways. Molecular genetics can reveal which glycoprotein processing enzymes are encoded by insect cells and establish the conditions under which these genes are expressed. Furthermore, genes isolated in these studies can be used to manipulate the expression of endogenous glycoprotein processing enzymes, which is one important approach to metabolic engineering. Molecular cloning of genes encoding glycoprotein processing enzymes is hindered by the fact that these enzymes are usually fotmd in only minute amounts in cells [32,33]. Thus, the first such gene, encoding 131,4-galactosyhransferase, was isolated from mammalian cells in 1986 [34]. T h e first insect cell genes were putative c~-mannosidase cDNAs isolated from Drosophila in 1995 [35,36] and the first lepidopteran insect cell genes were 0~-mannosidase cDNAs isolated from Sf9 cells in 1997 [37",38"]. Sequence analysis of the Sf9 cDNAs and biochemical studies of their protein products provided the first molecular genetic evidence that the Sf9 N-glycosylation pathway includes typical class I and II 0t-mannosidases. These results were consistent with previous structural data (above) and biochemical data demonstrating the presence of these enzymes in Sf9 extracts [39,40°,41]. Our ongoing studies on the natural substratc specificity of the enzyme encoded by the Sf9 ot-mannosidase I1 cDNA, however, suggest that it requires cobalt and acts preferentially on MansGlcNAc 2 (DL Jarvis and KW Moremen, unpublished data). This enzyme, therefore, is probably not the Sf9 c~-mannosidase II, but a functional homolog of an unusual 0~-mannosidase that mediates an alternate trimming pathway in knockout mice lacking 0~-mannosidase II [42,43"].
A comprehensive model of insect N-glycosylation Whereas most evidence indicates that insect cells can produce only N-linked glycans with high mannose or fucosylated trimannosyl core structures, some evidence argues against this conclusion. Some insect cells have Nacetylglucosaminyhransferase II, [~l,4-galactosyhransferase, and [31,4-N-acetylgalactosaminyltransferase activities, all of which are involved in producing complex N-linked glycans [28,29,44"]. Furthermore, some baculovirus-infected insect cells can produce recombinant glycoproteins with elongated trimannosyl core structures containing terminal GIcNAc or galactose [21°,45",46°], and one recombinant glycoprotein acquired complex biantennary N-linked glycans containing penultimate galactose and terminal sialic acid [47,48]. Finally, it has been reported that Drosophilaembryos contain sialic acid [49]. These findings demonstrate that the insect N-glycosylation pathway cannot be described in such simple terms. A more comprehensive model is needed to explain how insect cells can produce various types of N-linked glycans. The model proposed in Figure 1 presents insect cell N-glycosylation as a single pathway with multiple potential end-products. A critical feature of this model is that the structure of the N-linked glycan on a specific glycoprotein
produced by a specific insect cell under specific conditions is determined by the relative levels of competing processing activities and the structure of the glycoprotein being processed. Thus, this model includes the flexibility needed to explain how insect cells can produce various types of Nlinked glycans ranging from high mannose to complex. As production of complex, sialylatcd N-linked glycans is such a rare event in insect cells, we have speculated that it is protein-dependent and occurs only with specific recombinant proteins that are unusually poor substrates for the N-acetylglucosaminidase and/or unusually good substrates for the low-level glycosyltransferasc activities in these cells [24]. An important observation in the context of this model is that the levels of N-acetylgalactosaminyhransferase and galactosyltransferase activities in lepidopteran insect cells are reduced by baculovirus infection [44°].
E n g i n e e r i n g insect cell N-glycosylation pathways T h e inability of the baculovirus-insect cell expression system to routinely produce recombinant glycoproteins with complex N-linked glycans led to efforts to engineer the glycoprotein processing capabilities of this system. In the first example of this, a conventional baculovirus expression vector was used to introduce a human processing enzyme into the system [50"]. This vector expressed human N-acetylglucosaminyltransferase I under the control of the polyhedrin promoter, which significantly increased the level of N-acetylglucosaminyltransfcrase I activity during infection. Furthermore, insect cells coinfected with this vector plus another encoding influenza hemagglutinin produced recombinant hemagglutinin with a higher proportion of N-linked glycans containing terminal GIcNAc. These results demonstrated that overexpression of N-acetylglucosaminyhransferase I could extend thc glycoprotein processing capabilities of baculovirus-infected insect cells. Presumabl3; the N-acetylglucosaminyhransferase I activity in these cells had been increased enough to overcome the competing N-acetylglucosaminidase activit'~, which allowed these cells to produce a more complex N-linked glycan. Meanwhile, a new type of vector was specifically created to facilitate metabolic engineering in the baculovirusinsect cell system [51"]. Unlike conventional baculovirus vectors, which express foreign proteins during the very late phase of infection, these vectors were designed to express glycoprotein-processing enzymes immediately after infection. If the enzyme was active and could participate in the insect cell N-glycosylation pathway, it would contribute more efficiently to the processing of a recombinant glycoprotein expressed later in infection. This approach was first demonstrated with an 'immediate early' baculovirus vector designed to express mammalian [31,4-galactosyltransferase under the control of a baculoviral iel promoter [52"]. 131,4-galactosyltransferase was used because most evidence suggested that insect cells lacked this activity and the iel promoter was used
Engineering N-glycosylation pathways in the baeulovirus-insect cell system Jarvis, Kawar and Hollister
because it is active immediately after and throughout baculovirus infection [53]. This vector produced active 131,4-galactosyltransferase soon after infection, as expected, and led to the production of a baculovirus-encoded structural glycoprotein, gp64, with N-linked glycans containing galactose, gp64 was used as a model for this study because previous work had demonstrated that it is produced during the late phase of infection, that it has trimmed N-linked glycans with no galactose or sialic acid when produced by lepidopteran insect cells, and that it can acquire complex N-linked glycans when produced by mammalian cells [24,54]. Genetic transformation of the host cell is a more direct approach that can be used to engineer N-glycosylation in the baculovirus-insect cell system. In essence, it involves the permanent addition of a constitutively expressible gene encoding a mammalian glycoprotein-processing enzyme to an insect cell genome. Previous studies have described iel-based expression plasmids and coselection methods that can be used for this purpose [51",55,56]. These tools were used to produce a stably-transformed Sf9 cell derivative that constitutively expresses mammalian 131,4-galactosyltransferase [57°']. Genetic analysis of this new cell line, S~4GalT, showed that it has multiple copies of an iel-promoted mammalian ~l,4-galactosyltransferase gene integrated at a single genomic site. S ~ 4 G a l T cells supported normal levels of baculovirus replication and were able to produce gp64 with N-linked glycans that contained terminal galactose. Sf~4GalT cells also produced a galactosylated form of tissue plasminogen activator when infected with a conventional baculovirus vector encoding this human glycoprotein under the control of the polyhe&in promoter. T h e most recent efforts to engineer N-glycosylation in the baculovirus-insect cell system have combined the use of immediate early baculovirus vectors and stably-transformed insect cells (N-S Seo, JR Hollister, D L Jarvis, unpublished data). T h e basic idea was that this approach could be used to introduce two different mammalian glycoprotein processing enzymes into the system and extend the N-glycosylation pathway even further. An immediate early baculovirus vector designed to express mammalian oc2,6-sialyltransferase early in infection was isolated and used to infect S ~ 4 G a l T cells. Remarkably, the gp64 produced by this unique virus-host combination had N-linked glycans that contained both 13-1inked galactose and terminal oc2,6-1inked sialic acid.
