Expression of a functional Drosophila melanogasterN-acetylneuraminic acid (Neu5Ac) phosphate synthase gene: evidence for endogenous sialic acid biosynthetic ability in insects

Kim, Kil-Dong; Lawrence, Shawn M.; Park, Jung; Pitts, Lee; Vann, Willie F.; Betenbaugh, Michael J.; Palter, Karen B.

doi:10.1093/glycob/12.2.73

Cited by 53 publications

(53 citation statements)

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“…This search revealed a putative nucleotide sugar transporter gene, which had already been identified by at least three other groups [29][30][31], and was identical to one reported by Kim et al [28]. We subsequently expressed this gene and performed genetic complementation and nucleotide sugar transport assays to directly examine the function of the gene product.…”

supporting

confidence: 56%

“…One way to gain additional knowledge is to use bioinformatics to analyze the Drosophila genome for genes involved in glycoprotein biosynthesis. Recently, this approach provided some interesting results that are sure to stimulate further discussion on this topic [26][27][28].…”

Section: Discussionmentioning

confidence: 92%

“…In three recent studies, this approach was used to search the D. melanogaster genome for genes encoding proteins involved in sialic acid biosynthesis or glycoprotein sialylation. No relevant genes were identified in the first study [26], a putative sialyltransferase gene was identified in the second [27], and putative N-acetylneuraminic acid phosphate synthase, sialyltransferase, and CMP-sialic acid/UDP-galactose transporter genes were identified in the third [28].…”

mentioning

confidence: 99%

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Expression and functional characterization of a nucleotide sugar transporter from Drosophila melanogaster: relevance to protein glycosylation in insect cell expression systems

Aumiller

Jarvis

2002

Protein Expression and Purification

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Insect cells are used routinely to express recombinant mammalian glycoproteins. However, insect protein glycosylation pathways are not well understood and appear to differ from those of mammalian cells. One way to more clearly evaluate the protein glycosylation potential of insect cells is to use the Drosophila melanogaster genome to identify genes that might encode relevant functions. These genes can then be expressed and the functions of the gene products directly evaluated by biochemical assays. In this study, we used this approach to determine the function of a putative Drosophila nucleotide sugar transporter gene. The results showed that this gene encodes a protein that can transport UDP-galactose, but not CMP-sialic acid. Thus, Drosophila encodes at least some of the infrastructure needed to produce glycoproteins with complex glycans, but this particular gene product does not directly support glycoprotein sialylation. These findings are relevant to insect cell biology and to an informed consideration of insect cell expression systems as tools for recombinant glycoprotein production. KeywordsGlycoproteins; Insect expression systems; Drosophila; Protein glycosylation; Nucleotide sugar transporter The development of baculovirus-insect cell [1,2] and constitutive insect cell [3,4] expression systems has stimulated great interest in the nature of the protein glycosylation and glycan processing machinery in these cells. Currently, it seems clear that there is at least one major difference between insect and mammalian protein glycosylation pathways. Specifically, insect cells appear to be unable to produce sialylated glycoproteins (reviewed in [5][6][7][8]). The earliest indications of this difference came from a comprehensive survey, which showed that insects had no detectable sialic acids [9]. This finding was extended by biochemical studies, which showed that insect cells had no detectable sialyltransferase activity and produced no detectable sialylated N-glycans [10][11][12][13]. More recent studies have shown that the major Nglycans produced by insect cells are high mannose or paucimannose structures and their major O-glycans are monosaccharides or core 1 disaccharides with no terminal sialic acids (reviewed in [5][6][7][8]). In addition, recent studies have confirmed the absence of detectable sialyltransferase activity and CMP-sialic acids in insect cells [14,15] insects. Two transgenic lepidopteran insect cell lines engineered to express mammalian β1,4-galactosyltransferase and α2,6-sialyltransferase genes produced sialylated glycoproteins, indicating that they must be able to produce CMP-sialic acid and transport it into the Golgi apparatus [23,24]. Finally, three lepidopteran insect cell lines were able to produce sialylated recombinant glycoproteins when treated with a βN-acetylglucosaminidase inhibitor, indicating that they have all the machinery needed to produce complex, terminally sialylated N-glycans when the action of this putative N-glycan trimming enzyme is inhibited [25]. Construction ...

