Mucin‐type glycoprotein from <i>Drosophila melanogaster</i> embryonic cells: characterization of carbohydrate component

Kramerov, Andrei A.; Arbatsky, Nikolay P.; Rozovsky, Ya. M.; Mikhaleva, Elena A.; Polesskaya, Oksana; Гвоздев, В. А.; Shibaev, V. N.

doi:10.1016/0014-5793(95)01444-6

Cited by 44 publications

(33 citation statements)

References 31 publications

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“…17). Interestingly, the Drosophila melanogaster "mucin-D" glycoprotein contains a large amount (40% by mass) of Gal␤1-3GalNAc that renders it highly resistant to protease action (38), consistent with our data showing that the same disaccharide alone (generated after treatment of Lp(a) with sialidase) provided resistance to thermolysin digestion.…”

Section: Discussionsupporting

confidence: 91%

Structural Elucidation of the N- andO-Glycans of Human Apolipoprotein(a)

Garner

Merry

Royle

et al. 2001

Journal of Biological Chemistry

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Apolipoprotein(a) (apo(a)) is a multikringle domain glycoprotein that exists covalently linked to apolipoprotein B100 of low density lipoprotein, to form the lipoprotein(a) (Lp(a)) particle, or as proteolytic fragments. Elevated plasma concentrations of apo(a) and its fragments may promote atherosclerosis, but the underlying mechanisms are incompletely understood. The factors influencing apo(a) proteolysis are also uncertain. Here we have used exoglycosidase digestion and mass spectrometry to sequence the Asn (N)-linked and Ser/Thr (O)-linked oligosaccharides of human apo(a). We also assessed the potential role of apo(a) O-glycans in protecting thermolysin-sensitive regions of the polypeptide. Apo(a) contained two major N-glycans that accounted for 17% of the total oligosaccharide structures. The N-glycans were complex biantennary structures present in either a mono-or disialylated state. The O-glycans were mostly (80%) represented by the monosialylated core type 1 structure, NeuNAc␣2-3Gal␤1-3GalNAc, with smaller amounts of disialylated and non-sialylated O-glycans also detected. Removal of apo(a) O-glycans by sialidase and O-glycosidase treatment dramatically increased the sensitivity of the polypeptide to thermolysin digestion. These studies provide the first direct sequencing data for apo(a) glycans and indicate a novel function for apo(a) O-glycans that is potentially related to the atherogenicity of Lp(a).Lipoprotein(a) (Lp(a)) 1 consists of a low density lipoprotein (LDL) particle covalently linked through a single disulfide bond to apolipoprotein(a) (apo(a)) (1). Apo(a) contains multikringle domains that are homologous to plasminogen kringles IV and V (2). This homology is thought to underlie the atherogenicity of Lp(a) because apo(a) and its naturally occurring proteolytic fragments compete with plasminogen for fibrin(ogen) binding (3, 4). The predicted apo(a)-mediated decrease in fibrinolysis may contribute to the accumulation of fibrin at sites of atherosclerotic lesion development. Apo(a) also exhibits additional proatherogenic properties including binding to fibronectin (5) and decorin (6), direct inhibition of tissue-type plasminogen activator 1 action (7), and modulation of fibrinolysis after cellsurface binding (8,9).The concentration of plasma and urinary apo(a) proteolytic fragments is increased concomitantly with elevated plasma Lp(a) levels (10, 11). In some cases, the apo(a) fragments have a higher atherogenic potential than the intact Lp(a). For example, when the apo(a) moiety of Lp(a) is cleaved by protease to generate "mini-Lp(a)" (12), the resulting particle binds fibrinogen (12) and fibronectin (13) more avidly than undigested Lp(a). Because apo(a) fragments have potentially atherogenic properties and because they are also known to accumulate in atherosclerotic lesions (14), it is important to understand the factors that regulate apo(a) proteolysis. Previous studies have focused on the role of serine proteases and metalloproteinase as relevant enzymes (5, 12, 15, 16); however, the factor...