Conclusions Recent studies have shown that N-glycosylation can be engineered to improve the foreign glycoprotein processing capabilities of the baculovirus-insect cell system. T h e addition of two mammalian processing enzymes allows this system to produce a 'mammalianized' foreign glycoprotein with sialylated complex N-linked glycans. Further consideration of these findings can provide new insight on insect
531
N-glycosylation and reveal future directions for metabolic engineering efforts in this system. All published attempts to engineer insect glycosylation pathways have involved the introduction of mammalian glycosyltransferases. T h e success of these efforts indicates that the other requirements for the action of these enzymes can be met by insect cells. T h e s e requirements include the proper spatial and temporal subcellular distributions of the transferases, the ability to synthesize and properly localize nucleotide sugar pools, and the ability to produce the requisite N-linked glycan acceptors. It seems unlikely that insect cells would have all these capabilities if they never produced complex N-linked glycans. Thus, their ability to produce complex N-linked glycans after the mere introduction of heterologous transferases supports the idea that insect cells already have the potential to produce complex glycans. As proposed in our working model of this pathway (Figure 1), the extremely low frequency of complex N-glycosylation in insect cells might be explained by the absence of sufficient levels of terminal transferases to compete with the higher levels of exoglycosidases in these cells. This predicts that the frequency of complex glycosylation would be increased by increasing transferase expression levels, which was the observed result. T h e s e considerations lead us to speculate further that the ability to carry out complex N-glycosylation might be essential during some specific stage(s) of insect development. Perhaps insects have genes encoding terminal glycosyltransferases, but they are expressed only during certain development stages and/or in certain tissues. T h e s e genes could be silent or poorly expressed during other stages of development, in nonspecialized tissues, and in established insect cell lines. Experiments designed to test this hypothesis will be a fruitful direction for future basic research on insect N-glycosylation. An important direction for future metabolic engineering efforts will be to manipulate the expression of endogenous insect cell glycoprotein-processing enzymes. One attractive idea is to repress the N-acetylglucosaminidase, which interferes with the production of complex N-glycans [45°]. Another is to induce endogenous processing enzymes, such as oc-mannosidases and N-acetylglucosaminyltransferases, which might be rate-limiting for efficient N-glycosylation of highly-expressed recombinant glycoproteins. Finally, it will be important to develop ways to induce the pathways responsible for biosynthesis and transport of nucleotide sugars in insect cells. These goals will require new information and tools derived from basic studies of insect cells at the molecular genetic level. Therefore, it will be important to extend the use of molecular genetics to study insect glycosylation pathways in the future.
Acknowledgements We thank Joel and Nancy Shaper (Johns Hopkins University), Jim Paulson (Cytel Corporation), and Karen Colley (University of Illinois at Chicago) for providing cDNAs and/or antibodies for our studies. We are grateful to NIH (GM49734) and NSF (BES-9814157) for financial support of our work.
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Expression vectors and delivery systems
References and recommended reading Papers of particular interest, published within the annual period of review, have been highlighted as: • of special interest °" of outstanding interest 1.
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MarzL, Altmann F, Staudacher E, Kubelka V: Protein glycosylation in insects. In Glycoproteins. Edited by Montreuil J, Vliegenthart J FG, Schachter H. Amsterdam: Elsevier; 1995:543-563.
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21. Hsu TA, Takahashi N, Tsukamoto ¥, Kato K, Shimada I, Masuda K, * Whiteley EM, Fan JQ, Lee YC, Betenbaugh MJ: Differential N-glycan patterns of secreted and intracellular IgG produced in Trichoplusia ni cells. J Bio/Chem 199?, 272:9062-9070. This paper demonstrates that murine IgG produced in baculovirus-infected High Five cells has N-linked glycans with elongated trimannosyl core structures, supporting the idea that insect cells can produce complex N-linked glycans. This study was an exceptionally thorough example of direct and quantitative structural analyses of the N-linked glycans isolated from a baculovirus-expressed recombinant glycoprotein. 22. Yeh JC, Seals JR, Murphy Cl, van Halbeek H, Cummings RD: Sitespecific N-glycosylation and oligosaccharide structures of recombinant HIV-1 gp120 derived from a baculovirus expression system. Biochemistry 1993, 32:11087-11099. 23. Knepper TP, Arbogast B, Schreurs J, Deinzer ML: Determination of the glycosylaUon patterns, disulfide linkages, and protein heterogeneities of baculovirus-expressed mouse interleukin-3 by mass spectrometry. Biochemistry 1992, 31:11651-11659. 24. JarvisDL, Finn EE: Biochemical analysis of the N-glycosylation pathway in baculovirus-infected lepidopteran insect cells. Virology 1995, 212:500-511. 25. Manneberg M, Friedlein A, Kurth H, Lahm HW, Fountoulakis M: Structural analysis and localization of the carbohydrate moieties of a soluble human interferon gamma receptor produced in baculovirus-infected insect cells. Protein Sci 1994, 3:30-38. 26. Wathen MW, Aeed PA, Elhammer AP: Characterization of oligosaccharide structures on a chimeric respiratory syncytial virus protein expressed in insect cell line Sf9. Biochemistry 1991, 30:2863-2868. 27. Chen W, Bahl OP: Recombinant carbohydrate and selenomethionyl variants of human choriogonadotropin. J Biol Chern 1991,266:8192-8197 28. Velardo MA, Bretthauer RK, Boutaud A, Reinhold B, Reinhold VN, Castellino FJ: The presence of UDP-N-acetylglucosamine:alpha-3D-mannoside beta 1,2-N-acetylglucosaminyltransferase I activity in Spodoptera frugiperda cells (IPLB-SF-21AE) and its enhancement as a result of baculovirus infection. J Bio/Chem 1993, 268:17902-17907. 29. Altmann F, Kornfeld G, Dalik T, Staudacher E, Glossl J: Processing of asparagine-linked oligosaccharides in insect cells. Nacetylglucosaminyltransferase I and II activities in cultured lepidopteran cells. Glycobiology 1993, 3:619-625. 30. Licari PJ, Jarvis DL, Bailey JE: Insect cell hosts for baculovirus expression vectors contain endogenous exogiycosidase activity. Biotechno/Prog 1993, 9:146-152. 31. Altmann F, Schwihla H, Staudacher E, Glossl J, Marz L: Insect cells contain an unusual, membrane-bound ~-N-acetylglucosaminidase probably involved in the processing of protein N-glycans. J Biol Chern 1995, 270:17344-17349. 32. Field MC, Wainwright U: Molecular cloning of eukaryotic glycoprotein and glycolipid glycosyltransferases: a survey. Glycobiology 1995, 5:463-472. 33. Fukuda M, Bierhuizen MFA, Nakayama J: Expression cloning of glycosyltransferases. Glycobiology 1996, 6:683-689. 34. Shaper NL, Shaper JH, Mouth JL, Fox JL, Chang H, Kirsch IR, Hollis GF: Bovine galactosyltransferase: identification of a clone by direct immunological screening of a cDNA expression library. Proc Nat/Acad Sci USA 1986, 83:1573-1577.
