Targeting of active rat α2,3-sialyltransferase to the yeast cell wall by the aid of the hsp 150Δ-carrier: toward synthesis of sLe<sup>x</sup>-decorated L-selectin ligands

Mattila, Pirkko; Joutsjoki, Vesa; Kaitera, Eero; Majuri, Marja‐Leena; Niittymäki, Jaana; Saris, Nina; Maaheimo, Hannu; Renkonen, Ossi; Renkonen, Risto; Makarow, Marja

doi:10.1093/glycob/6.8.851

Cited by 13 publications

(26 citation statements)

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“…Finally, a number of publications report the functional expression of sialyltransferases in the yeast cell wall [183][184][185]. When these cells were provided with CMP-sialic acid and an acceptor substrate, sialic acid transfer could be demonstrated.…”

Section: Conversion Of Yeast Hyperglycosylation To Homogeneous Glycansmentioning

confidence: 99%

N-glycosylation Engineering of Biopharmaceutical Expression Systems

Jacobs

Callewaert²

2009

CMM

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N-glycosylation, the enzymatic coupling of oligosaccharides to specific asparagine residues of nascent polypeptide chains, is one of the most widespread post-translational modifications. Following transfer of an N-glycan precursor in the ER, this structure is further modified by a number of glycosidases and glyco-syltransferases in the ER and the Golgi complex. The processing reactions occurring in the ER are highly conserved between lower and higher eukaryotes. In contrast, the reactions that take place in the Golgi complex are species- and cell type-specific. Due to its non-template driven nature, glycoproteins typically occur as a mixture of glycoforms. Since N-glycans influence circulation half-life, tissue distribution, and biological activity each glycoform has its own pharmacokinetic, pharmacodynamic and efficacy profile. Moreover, modification of glycoproteins with non-human oligosaccharides can result in undesired immunogenicity. Therefore, engineering of the N-glycosylation pathway of most currently used heterologous protein expression systems (bacteria, mammalian cells, insect cells, yeasts and plants) is actively pursued by several academic and industrial laboratories. These research efforts are in the first place directed at humanizing the N-glycosylation pathway and eliminating immunogenic glycotopes. Moreover, one wants to establish new structure-function relationships of different glycoforms, which helps to decreasing the complexity of the N-glycan repertoire towards one defined N-glycan structure. In this review, we discuss the most important recent milestones in the glycoengineering field.

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Section: Conversion Of Yeast Hyperglycosylation To Homogeneous Glycansmentioning

confidence: 99%

N-glycosylation Engineering of Biopharmaceutical Expression Systems

Jacobs

Callewaert²

2009

CMM

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“…Mammalian glycosyltransferases can be produced in yeast cells, which are able to secrete proteins and can be grown to high cell densities in inexpensive growth media. However, mammalian proteins usually cannot exit the yeast endoplasmic reticulum (ER) due to misfolding [17–22]. This secretion block can be overcome by fusing the heterologous protein to the Hsp150Δ carrier, which helps the heterologous protein to acquire its proper conformation [23–26].…”

Section: Introductionmentioning

confidence: 99%

“…The Hsp150Δ carrier consists of an N‐terminal fragment of Hsp150, a glycoprotein of S. cerevisiae , which is secreted rapidly and efficiently to the yeast culture medium [27,28]. We have earlier been able to express the catalytic domain of rat α‐2,3‐sialyltransferase (ST3Ne) as an Hsp150Δ fusion protein (Hsp150Δ–ST3Ne) in S. cerevisiae and P. pastoris [18,19,29]. The glycosyltransferase portion was found to acquire a catalytically active, disulfide‐bonded and N‐glycosylated form in the yeast ER, and Hsp150Δ–ST3Ne was transported to the exterior of the cells.…”

Section: Introductionmentioning

confidence: 99%

“…The glycosyltransferase portion was found to acquire a catalytically active, disulfide‐bonded and N‐glycosylated form in the yeast ER, and Hsp150Δ–ST3Ne was transported to the exterior of the cells. However, the fusion protein was not secreted to the culture medium, but remained attached to the porous cell wall [18,19,29]. Thus, intact yeast cells could be used as an enzyme source for the synthesis of sialyl‐α‐2,3‐ N ‐acetyllactosamine from CMP‐NeuNAc and N ‐acetyllactosamine [18,19,29].…”

Section: Introductionmentioning

confidence: 99%

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Co-expression of two mammalian glycosyltransferases in the yeast cell wall allows synthesis of sLex