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supporting

confidence: 56%

Section: Discussionmentioning

confidence: 92%

mentioning

confidence: 99%

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Expression and functional characterization of a nucleotide sugar transporter from Drosophila melanogaster: relevance to protein glycosylation in insect cell expression systems

Aumiller

Jarvis

2002

Protein Expression and Purification

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“…Fst is upregulated during cold stress (Goto, 2001). Sas has been implicated in ice binding and response to freezing (Kim et al, 2002a). desat2 is involved in cuticular hydrocarbon biosynthesis (Coyne et al, 1999), and Cosmopolitan and Zimbabwe alleles of this locus vary in their cold tolerance (Greenberg et al, 2003).…”

Section: Discussionmentioning

confidence: 99%

Quantitative trait loci for thermotolerance phenotypes in Drosophila melanogaster

2006

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For insects, temperature is a major environmental variable that can influence an individual's behavioral activities and fitness. Drosophila melanogaster is a cosmopolitan species that has had great success in adapting to and colonizing diverse thermal niches. This adaptation and colonization has resulted in complex patterns of genetic variation in thermotolerance phenotypes in nature. Although extensive work has been conducted documenting patterns of genetic variation, substantially less is known about the genomic regions or genes that underlie this ecologically and evolutionarily important genetic variation. To begin to understand and identify the genes controlling thermotolerance phenotypes, we have used a mapping population of recombinant inbred (RI) lines to map quantitative trait loci (QTL) that affect variation in both heat-and cold-stress resistance. The mapping population was derived from a cross between two lines of D. melanogaster (Oregon-R and 2b) that were not selected for thermotolerance phenotypes, but exhibit significant genetic divergence for both phenotypes. Using a design in which each RI line was backcrossed to both parental lines, we mapped seven QTL affecting thermotolerance on the second and third chromosomes. Three of the QTL influence cold-stress resistance and four affect heat-stress resistance. Most of the QTL were trait or sex specific, suggesting that overlapping but generally unique genetic architectures underlie resistance to low-and high-temperature extremes. Each QTL explained between 5 and 14% of the genetic variance among lines, and degrees of dominance ranged from completely additive to partial dominance. Potential thermotolerance candidate loci contained within our QTL regions are identified and discussed.

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“…This limitation reflects a fundamental difference between the N-glycan processing pathways of lepidopteran insect and mammalian cells, as most evidence indicates that the former lack functional levels of sialyltransferases (Butters et al, 1981;Stollar et al, 1976) and CMP-sialic acids (Hooker et al, 1999;Tomiya et al, 2001). Clearly, there are some published results that raise questions about the overall generality of this conclusion as it applies to lepidopteran (Davidson et al, 1990;Joosten et al, 2003;Shuler, 2003a, 2003b;Joshi et al, 2001;Palomares et al, 2003;Watanabe et al, 2001) and other (Kim et al, 2002;Koles et al, 2004) insect systems. However, the fact remains that most recombinant N-glycoproteins produced by the most commonly used baculovirus-insect cell combinations are not terminally sialylated.…”

Section: Discussionmentioning

confidence: 99%

Isolation and analysis of a baculovirus vector that supports recombinant glycoprotein sialylation by SfSWT‐1 cells cultured in serum‐free medium

Hill¹,

Aumiller²,

Shi³

et al. 2006

Biotech & Bioengineering

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The inability to sialylate recombinant glycoproteins is a critical limitation of the baculovirusinsect cell expression system. This limitation is due, at least in part, to the absence of detectable sialyltransferase activities and CMP-sialic acids in the insect cell lines routinely used as hosts in this system. SfSWT-1 is a transgenic insect cell line encoding five mammalian glycosyltransferases, including sialyltransferases, which can contribute to sialylation of recombinant glycoproteins expressed by baculovirus vectors. However, sialylation of recombinant glycoproteins requires culturing SfSWT-1 cells in the presence of fetal bovine serum or another exogenous source of sialic acid. To eliminate this requirement and extend the utility of SfSWT-1 cells, we have isolated a new baculovirus vector, AcSWT-7B, designed to express two mammalian enzymes that can convert N-acetylmannosamine to CMP-sialic acid during the early phase of infection. AcSWT-7B was also designed to express a model recombinant glycoprotein during the very late phase of infection. Characterization of this new baculovirus vector showed that it induced high levels of intracellular CMP-sialic acid and sialylation of the recombinant Nglycoprotein upon infection of SfSWT-1 cells cultured in serum-free medium supplemented with N-acetylmannosamine. In addition, co-infection of SfSWT-1 cells with AcSWT-7B plus a conventional baculovirus vector encoding human tissue plasminogen activator resulted in sialylation of this recombinant N-glycoprotein under the same culture conditions. These results demonstrate that AcSWT-7B can be used in two different ways to support recombinant Nglycoprotein sialylation by SfSWT-1 cells in serum-free medium. Thus, AcSWT-7B can be used to extend the utility of this previously described transgenic insect cell line for recombinant sialoglycoprotein production.

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Expression of a functional Drosophila melanogasterN-acetylneuraminic acid (Neu5Ac) phosphate synthase gene: evidence for endogenous sialic acid biosynthetic ability in insects

Cited by 53 publications

References 65 publications

Expression and functional characterization of a nucleotide sugar transporter from Drosophila melanogaster: relevance to protein glycosylation in insect cell expression systems

Expression and functional characterization of a nucleotide sugar transporter from Drosophila melanogaster: relevance to protein glycosylation in insect cell expression systems

Quantitative trait loci for thermotolerance phenotypes in Drosophila melanogaster

Isolation and analysis of a baculovirus vector that supports recombinant glycoprotein sialylation by SfSWT‐1 cells cultured in serum‐free medium

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