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35. Kerscher S, Albert S, Wucherpfennig D, Heisenberg M, Schneuwly S: Molecular and genetic analysis of the Drosophila mas-1 (mannosidase-1) gone which encodes a glycoprotein processing alpha 1,2-mannosidase. Dev Biol 1995, 168:613-626.
18. Grabenhorst E, Hofer B, Nimtz M, Jager V, Conradt HS: Biosynthesis and secretion of human interleukin 2 glycoprotein variants from baculovirus-infected Sf21 cells. Characterization of polypeptides and posttranslational modifications. Eur J Biochem 1993, 215:189-19?.
36. Foster JM, Yudkin B, Lockyer AE, Roberts DB: Cloning and sequence analysis of Gmll, a Drosophila melanogaster homologue of the cDNA encoding murine Golgi alphamannosidase II. Gone 1995, 154:188-186.
19. Hogeland KE Jr, Deinzer ML: Mass spectrometric studies on the Nlinked oligosaccharides of baculovirus-expressed mouse interleukin-3. Bio/Mass Spec 1994, 23:218-224. 20. VossT, Ergulen E, Ahorn H, Kubelka V, Sugiyama K, Maurer-Fogy I, Glossl J: Expression of human interferon omega 1 in Sf9 cells. No evidence for complex-type N-linked glycosylaUon or sialylation. Eur J Biochem 1993, 217:913-919.
37. Kawar Z, Herscovics A, Jarvis DL: Isolation and characterization of • an alpha 1,2-mannosidase cDNA from the lepidopteran insect cell line Sf9. Glycobiology 1997, 7:433-443. This paper presented the first molecular genetic evidence that the N-glycosylation pathway of lepidopteran insect cells includes class I (z-mannosidases. The sequence of an Sf9 class I o~-mannosidase cDNA was presented and the biochemical properties of the cDNA product were examined. The results indicated that Sf9 cells encode a typical class I (x-mannosidase.
Engineering N-glycosylation pathways in the baculovirus-insect cell system Jarvis, Kawar and Hollister
38. Jarvis DL, Bohlmeyer DA, Liao YF, Lomax KK, Merkle RK, Weinkauf C, • Moremen KW: Isolation and characterization of a class II alphamannosidase cDNA from lepidopteran insect cells. G/ycobio/ogy 1997, 7:113-127 This paper describes the first cDNA clone encoding a lepidopteran insect cell glycoprotein-processing enzyme. DNA sequence analysis and biochemical characterization of this cDNA product suggested that it encoded a typical class II o(-mannosidase. Subsequent data, however, suggest that it might encode a member of a new class of (z-mannosidases that trim mannose residues directly from Man5GIcNAc 2 (see references [42,43*]).
core glycans containing terminal GIc.NAc and galactose. This work provided another excellent example of direct and quantitative structural analyses of the N-linked glycans from a recombinant protein produced in baculovirusinfected insect cells. 47.
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39. AItmann F, Marz L: Processing of asparagine-linked oligosaccharides in insect cells: evidence for o~-mannosidase II. G/ycoconj J 1995, 12:150-155. 40. Ren J, Oastellino FJ, Bretthauer RK: Purification and properties of • alpha-mannosidase II from Golgi-like membranes of baculovirusinfected Spodoptera frugiperda (IPLB-SF-21 AE) cells. Biochem J 1997, 15:951 +956. A class II o~-mannosidase was purified to homogeneity from Sf9 cells and its biochemical properties were characterized. This enzyme converted GIcNAcMan5GIcNAc 2 to GIcNAcMan3GIcNAc2, as expected of a class II cemannosidase. These data supported the idea that the Sf9 cell N-glycosylation pathway includes a typical class II cemannosidase, distinct from the unusual cc-mannosidase encoded by the cDNA described in reference [38°]. 41.
42.
Ren J, Bretthauer RK, Castellino FJ: Purification and properties of a Golgi-derived (alpha 1,2)-rnannosidase-I from baculovirusinfected lepidopteran insect cells (IPLB-SF21AE) with preferential activity toward rnannose 6-N-acetylglucosarnine 2. Biochemistry 1995, 34:2489-2495. Bonay P, Hughes RC: Purification and characterization of a novel bread-specificity (alpha t - 2 , alpha 1 - 3 and alpha 1-6) mannosidase from rat liver. Eur J Biochem 1991, 197:229-238.
