Molecular Cloning and Functional Characterization of a Lepidopteran Insect β4-N-Acetylgalactosaminyltransferase with Broad Substrate Specificity, a Functional Role in Glycoprotein Biosynthesis, and a Potential Functional Role in Glycolipid Biosynthesis

Vadaie, Nadia; Jarvis, Donald L.

doi:10.1074/jbc.m404925200

Cited by 45 publications

(42 citation statements)

References 99 publications

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“…The capacity of insect cells to synthesize complex N-glycans has been a matter of some controversy. Indeed, recent studies show that the genome of Drosophila encodes two N-acetylgalactosaminyltransferases (62) and a sialyltransferase (63); whereas Trichoplusia ni has a proven GalNAc-transferase that could in theory participate in the synthesis of complex N-glycans (64). However, to date there is no published evidence for such structures in adult flies (although some complex structures such as fucosylated LacdiNAc are found on bee venom glycoproteins (9)).…”

Section: (Df(2r)achimentioning

confidence: 99%

The Drosophila fused lobes Gene Encodes an N-Acetylglucosaminidase Involved in N-Glycan Processing

Léonard

Rendić

Rabouille

et al. 2006

Journal of Biological Chemistry

149

152

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Most processed, e.g. fucosylated, N-glycans on insect glycoproteins terminate in mannose, yet the relevant modifying enzymes require the prior action of N-acetylglucosaminyltransferase I. This led to the hypothesis that a hexosaminidase acts during the course of N-glycan maturation. To determine whether the Drosophila melanogaster genome indeed encodes such an enzyme, a cDNA corresponding to fused lobes (fdl), a putative ␤-N-acetylglucosaminidase with a potential transmembrane domain, was cloned. When expressed in Pichia pastoris, the enzyme exhibited a substrate specificity similar to that previously described for a hexosaminidase activity from Sf-9 cells, i.e. it hydrolyzed exclusively the GlcNAc residue attached to the ␣1,3-linked mannose of the core pentasaccharide of N-glycans. It also hydrolyzed p-nitrophenyl-N-acetyl-␤-glucosaminide, but not chitooligosaccharides; in contrast, Drosophila HEXO1 and HEXO2 expressed in Pichia cleaved both these substrates but not N-glycans. The localization of recombinant FDL tagged with green fluorescent protein in Drosophila S2 cells by immunoelectron microscopy showed that this enzyme transits through the Golgi, is present on the plasma membrane and in multivesicular bodies, and is secreted. Finally, the N-glycans of two lines of fdl mutant flies were analyzed by mass spectrometry and reversed-phase high-performance liquid chromatography. The ratio of structures with terminal GlcNAc over those without (i.e. paucimannosidic N-glycans) was drastically increased in the fdldeficient flies. Therefore, we conclude that the fdl gene encodes a novel hexosaminidase responsible for the occurrence of paucimannosidic N-glycans in Drosophila.Insect cells are considered to be an interesting alternative to mammalian, yeast, or bacterial cells for the expression of recombinant proteins (1, 2), because the potentially high levels of expression are associated with the ability to perform many eukaryotic post-translational modifications. Nevertheless, glycoproteins produced in insects and insect cells have been mainly found to carry paucimannosidic N-glycans, i.e. glycans consisting of the pentasaccharide core with or without ␣1,6-and/or ␣1,3-linked fucose (1, 3-5). In addition to the possible presence of the immunogenic core ␣1,3-linked fucose, the lack of complex type N-glycans with complete, sialylated antennae as found on mammalian glycoproteins makes insect cells unsuitable for the production of many therapeutic glycoproteins (6).Only in a few cases have substitutions of the terminal mannose residues of insect N-glycans been described. The most common substitution is the presence of terminal GlcNAc on the ␣1,3-linked mannosyl residue (GlcNAc-1) 3 added by GlcNAc-transferase I, although in a few cases further modifications such as galactose, N-acetylgalactosamine, fucose, and aminoethylphosphonate (i.e. phosphorylethanolamine) have been found linked to this GlcNAc residue (7-11). The occurrence of larger and sialylated N-glycans has been reviewed elsewhere (1, 4). In most instances, howe...

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Section: (Df(2r)achimentioning

confidence: 99%

The Drosophila fused lobes Gene Encodes an N-Acetylglucosaminidase Involved in N-Glycan Processing

Léonard

Rendić

Rabouille

et al. 2006

Journal of Biological Chemistry

149

152

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“…For instance, Tn-5B1-4 cells contain only 10z of the GalT activity detected in CHO cells, and Sf9 cells contain no detectable levels of the GalT activity (41). Interestingly, T. ni (42) and Drosophila (43) possess orthologous genes to human b4GalTs; however, the encoded gene products do not catalyze e‹cient transfer of Gal from UDP-Gal to acceptor substrates. Instead, the insect enzyme catalyzes the transfer of GalNAc from UDP-GalNAc to acceptors more e‹ciently (42,43).…”