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Section: Discussionsupporting

confidence: 91%

Structural Elucidation of the N- andO-Glycans of Human Apolipoprotein(a)

Garner

Merry

Royle

et al. 2001

Journal of Biological Chemistry

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“…However, these results did confirm that Drosophila encodes a functional UDP-galactose transporter, which is consistent with the ability of this organism to produce galactosylated glycoconjugates including O-glycoproteins [55], glycosaminoglycans [56], and glycolipids [57]. Thus, this study contributed to our growing knowledge of the machinery available for glycoconjugate processing in insect cell systems, which is needed for informed consideration of these systems as tools for recombinant glycoprotein production.…”

Section: Discussionsupporting

confidence: 78%

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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“…Structural assignments presented here are based on multiple lines of evidence, including extensive NSI-MS n , composition and linkage analysis by GC-MS of permethylated oligosaccharide alditols or of partially methylated alditol acetates, and exoglycosidase sensitivity. The core 1 disaccharide, Gal␤1-3GalNAc, has been identified previously in Drosophila tissues and cultured Drosophila cells; it is released from Drosophila proteins by the enzyme O-glycanase, which has a strict anomeric specificity for Gal␤1-3GalNAc, and is recognized by structurally specific lectin and antibody probes (34,35,37,40,60). Combined with the fragmentation and composition data presented here, the core 1 disaccharide structure can be assigned with confidence as expected.…”

Section: Recovery Of O-linked Glycans From the Drosophila Embryo-mentioning

confidence: 73%

The Diversity of O-Linked Glycans Expressed during Drosophila melanogaster Development Reflects Stage- and Tissue-specific Requirements for Cell Signaling

Aoki

Porterfield

Lee

et al. 2008

Journal of Biological Chemistry

132

160

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Appropriate glycoprotein O-glycosylation is essential for normal development and tissue function in multicellular organisms. To comprehensively assess the developmental and functional impact of altered O-glycosylation, we have extensively analyzed the nonglycosaminoglycan, O-linked glycans expressed in Drosophila embryos. Through multidimensional mass spectrometric analysis of glycans released from glycoproteins by ␤-elimination, we detected novel as well as previously reported O-glycans that exhibit developmentally modulated expression. The core 1 mucin-type disaccharide (Gal␤1-3GalNAc) is the predominant glycan in the total profile. HexNAcitol, hexitol, xylosylated hexitol, and branching extension of core 1 with HexNAc (to generate core 2 glycans) were also evident following release and reduction. After Gal␤1-3GalNAc, the next most prevalent glycans were a mixture of novel, isobaric, linear, and branched forms of a glucuronyl core 1 disaccharide. Other less prevalent structures were also extended with HexA, including an O-fucose glycan. Although the expected disaccharide product of the Fringe glycosyltransferase, (GlcNAc␤1-3)fucitol, was not detectable in whole embryos, mass spectrometry fragmentation and exoglycosidase sensitivity defined a novel glucuronyl trisaccharide as GlcNAc␤1-3(GlcA␤1-4)fucitol. Consistent with the spatial distribution of the Fringe function, the GlcAextended form of the Fringe product was enriched in the dorsal portion of the wing imaginal disc. Furthermore, loss of Fringe activity reduced the prevalence of the O-Fuc trisaccharide. Therefore, O-Fuc glycans necessary for the modulation of important signaling events in Drosophila are, as in vertebrates, substrates for extension beyond the addition of a single HexNAc.

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Mucin‐type glycoprotein from Drosophila melanogaster embryonic cells: characterization of carbohydrate component

Cited by 44 publications

References 31 publications

Structural Elucidation of the N- andO-Glycans of Human Apolipoprotein(a)

Structural Elucidation of the N- andO-Glycans of Human Apolipoprotein(a)

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

The Diversity of O-Linked Glycans Expressed during Drosophila melanogaster Development Reflects Stage- and Tissue-specific Requirements for Cell Signaling

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