43. •
Chui D, Oh-Eda M, Liao YF, Panneerselvam K, Lal A, Marek KW, Freeze HH, Moremen KW, Fukuda MN, Marth JD: Alphamannosidase-II deficiency results in dyserythropoiesis and unveils an alternate pathway in oligosaccharide biosynthesis. Ceil 1997, 90:157-167. This paper shows that an unusual mammalian cz-mannosidase, perhaps the one described in reference [42], can function in N-glycosylation. Emerging evidence suggests that the enzyme encoded by the Sf9 cDNA described in reference [38 °] might play a similar role in insect N-glycosylation. 44. van Die I, van Tetering A, Bakker H, van den Eijnden DH, Joziasse DH: • Glycosylation in lepidopteran insect cells: identification of a I}1,4N-acetylgalactosaminyltransferase involved in the synthesis of complex-type oligosaccharide chains. G/ycobio/ogy 1996, 6:157-164. This paper provided the first enzymatic data indicating that lepidopteran insect cells have N-acetylgalactosaminykransferase and galactosyltransferase activities. The authors found that baculovirus infection decreased the levels of these transferase activities, but not the levels of competing exoglycosidases. Thus, they concluded that insect cells have the enzymatic potential to produce complex N-glycans, but usually fail to do so because baculovirus infection decreases the transferase activities found in these cells. 45. Wagner R, Geyer H, Geyer R, Klenk H-D: N-acetyl-~• glucosaminidase accounts for differences in glycosylation of influenza virus hernagglutinin expressed in insect cells from a baculovirus vector. J Viro! 1996, 70:4103-4109. This paper explained why one specific lepidopteran insect cell line routinely produces glycoproteins with more extended N-linked glycans. The authors found that E. acrea cells lack the N-acetylglucosaminidase activity found in other insect cell lines, for example, Sf9. This observation revealed the competitive nature of the N-acetylglucosaminyltransferase I and N-acetylglucosaminidase activities in insect cells. It also led the authors to conclude that insect cells have a conventional N-glycosylation pathway that produces GIcNAcMan3GIcNAc2, which is usually converted to a trimannosyl core in insect cells which have N-acetylglucosaminidase activity. 46. Ogonah OW, Freedman RB, Jenkins N, Patel K, Rooney B: Isolation • and characterization of an insect cell line able to perform complex N-linked glycosylation on recombinant proteins. Bio/Techno/ogy 1996, 14:197-202. This paper provided the first published evidence that E. acrea cells could produce recombinant glycoproteins with extended N-linked trimannosyl
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49. Roth J, Kempf A, Reuter G, Schauer R, Gehring W: Occurence of sialic acids in Drosophila melanogaster. Science 1992, 256:673675. 50. Wagner R, Liedtke S, Kretzschmar E, Geyer H, Geyer R, Klenk HD: •. Elongation of the N-glycans of fowl plague virus hernagglutinin expressed in Spodoptera frugiperda (sfg) cells by coexpression of human ~l,2-N-acetylglucosaminyltransferase I. G/ycobio/ogy 1996, 6:165-175. This was the first published example of engineering N-glycosylation in the baculovirus-insect system. The authors' approach was to use a conventional baculovirus vector to introduce a mammalian glycosyltransferase into the system. When Sf9 cells were coinfected with another baculovirus vector encoding a recombinant glycoprotein, that glycoprotein acquired extended N-linked trimannosyl core structures containing terminal GIcNAc. 51. Jarvis DL, Weinkauf C, Guarino LA: Immediate early baculovirus • vectors for foreign gene expression in transformed or infected insect cells. Protein Expr Purif 1996, 8:191-203. This paper described the construction and characterization of new baculovirus vectors designed to express foreign genes during the early phases of baculovirus infection or in uninfected insect cells. These vectors are ideal tools for metabolic engineering of protein processing pathways in the baculovirus-insect cell system. 52. Jarvis DL, Finn EE: Modifying the insect cell N-glycosylation • o pathway with immediate early baculovirus expression vectors. Nat Biotechnol 1996, 14:1288-1292. This was the first published example of using novel immediate early baculovirus vectors to engineer N-glycosylation in the baculovirus-insect cell system. A recombinant baculovirus containing a mammalian glycosyltransferase cDNA under the control of a baculoviral early promoter was isolated. Infection of insect cells with this virus resulted in expression of the glycosyltransferase early in infection and extended N-glycosylation of gp64, a foreign glycoprotein expressed later in infection. 53.
Guarino LA, Summers MD: Nucleotide sequence and temporal expression of a baculovirus regulatory gene. J Virol 1987, 61:2091-2099.
54. Jarvis DL, Garcia A Jr: Biosynthesis and processing of the Autographa californica nuclear polyhedrosis virus gp64 protein. Virology 1994, 205:300-313. 55. Jarvis DL, Fleming JA, Kovacs GR, Summers MD, Guarino LA: Use of early baculovirus promoters for continuous expression and efficient processing of foreign gene products in stably transformed lepidopteran cells. Bio/Technology 1990, 8:950-955. 56. Jarvis DL, Guarino LA: Continuous foreign gene expression in transformed lepidopteran insect cells. In Bacu/ovirus Expression Protocols. Edited by Richardson CD. Clifton, NJ: Humana Press; 1995:187-202. 57. o.
Hollister JR, Shaper JH, Jarvis DL: Stable expression of mammalian 131,4-galactosyltransferase extends the N-glycosylation pathway in insect cells. G/ycobio/ogy 1997, 8:473-490. This paper describes the isolation and characterization of a new Sf9-derived cell line stably-transformed to provide extended processing of N-linked glycoproteins expressed by baculovirus vectors. This is the first example of engineering the insect cell N-glycosylation pathway by direct modification of an insect cell genome. The SF~4GalT cells produced more extensively processed foreign glycoproteins, including a human gene product that was expressed under polyhedrin control during the very late phase of infection by a conventional baculovirus vector.
Engineering N-glycosylation pathways in the baculovirus-insect cell system Donald L Jarvis, Ziad S Kawar and Jason R Hollister The inability to produce eukaryotic glycoproteins with complex N-linked glycans is a major limitation of the baculovirus-insect cell expression system. Recent studies have demonstrated that metabolic engineering can be used to extend the glycoprotein processing capabilities of lepidopteran insect cells. This approach is being used to develop new baculovirusinsect cell expression systems that can produce more authentic recombinant glycoproteins and obtain new information on insect N-glycosylation pathways.