Section: Processing Enzymes In the Golgimentioning

confidence: 99%

“…Instead, the insect enzyme catalyzes the transfer of GalNAc from UDP-GalNAc to acceptors more e‹ciently (42,43). Indeed, when a soluble form of a S. frugiperda a-mannosidase I was expressed in Sf9 cells with a T. ni ortholog of human b4GalT, N-glycans modiˆed with a terminal GalNAc, but not Gal, were synthesized (42).…”

Section: Processing Enzymes In the Golgimentioning

confidence: 99%

“…Some insects, such as T. ni (42) and Drosophila (43) possess orthologs of human b4GalTs; however, unlike human b4GalT1, the insect enzymes do not e‹ciently transfer Gal to the expected acceptor substrate. Therefore, a mammalian b4GalT has been expressed in insect cells in a number of studies.…”

Section: E-2 Expression Of Galactosylated N-glycansmentioning

confidence: 99%

“…Interestingly, T. ni (42) and Drosophila (43) possess orthologous genes to human b4GalTs; however, the encoded gene products do not catalyze e‹cient transfer of Gal from UDP-Gal to acceptor substrates. Instead, the insect enzyme catalyzes the transfer of GalNAc from UDP-GalNAc to acceptors more e‹ciently (42,43). Indeed, when a soluble form of a S. frugiperda a-mannosidase I was expressed in Sf9 cells with a T. ni ortholog of human b4GalT, N-glycans modiˆed with a terminal GalNAc, but not Gal, were synthesized (42).…”

Section: Processing Enzymes In the Golgimentioning

confidence: 99%

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Humanization of recombinant glycoproteins expressed in insect cells

Tomiya¹

2009

TIGG

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Many of the most valuable recombinant DNA products, including monoclonal antibodies, are secreted glycoproteins, which contain N-glycans. The biochemical nature of these attached glycans depends on the characteristics of the host cell used, the protein synthesized, and the culture environment. The capacity to produce functional glycoproteins with desired glycosylation patterns depends on the presence of proper cellular enzymatic machinery required for the processing of N-glycans. It is well recongnized that expression of therapeutic proteins using insect cells in combination with the baculovirus-expression system has potential advantages in terms of the ease of large scale, low cost production of heterologous glycoproteins. However, the glycoforms expressed in several commonly used insect cell lines are markedly diŠerent from those expressed in human cells. As N-glycans often aŠect the in vivo biological activity, pharmacokinetics, and several other properties of glycoproteins, the glycoforms produced in this system have become a major concern in the last few years. Accordingly, considerable eŠort has been invested in the past decade in order to gain a better understanding of the processing of N-glycans in insect cells that generate diŠerent glycoforms, and also to overcome the bottlenecks to producing glycoproteins with humanized Nglycans that are more suitable for therapeutic use.

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Glycoproteins

Merry¹,

Astrautsova²

2010

Encyclopedia of Life Sciences

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Glycoproteins are proteins that contain covalently bound oligosaccharides (glycans). Although there are structural features of N ‐ and O ‐linked glycans common to all eukaryotes, species‐specific glycosylation reactions create a wide diversity of structures. This is particularly relevant to cell surface glycoproteins although intracellular and even nuclear proteins may be glycosylated. The factors controlling glycosylation are complex and include the protein sequence and structure, specificity of relevant transferase enzymes, availability of donor sugars and other environmental factors. These may all interact so it is not surprising that there is great variety in glycosylation even on the same protein. Glycosylation of proteins is important in such areas as development and interaction with pathogens and provides a means of modifying the function of proteins that is not directly dependent on their deoxyribonucleic acid (DNA) template. Interest in the field is growing and advances in analytical technology now make the field accessible to a wider community. Key Concepts: Proteins are frequently modified posttranslationally, the most frequent modification being glycosylation. Glycosylation may play an important role in protein folding through interaction with the chaperones calnexin and calreticulin. It has been calculated that as many as 70% of all proteins may be glycosylated. The glycosylation of a protein may vary extensively depending on the cell in which it is produced which may enable the same protein to function in different ways depending where it is expressed. On the cell surface, glycoprotein glycans are frequently involved in cell–cell interactions. Glycosylation of the same protein may change during development or in maturation, for example in cells of the immune system. Glycosylation differs between species even if they are closely related, for example between great apes and man. Analysis of glycosylation has progressed in recent years and is now almost a routine procedure. Particular features of glycosylation may only have functional significance in a particular time and place but can be widely distributed which can make assignment of function to glycan structure difficult.

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Molecular Cloning and Functional Characterization of a Lepidopteran Insect β4-N-Acetylgalactosaminyltransferase with Broad Substrate Specificity, a Functional Role in Glycoprotein Biosynthesis, and a Potential Functional Role in Glycolipid Biosynthesis

Cited by 45 publications

References 99 publications

The Drosophila fused lobes Gene Encodes an N-Acetylglucosaminidase Involved in N-Glycan Processing

The Drosophila fused lobes Gene Encodes an N-Acetylglucosaminidase Involved in N-Glycan Processing

Humanization of recombinant glycoproteins expressed in insect cells

Glycoproteins

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