Salo¹,

Sievi²,

Suntio³

et al. 2005

FEMS Yeast Research

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Interactions between selectins and their oligosaccharide-decorated counter-receptors play an important role in the initiation of leukocyte extravasation in inflammation. L-selectin ligands are O-glycosylated with sulphated sialyl Lewis X epitopes (sulpho-sLex). Synthetic sLex oligosaccharides have been shown to inhibit adhesion of lymphocytes to endothelium at sites of inflammation. Thus, they could be used to prevent undesirable inflammatory reactions such as rejection of organ transplants. In vitro synthesis of sLex glycans is dependent on the availability of recombinant glycosyltransferases. Here we expressed the catalytic domain of human alpha-1,3-fucosyltransferase VII in the yeasts Saccharomyces cerevisiae and Pichia pastoris. To promote proper folding and secretion competence of this catalytic domain in yeast, it was fused to the Hsp150 delta carrier, which is an N-terminal fragment of a secretory glycoprotein of S. cerevisiae. In both yeasts, the catalytic domain acquired an active conformation and the fusion protein was externalised, but remained mostly attached to the cell wall in a non-covalent fashion. Incubation of intact S. cerevisiae or P. pastoris cells with GDP-[14C]fucose and sialyl-alpha-2,3-N-acetyllactosamine resulted in synthesis of radioactive sLex, which diffused to the medium. Finally, we constructed an S. cerevisiae strain co-expressing the catalytic domains of alpha-2,3-sialyltransferase and alpha-1,3-fucosyltransferase VII, which were targeted to the cell wall. When these cells were provided with N-acetyllactosamine, CMP-sialic acid and GDP-[14C]fucose, radioactive sLex was produced to the medium. These data imply that yeast cells can provide a self-perpetuating source of fucosyltransferase activity immobilized in the cell wall, useful for the in vitro synthesis of sLex.

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“…The molecular mechanism of Pir protein binding to the cell wall remains unclear (12,25), but Pir protein sequences possess binding potential irrespective of the fusion site, i.e., at either terminus of the target protein (32,51). This property is useful for immobilizing glycosyltransferases.…”

mentioning

confidence: 99%

Construction of a Library of Human Glycosyltransferases Immobilized in the Cell Wall ofSaccharomyces cerevisiae

Shimma¹,

Saito²,

Oosawa³

et al. 2006

Appl Environ Microbiol

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Fifty-one human glycosyltransferases were expressed in Saccharomyces cerevisiae as immobilized enzymes and were assayed for enzymatic activities. The stem and catalytic regions of sialyl-, fucosyl-, galactosyl-, N-acetylgalactosaminyl-, and N-acetylglucosaminyltransferases were fused with yeast cell wall Pir proteins, which anchor glycosyltransferases at the yeast cell wall glucan. More than 75% of expressed recombinant glycosyltransferases retained their enzymatic activities in the yeast cell wall fraction and will be used as a human glycosyltransferase library. In increasing the enzymatic activities of immobilized glycosyltransferases, several approaches were found to be effective. Additional expression of yeast protein disulfide isomerase increased the expression levels and activities of polypeptide N-acetylgalactosaminyltransferases and other glycosyltransferases. PIR3 and/or PIR4 was more effective than PIR1 as a cell wall anchor when the Pirglycosyltransferase fusions were expressed under the control of the constitutive glyceraldehyde-3-phosphate dehydrogenase promoter. Oligosaccharides such as Lewis x, Lewis y, and H antigen were successfully synthesized using this immobilized glycosyltransferase library, indicating that the Pir-fused glycosyltransferases are useful for the production of various human oligosaccharides.Most oligosaccharides exist as glycoproteins and glycolipids, many of which are localized at the cell surface and are involved in biologically important processes such as cellular adhesion, molecular recognition, and signal transduction (58). The oligosaccharides are extremely rich in structural variations, and isomers exhibit different branching structures, anomeric configurations, and linkage positions; these vary widely with species, tissue, and degree of cellular differentiation. To clarify the structure-function relationship of oligosaccharides, the synthesis and remodeling of oligosaccharides are required. In order to synthesize complex oligosaccharides on proteins and lipids or in living cells, enzymatic methods are more suitable than chemical methods, since degradation or denaturation of active proteins, lipids, and living cells can be avoided. Human-derived enzymes are particularly ideal for the synthesis of mammalian oligosaccharides, such as sialylated and branched glycans, because these reactions are difficult to reproduce with either chemical methods or bacterial enzymes (19).At present, nearly 200 human glycosyltransferase genes are known and are available for use. These are indispensable for enzymatic synthesis of human-type oligosaccharides (41). However, it is important to make these resources suitable for application as enzyme sources. Immobilized enzymes enable the recovery of valuable enzymes for repeated usage and have several advantages for industrial applications; they are usually more stable than free enzymes and can be applied via automated bioreactor-packed columns.

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Targeting of active rat α2,3-sialyltransferase to the yeast cell wall by the aid of the hsp 150Δ-carrier: toward synthesis of sLe^x-decorated L-selectin ligands

Cited by 13 publications

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N-glycosylation Engineering of Biopharmaceutical Expression Systems

N-glycosylation Engineering of Biopharmaceutical Expression Systems

Co-expression of two mammalian glycosyltransferases in the yeast cell wall allows synthesis of sLex

Construction of a Library of Human Glycosyltransferases Immobilized in the Cell Wall ofSaccharomyces cerevisiae

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Targeting of active rat α2,3-sialyltransferase to the yeast cell wall by the aid of the hsp 150Δ-carrier: toward synthesis of sLex-decorated L-selectin ligands

Cited by 13 publications

References 0 publications

N-glycosylation Engineering of Biopharmaceutical Expression Systems

N-glycosylation Engineering of Biopharmaceutical Expression Systems

Co-expression of two mammalian glycosyltransferases in the yeast cell wall allows synthesis of sLex

Construction of a Library of Human Glycosyltransferases Immobilized in the Cell Wall ofSaccharomyces cerevisiae

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Product

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Targeting of active rat α2,3-sialyltransferase to the yeast cell wall by the aid of the hsp 150Δ-carrier: toward synthesis of sLe^x-decorated L-selectin ligands