Addresses Department of Molecular Biology, University of Wyoming, PO Box 3944, Laramie, Wyoming 82071-3944, USA Correspondence: Donald J Jarvis; e-mail: [email protected]
Current Opinion in Biotechnology 1998, 9:528-533 http://biomednet.com/elecref/0958166900900528 :c: Current Biology Ltd ISSN 0958-1669
Abbreviations GIc
glucose
GIcNAc N-acetylglucosamine Man mannose Introduction Recombinant baculoviruses are frequently used to prodt,ce foreign proteins in lepidopteran insect cells, which usually serve as the laboratory hosts for these viruses [1,2]. One important feature of the bacuh)virus-inscct cell system is that it can produce glycosylated proteins. (;lycosylation is a comnqon covalent chemical m()dification that can influence many prntein properties, including intracelh, lar trafficking, biological function, and biochemical stabilit> Most evidence, however, suggests that the protein glycosylation pathways of lcpidopteran insect cells differ from these of higher et,karyotes [3",4]. Recent studies have described metabolic engineering apprnaches that can be used to extend the glycoprotein processing capabilities of the bacuh)virus-insect cell system. In this review, we will summarize some of the evidence leading to our current understanding of insect N-glycosylation, present a comprehensive model of insect N-g;lycosylation, and discuss current efforts to improve N-glycosylation in the baculovirus-inscct cell expression system. Protein N-glycosylation T h e protein N-glycosylation reactions of mammalian cells have been reviewed [5-7] and are summarized in Figure 1. T h e mammalian N-glycosylation pathway begins with the transfer of a preassembled oligosaccharide, Glc.~-Mang-GlcNAc z, to a nascent polypeptide. This is followed by trimming of the glucose residues to produce a 'high mannose' glycan, MangGlcNAc z. Some
N-linked glycans are processed no further and rcmain in this high mannose form. Others are processed by class I o~mannosidases, which remove four mannose residues to produce lklansGlcNAcz, and N-acetylglucosaminyltransferase I, which adds a terminal GIcNAc residue to produce GIcNAcMansGlcNAc z. Addition of the GIcNAc permits o~-mannnsidase II to remove two morc mannosc rcsiducs to produce GIcNAcMan~GIcNAcz, a trimmed intermediate that can be converted to 'complex' glycans by various glycosyltransferases, including N-acetylglucosaminyltransfcrases, fucosyltransferases, galactnsyhransferascs, N-acetylgalactosaminyhransferases, and sialvltransfcrases. N - g l y c o s y l a t i o n in insect cells Historically, two diffcrent approaches have been used to study N-glycosylation in insect cells. One was to determine the structures of N-linked glycans on glycoproteins produced by insect cells. T h e other was to assay insect cell extracts for specific glycoprotein processing activities. Recently, these biochemical approaches have been supplcmented by a molecular genetic approach. Selected biochemical studies An early survey suggested that invertebrates lacked sialic acids [81. lmtcr, it was shown that Sindbis virus glycoproreins were sialylated by mammalian but not mosquito cells, anti it was found that mosquito cell extracts lacked both sialic acid and siaMtransferase activity [9]. Structural analyses of native mosquito cell-surface glycoproteins suggested that they had only high mannose N-linked glycans [10] and activity assays indicatcd that these cells lacked N-acetylglucosaminyltransfcrascs, galactosyhransferases, and sialyltransferases [1 1]. Subseqt,ent structural studies revealed that dipteran insect cells could produce the trimannosvl core structure, Man.~GlcNAc z, but not complex N-linked glycans [12]. Together, these observations led to the general conclusion that insect cells can produce N-linked glycoproteins with high mannose glycans and trim them to produce trimannosvl core structures, but they lack the glycosyhransferases needed to convcrt the trimmed glycans to complex structures. This conclusion was derived mainly from studies of dipteran insect cells, but structural studies of N-linked glycans from native lepidopteran insect cell glycoproteins suggested that these cells had the same capabilities [13-16]. Further information on protein N-glycosylation in lepidopteran insect cells came with the developnaent of baculovims expression vectors, which led to widespread use of these cells for recombinant glycoprotein production. One of the first direct analyses of the N-linked glycans from a recombinant glycoprotein produced by the baeulovirt,s-insect cell system demonstrated that they had high mannose or
Engineering N-glycosylation pathways in the baculovirus-insect cell system Jarvis, Kawar and Hollister
529
Figure 1
.u , C)-~
~ A S N
~
[~ ~z-mannosidaseI (RER) [I o~-mannosidasel(Golgi)
=Fucose
O = Galactose O = G.atNAc.,
L-.r @ A S N
~
N-aeetylglucosaminyltransferaseI ASN H cz-mannosidaseII ~7 Fucosyltransferases
, N-acetylglucosaminidase
[ ~
~ A S N ~ ~
, N-acetylglucosaminyl transferase II ~k~
~
~ A S N
Trimannosyl core
NialAy/tetaYIngs faleaCts°eSY It ransferas ~ / / /
~
Current Opinion in Biotechnology
A
S
N
ASN
~
~
Giall~l~trO::lf~raans::rases
A
S
N
Complex N-linked glycans
A comprehensive working model of insect N-glycosylation pathways. The model proposes that there are multiple branches for the processing of Nlinked glycans on newly synthesized insect glycoproteins. Depending upon the levels of competing processing enzyme activities in a specific insect cell type under a specific set of conditions, high mannose, trimannosyl core, or complex N-linked glycans may be produced. We also propose that the net result of the N-glycosylation pathway is strongly influenced by the structural characteristics of the glycoprotein being processed. This model stems from observations described in many different studies from many different labs, including the representative studies cited in this review, as well as additional studies cited in references [3 °] and [4]. GalNAc, N-acetylgalactosamine; RER, rough endoplasmic reticulum.
od,6-fucosylated trimannosyl core structures [17]. Subsequent structural analyses of N-linked glycans from other recombinant glycoproteins produced by this system provided analogous results [18-20,Z1",22-27], The inability to produce complex N-linked glycans suggested that bacuh)virus-infected lepidopteran insect cells lacked glycosyltransferases. If they lacked all glycosyltransferases, however, including N-acetylglucosaminyltransferase I, these cells would be unable to convert MansGlcNAc 2 to GlcNAcMansGlcNAc 2, which is required for o~-mannosidase II to complete the trimming reactions [5,6]. Thus, insect cells would need a different way to produce trimannosyl core structures. Ultimately, N-acetylglucosaminyl-
transferase 1 activity was found in lepidopteran insect cells [28,29] and this indicated that mannose trimming could proceed by the conventional pathway. However, this still failed to explain how terminal GIcNAc was removed from GIcNAcMan,~GIcNAc2 to produce the trimannosyl core. A possible explanation arose from the finding that insect cells have high levels of N-acetylglucosaminidase activity, which could remove terminal GlcNAc in a degradative fashion [30]. A more elegant explanation arose with the demonstration that some lepidopteran insect cells have a membranebound N-acetylglucosaminidase that might fimction as a processing enzyme that converts GIcNAcMan3GIcNAc z to Man.~GlcNAc z [31].
530 Expressionvectorsand deliverysystems
Molecular genetic studies More recentl'~, molecular genetics has been used to study insect cell N-glycosylation pathways. Molecular genetics can reveal which glycoprotein processing enzymes are encoded by insect cells and establish the conditions under which these genes are expressed. Furthermore, genes isolated in these studies can be used to manipulate the expression of endogenous glycoprotein processing enzymes, which is one important approach to metabolic engineering. Molecular cloning of genes encoding glycoprotein processing enzymes is hindered by the fact that these enzymes are usually fotmd in only minute amounts in cells [32,33]. Thus, the first such gene, encoding 131,4-galactosyhransferase, was isolated from mammalian cells in 1986 [34]. T h e first insect cell genes were putative c~-mannosidase cDNAs isolated from Drosophila in 1995 [35,36] and the first lepidopteran insect cell genes were 0~-mannosidase cDNAs isolated from Sf9 cells in 1997 [37",38"]. Sequence analysis of the Sf9 cDNAs and biochemical studies of their protein products provided the first molecular genetic evidence that the Sf9 N-glycosylation pathway includes typical class I and II 0t-mannosidases. These results were consistent with previous structural data (above) and biochemical data demonstrating the presence of these enzymes in Sf9 extracts [39,40°,41]. Our ongoing studies on the natural substratc specificity of the enzyme encoded by the Sf9 ot-mannosidase I1 cDNA, however, suggest that it requires cobalt and acts preferentially on MansGlcNAc 2 (DL Jarvis and KW Moremen, unpublished data). This enzyme, therefore, is probably not the Sf9 c~-mannosidase II, but a functional homolog of an unusual 0~-mannosidase that mediates an alternate trimming pathway in knockout mice lacking 0~-mannosidase II [42,43"].
A comprehensive model of insect N-glycosylation Whereas most evidence indicates that insect cells can produce only N-linked glycans with high mannose or fucosylated trimannosyl core structures, some evidence argues against this conclusion. Some insect cells have Nacetylglucosaminyhransferase II, [~l,4-galactosyhransferase, and [31,4-N-acetylgalactosaminyltransferase activities, all of which are involved in producing complex N-linked glycans [28,29,44"]. Furthermore, some baculovirus-infected insect cells can produce recombinant glycoproteins with elongated trimannosyl core structures containing terminal GIcNAc or galactose [21°,45",46°], and one recombinant glycoprotein acquired complex biantennary N-linked glycans containing penultimate galactose and terminal sialic acid [47,48]. Finally, it has been reported that Drosophilaembryos contain sialic acid [49]. These findings demonstrate that the insect N-glycosylation pathway cannot be described in such simple terms. A more comprehensive model is needed to explain how insect cells can produce various types of N-linked glycans. The model proposed in Figure 1 presents insect cell N-glycosylation as a single pathway with multiple potential end-products. A critical feature of this model is that the structure of the N-linked glycan on a specific glycoprotein
produced by a specific insect cell under specific conditions is determined by the relative levels of competing processing activities and the structure of the glycoprotein being processed. Thus, this model includes the flexibility needed to explain how insect cells can produce various types of Nlinked glycans ranging from high mannose to complex. As production of complex, sialylatcd N-linked glycans is such a rare event in insect cells, we have speculated that it is protein-dependent and occurs only with specific recombinant proteins that are unusually poor substrates for the N-acetylglucosaminidase and/or unusually good substrates for the low-level glycosyltransferasc activities in these cells [24]. An important observation in the context of this model is that the levels of N-acetylgalactosaminyhransferase and galactosyltransferase activities in lepidopteran insect cells are reduced by baculovirus infection [44°].
E n g i n e e r i n g insect cell N-glycosylation pathways T h e inability of the baculovirus-insect cell expression system to routinely produce recombinant glycoproteins with complex N-linked glycans led to efforts to engineer the glycoprotein processing capabilities of this system. In the first example of this, a conventional baculovirus expression vector was used to introduce a human processing enzyme into the system [50"]. This vector expressed human N-acetylglucosaminyltransferase I under the control of the polyhedrin promoter, which significantly increased the level of N-acetylglucosaminyltransfcrase I activity during infection. Furthermore, insect cells coinfected with this vector plus another encoding influenza hemagglutinin produced recombinant hemagglutinin with a higher proportion of N-linked glycans containing terminal GIcNAc. These results demonstrated that overexpression of N-acetylglucosaminyhransferase I could extend thc glycoprotein processing capabilities of baculovirus-infected insect cells. Presumabl3; the N-acetylglucosaminyhransferase I activity in these cells had been increased enough to overcome the competing N-acetylglucosaminidase activit'~, which allowed these cells to produce a more complex N-linked glycan. Meanwhile, a new type of vector was specifically created to facilitate metabolic engineering in the baculovirusinsect cell system [51"]. Unlike conventional baculovirus vectors, which express foreign proteins during the very late phase of infection, these vectors were designed to express glycoprotein-processing enzymes immediately after infection. If the enzyme was active and could participate in the insect cell N-glycosylation pathway, it would contribute more efficiently to the processing of a recombinant glycoprotein expressed later in infection. This approach was first demonstrated with an 'immediate early' baculovirus vector designed to express mammalian [31,4-galactosyltransferase under the control of a baculoviral iel promoter [52"]. 131,4-galactosyltransferase was used because most evidence suggested that insect cells lacked this activity and the iel promoter was used
Engineering N-glycosylation pathways in the baeulovirus-insect cell system Jarvis, Kawar and Hollister
because it is active immediately after and throughout baculovirus infection [53]. This vector produced active 131,4-galactosyltransferase soon after infection, as expected, and led to the production of a baculovirus-encoded structural glycoprotein, gp64, with N-linked glycans containing galactose, gp64 was used as a model for this study because previous work had demonstrated that it is produced during the late phase of infection, that it has trimmed N-linked glycans with no galactose or sialic acid when produced by lepidopteran insect cells, and that it can acquire complex N-linked glycans when produced by mammalian cells [24,54]. Genetic transformation of the host cell is a more direct approach that can be used to engineer N-glycosylation in the baculovirus-insect cell system. In essence, it involves the permanent addition of a constitutively expressible gene encoding a mammalian glycoprotein-processing enzyme to an insect cell genome. Previous studies have described iel-based expression plasmids and coselection methods that can be used for this purpose [51",55,56]. These tools were used to produce a stably-transformed Sf9 cell derivative that constitutively expresses mammalian 131,4-galactosyltransferase [57°']. Genetic analysis of this new cell line, S~4GalT, showed that it has multiple copies of an iel-promoted mammalian ~l,4-galactosyltransferase gene integrated at a single genomic site. S ~ 4 G a l T cells supported normal levels of baculovirus replication and were able to produce gp64 with N-linked glycans that contained terminal galactose. Sf~4GalT cells also produced a galactosylated form of tissue plasminogen activator when infected with a conventional baculovirus vector encoding this human glycoprotein under the control of the polyhe&in promoter. T h e most recent efforts to engineer N-glycosylation in the baculovirus-insect cell system have combined the use of immediate early baculovirus vectors and stably-transformed insect cells (N-S Seo, JR Hollister, D L Jarvis, unpublished data). T h e basic idea was that this approach could be used to introduce two different mammalian glycoprotein processing enzymes into the system and extend the N-glycosylation pathway even further. An immediate early baculovirus vector designed to express mammalian oc2,6-sialyltransferase early in infection was isolated and used to infect S ~ 4 G a l T cells. Remarkably, the gp64 produced by this unique virus-host combination had N-linked glycans that contained both 13-1inked galactose and terminal oc2,6-1inked sialic acid.
Conclusions Recent studies have shown that N-glycosylation can be engineered to improve the foreign glycoprotein processing capabilities of the baculovirus-insect cell system. T h e addition of two mammalian processing enzymes allows this system to produce a 'mammalianized' foreign glycoprotein with sialylated complex N-linked glycans. Further consideration of these findings can provide new insight on insect
531
N-glycosylation and reveal future directions for metabolic engineering efforts in this system. All published attempts to engineer insect glycosylation pathways have involved the introduction of mammalian glycosyltransferases. T h e success of these efforts indicates that the other requirements for the action of these enzymes can be met by insect cells. T h e s e requirements include the proper spatial and temporal subcellular distributions of the transferases, the ability to synthesize and properly localize nucleotide sugar pools, and the ability to produce the requisite N-linked glycan acceptors. It seems unlikely that insect cells would have all these capabilities if they never produced complex N-linked glycans. Thus, their ability to produce complex N-linked glycans after the mere introduction of heterologous transferases supports the idea that insect cells already have the potential to produce complex glycans. As proposed in our working model of this pathway (Figure 1), the extremely low frequency of complex N-glycosylation in insect cells might be explained by the absence of sufficient levels of terminal transferases to compete with the higher levels of exoglycosidases in these cells. This predicts that the frequency of complex glycosylation would be increased by increasing transferase expression levels, which was the observed result. T h e s e considerations lead us to speculate further that the ability to carry out complex N-glycosylation might be essential during some specific stage(s) of insect development. Perhaps insects have genes encoding terminal glycosyltransferases, but they are expressed only during certain development stages and/or in certain tissues. T h e s e genes could be silent or poorly expressed during other stages of development, in nonspecialized tissues, and in established insect cell lines. Experiments designed to test this hypothesis will be a fruitful direction for future basic research on insect N-glycosylation. An important direction for future metabolic engineering efforts will be to manipulate the expression of endogenous insect cell glycoprotein-processing enzymes. One attractive idea is to repress the N-acetylglucosaminidase, which interferes with the production of complex N-glycans [45°]. Another is to induce endogenous processing enzymes, such as oc-mannosidases and N-acetylglucosaminyltransferases, which might be rate-limiting for efficient N-glycosylation of highly-expressed recombinant glycoproteins. Finally, it will be important to develop ways to induce the pathways responsible for biosynthesis and transport of nucleotide sugars in insect cells. These goals will require new information and tools derived from basic studies of insect cells at the molecular genetic level. Therefore, it will be important to extend the use of molecular genetics to study insect glycosylation pathways in the future.
Acknowledgements We thank Joel and Nancy Shaper (Johns Hopkins University), Jim Paulson (Cytel Corporation), and Karen Colley (University of Illinois at Chicago) for providing cDNAs and/or antibodies for our studies. We are grateful to NIH (GM49734) and NSF (BES-9814157) for financial support of our work.
532
Expression vectors and delivery systems
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17. Kuroda K, Geyer H, Geyer R, Doerfler W, Klenk HD: The oligosaccharides of influenza virus hemagglutinin expressed in insect cells by a baculovirus vector. Virology 1990, 174:418-429.
35. Kerscher S, Albert S, Wucherpfennig D, Heisenberg M, Schneuwly S: Molecular and genetic analysis of the Drosophila mas-1 (mannosidase-1) gone which encodes a glycoprotein processing alpha 1,2-mannosidase. Dev Biol 1995, 168:613-626.
18. Grabenhorst E, Hofer B, Nimtz M, Jager V, Conradt HS: Biosynthesis and secretion of human interleukin 2 glycoprotein variants from baculovirus-infected Sf21 cells. Characterization of polypeptides and posttranslational modifications. Eur J Biochem 1993, 215:189-19?.
36. Foster JM, Yudkin B, Lockyer AE, Roberts DB: Cloning and sequence analysis of Gmll, a Drosophila melanogaster homologue of the cDNA encoding murine Golgi alphamannosidase II. Gone 1995, 154:188-186.
19. Hogeland KE Jr, Deinzer ML: Mass spectrometric studies on the Nlinked oligosaccharides of baculovirus-expressed mouse interleukin-3. Bio/Mass Spec 1994, 23:218-224. 20. VossT, Ergulen E, Ahorn H, Kubelka V, Sugiyama K, Maurer-Fogy I, Glossl J: Expression of human interferon omega 1 in Sf9 cells. No evidence for complex-type N-linked glycosylaUon or sialylation. Eur J Biochem 1993, 217:913-919.
37. Kawar Z, Herscovics A, Jarvis DL: Isolation and characterization of • an alpha 1,2-mannosidase cDNA from the lepidopteran insect cell line Sf9. Glycobiology 1997, 7:433-443. This paper presented the first molecular genetic evidence that the N-glycosylation pathway of lepidopteran insect cells includes class I (z-mannosidases. The sequence of an Sf9 class I o~-mannosidase cDNA was presented and the biochemical properties of the cDNA product were examined. The results indicated that Sf9 cells encode a typical class I (x-mannosidase.
Engineering N-glycosylation pathways in the baculovirus-insect cell system Jarvis, Kawar and Hollister
38. Jarvis DL, Bohlmeyer DA, Liao YF, Lomax KK, Merkle RK, Weinkauf C, • Moremen KW: Isolation and characterization of a class II alphamannosidase cDNA from lepidopteran insect cells. G/ycobio/ogy 1997, 7:113-127 This paper describes the first cDNA clone encoding a lepidopteran insect cell glycoprotein-processing enzyme. DNA sequence analysis and biochemical characterization of this cDNA product suggested that it encoded a typical class II o(-mannosidase. Subsequent data, however, suggest that it might encode a member of a new class of (z-mannosidases that trim mannose residues directly from Man5GIcNAc 2 (see references [42,43*]).
core glycans containing terminal GIc.NAc and galactose. This work provided another excellent example of direct and quantitative structural analyses of the N-linked glycans from a recombinant protein produced in baculovirusinfected insect cells. 47.
Davidson DJ, Castellino FJ: Structures of the asparagine-289-1inked oligosaccharides assembled on recombinant human plasminogen expressed in a Mamestra brassicae cell line (IZDMBO503). Biochemistry 1991, 30:6689-6696.
48.
Davidson DJ, Fraser M J, Castellino FJ: Oligosaccharide processing in the expression of human plasminogen cDNA by lepidopteran insect (Spodoptera frugiperda) cells. Biochemistry 1990, 29:55845590.
39. AItmann F, Marz L: Processing of asparagine-linked oligosaccharides in insect cells: evidence for o~-mannosidase II. G/ycoconj J 1995, 12:150-155. 40. Ren J, Oastellino FJ, Bretthauer RK: Purification and properties of • alpha-mannosidase II from Golgi-like membranes of baculovirusinfected Spodoptera frugiperda (IPLB-SF-21 AE) cells. Biochem J 1997, 15:951 +956. A class II o~-mannosidase was purified to homogeneity from Sf9 cells and its biochemical properties were characterized. This enzyme converted GIcNAcMan5GIcNAc 2 to GIcNAcMan3GIcNAc2, as expected of a class II cemannosidase. These data supported the idea that the Sf9 cell N-glycosylation pathway includes a typical class II cemannosidase, distinct from the unusual cc-mannosidase encoded by the cDNA described in reference [38°]. 41.
42.
Ren J, Bretthauer RK, Castellino FJ: Purification and properties of a Golgi-derived (alpha 1,2)-rnannosidase-I from baculovirusinfected lepidopteran insect cells (IPLB-SF21AE) with preferential activity toward rnannose 6-N-acetylglucosarnine 2. Biochemistry 1995, 34:2489-2495. Bonay P, Hughes RC: Purification and characterization of a novel bread-specificity (alpha t - 2 , alpha 1 - 3 and alpha 1-6) mannosidase from rat liver. Eur J Biochem 1991, 197:229-238.
43. •
Chui D, Oh-Eda M, Liao YF, Panneerselvam K, Lal A, Marek KW, Freeze HH, Moremen KW, Fukuda MN, Marth JD: Alphamannosidase-II deficiency results in dyserythropoiesis and unveils an alternate pathway in oligosaccharide biosynthesis. Ceil 1997, 90:157-167. This paper shows that an unusual mammalian cz-mannosidase, perhaps the one described in reference [42], can function in N-glycosylation. Emerging evidence suggests that the enzyme encoded by the Sf9 cDNA described in reference [38 °] might play a similar role in insect N-glycosylation. 44. van Die I, van Tetering A, Bakker H, van den Eijnden DH, Joziasse DH: • Glycosylation in lepidopteran insect cells: identification of a I}1,4N-acetylgalactosaminyltransferase involved in the synthesis of complex-type oligosaccharide chains. G/ycobio/ogy 1996, 6:157-164. This paper provided the first enzymatic data indicating that lepidopteran insect cells have N-acetylgalactosaminykransferase and galactosyltransferase activities. The authors found that baculovirus infection decreased the levels of these transferase activities, but not the levels of competing exoglycosidases. Thus, they concluded that insect cells have the enzymatic potential to produce complex N-glycans, but usually fail to do so because baculovirus infection decreases the transferase activities found in these cells. 45. Wagner R, Geyer H, Geyer R, Klenk H-D: N-acetyl-~• glucosaminidase accounts for differences in glycosylation of influenza virus hernagglutinin expressed in insect cells from a baculovirus vector. J Viro! 1996, 70:4103-4109. This paper explained why one specific lepidopteran insect cell line routinely produces glycoproteins with more extended N-linked glycans. The authors found that E. acrea cells lack the N-acetylglucosaminidase activity found in other insect cell lines, for example, Sf9. This observation revealed the competitive nature of the N-acetylglucosaminyltransferase I and N-acetylglucosaminidase activities in insect cells. It also led the authors to conclude that insect cells have a conventional N-glycosylation pathway that produces GIcNAcMan3GIcNAc2, which is usually converted to a trimannosyl core in insect cells which have N-acetylglucosaminidase activity. 46. Ogonah OW, Freedman RB, Jenkins N, Patel K, Rooney B: Isolation • and characterization of an insect cell line able to perform complex N-linked glycosylation on recombinant proteins. Bio/Techno/ogy 1996, 14:197-202. This paper provided the first published evidence that E. acrea cells could produce recombinant glycoproteins with extended N-linked trimannosyl
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49. Roth J, Kempf A, Reuter G, Schauer R, Gehring W: Occurence of sialic acids in Drosophila melanogaster. Science 1992, 256:673675. 50. Wagner R, Liedtke S, Kretzschmar E, Geyer H, Geyer R, Klenk HD: •. Elongation of the N-glycans of fowl plague virus hernagglutinin expressed in Spodoptera frugiperda (sfg) cells by coexpression of human ~l,2-N-acetylglucosaminyltransferase I. G/ycobio/ogy 1996, 6:165-175. This was the first published example of engineering N-glycosylation in the baculovirus-insect system. The authors' approach was to use a conventional baculovirus vector to introduce a mammalian glycosyltransferase into the system. When Sf9 cells were coinfected with another baculovirus vector encoding a recombinant glycoprotein, that glycoprotein acquired extended N-linked trimannosyl core structures containing terminal GIcNAc. 51. Jarvis DL, Weinkauf C, Guarino LA: Immediate early baculovirus • vectors for foreign gene expression in transformed or infected insect cells. Protein Expr Purif 1996, 8:191-203. This paper described the construction and characterization of new baculovirus vectors designed to express foreign genes during the early phases of baculovirus infection or in uninfected insect cells. These vectors are ideal tools for metabolic engineering of protein processing pathways in the baculovirus-insect cell system. 52. Jarvis DL, Finn EE: Modifying the insect cell N-glycosylation • o pathway with immediate early baculovirus expression vectors. Nat Biotechnol 1996, 14:1288-1292. This was the first published example of using novel immediate early baculovirus vectors to engineer N-glycosylation in the baculovirus-insect cell system. A recombinant baculovirus containing a mammalian glycosyltransferase cDNA under the control of a baculoviral early promoter was isolated. Infection of insect cells with this virus resulted in expression of the glycosyltransferase early in infection and extended N-glycosylation of gp64, a foreign glycoprotein expressed later in infection. 53.
Guarino LA, Summers MD: Nucleotide sequence and temporal expression of a baculovirus regulatory gene. J Virol 1987, 61:2091-2099.
54. Jarvis DL, Garcia A Jr: Biosynthesis and processing of the Autographa californica nuclear polyhedrosis virus gp64 protein. Virology 1994, 205:300-313. 55. Jarvis DL, Fleming JA, Kovacs GR, Summers MD, Guarino LA: Use of early baculovirus promoters for continuous expression and efficient processing of foreign gene products in stably transformed lepidopteran cells. Bio/Technology 1990, 8:950-955. 56. Jarvis DL, Guarino LA: Continuous foreign gene expression in transformed lepidopteran insect cells. In Bacu/ovirus Expression Protocols. Edited by Richardson CD. Clifton, NJ: Humana Press; 1995:187-202. 57. o.
Hollister JR, Shaper JH, Jarvis DL: Stable expression of mammalian 131,4-galactosyltransferase extends the N-glycosylation pathway in insect cells. G/ycobio/ogy 1997, 8:473-490. This paper describes the isolation and characterization of a new Sf9-derived cell line stably-transformed to provide extended processing of N-linked glycoproteins expressed by baculovirus vectors. This is the first example of engineering the insect cell N-glycosylation pathway by direct modification of an insect cell genome. The SF~4GalT cells produced more extensively processed foreign glycoproteins, including a human gene product that was expressed under polyhedrin control during the very late phase of infection by a conventional baculovirus vector.