Recombinant Soluble β‐1,4‐Galactosyltransferases Expressed in <i>Saccharomyces cerevisiae</i>

Malissard, Martine; Borsig, Lubor; Marco, Stefania Di; Grütter, Markus G.; Kragl, Udo; Wandrey, Christian; Berger, Eric G.

doi:10.1111/j.1432-1033.1996.0340u.x

Cited by 48 publications

(26 citation statements)

References 61 publications

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“…Asialo-ovine submaxillary mucin was a gift from Dr. Robert L. Hill (16). Gal␤1,4GlcNAc-␤-pNP was generated enzymatically by the action of recombinant human ␤1,4-galactosyltransferase (17), and the Gal␤1,3GlcNAc-␤-pNP standard was purchased from Sigma. The HPLC system consisted of a Jour Research X-ACT degasser, two Waters 510 pumps, a Waters 717 autoinjector, a Jasco C0965 Column Heater (30°C), and a Waters 490 programmable multi-wavelength detector (214, 262, and 350 nm).…”

Section: Methodsmentioning

confidence: 99%

Genomic Cloning and Expression of Three Murine UDP-galactose: β-N-Acetylglucosamine β1,3-Galactosyltransferase Genes

Hennet

Dinter

Kuhnert

et al. 1998

Journal of Biological Chemistry

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Based on the detection of expressed sequence tags that are similar to known galactosyltransferase sequences, we have isolated three novel UDP-galactose:␤-N-acetylglucosamine ␤1,3-galactosyltransferase (␤3GalT) genes from a mouse genomic library. The three genes, named ␤3GalT-I, -II, and -III, encode type II transmembrane proteins of 326, 422, and 331 amino acids, respectively. The three proteins constitute a distinct subfamily as they do not share any sequence identity with other eucaryotic galactosyltransferases. Also, the entire protein-coding region of the three ␤3GalT genes was contained in a single exon, which contrasts with the genomic organization of the ␤1,4-and ␣1,3-galactosyltransferase genes. The three ␤3GalT genes were mainly expressed in brain tissue. The expression of the fulllength murine genes as recombinant baculoviruses in insect cells revealed that the ␤3GalT enzymes share the same acceptor specificity for ␤-linked GlcNAc, although they differ in their K m for this acceptor and the donor UDP-Gal. The identification of ␤3GalT genes emphasizes the structural diversity present in the galactosyltransferase gene family.

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Section: Methodsmentioning

confidence: 99%

Genomic Cloning and Expression of Three Murine UDP-galactose: β-N-Acetylglucosamine β1,3-Galactosyltransferase Genes

Hennet

Dinter

Kuhnert

et al. 1998

Journal of Biological Chemistry

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“…We chose to express the catalytic domain of human b4gal-T1 in E. coli despite the fact that much higher expression levels for b4gal-T have been achieved in S. cerevisiae (30 U : L 21 ) under conditions of optimized fedbatch fermentation [32] and in Sf9 cells [33]. The main reason for using E. coli as the expression system was to avoid additional heterogeneity due to O-glycosylation that occurs in both S. cerevisiae [20] and Sf9 cells. The cDNA encoding the catalytic domain of human b4gal-T1 was Table 4.…”

Section: Discussionmentioning

confidence: 99%

“…The standard assay was carried out as described previously [20]. Briefly, incorporation of [ ]Gal into free GlcNAc was measured.…”

Section: Enzyme Assays and Kinetic Analysismentioning

confidence: 99%

“…The enzyme harbours one N-linked glycan chain [18]) and a variety of O-linked glycan chains [19], all of them being highly heterogeneous. In a previous study [20], we described the expression of an N-deglycosylated form of human b4gal-T1 (Ndrgal-T1) in the yeast Saccharomyces cerevisiae. The purified Ndrgal-T1 (theoretical molecular mass 39 kDa) comigrated with the human b4gal-T1 (55 kDa) on SDS/PAGE and was resolved into only two bands by IEF.…”

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confidence: 99%

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Improving solubility of catalytic domain of human β‐1,4‐galactosyltransferase 1 through rationally designed amino acid replacements

Malissard¹,

Berger²

2001

European Journal of Biochemistry

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b-1,4-galactosyltransferase 1 (b4gal-T1, EC 2.4.1.38) transfers galactose from UDP-galactose to free N-acetyl-D-glucosamine or bound N-acetyl-D-glucosamine-R. Soluble b4gal-T1, purified from human milk has been refractory to structural studies by X-ray or NMR. In a previous study (Malissard et al. 1996, Eur. J. Biochem. 239, 340-348) we produced in the yeast Saccharomyces cerevisiae an N-deglycosylated form of soluble b4gal-T1 that was much more homogeneous than the human enzyme, as it displayed only two isoforms when analysed by IEF as compared to 13 isoforms for the native b4gal-T1. The propensity of recombinant b4gal-T1 to aggregate at concentrations . 1 mg : mL 21 prevented structural and biophysical studies. In an attempt to produce a b4gal-T1 form suitable for structural studies, we combined site-directed mutagenesis and heterologous expression in Escherichia coli. We produced a mutated form of the catalytic domain of b4gal-T1 (sfb4gal-T1mut) in which seven mutations were introduced at nonconserved sites (A155E, N160K, M163T, A168T, T242N, N255D and A259T). Sfb4gal-T1mut was shown to be much more soluble than b4gal-T1 expressed in S. cerevisiae (8.5 mg : mL 21 vs. 1 mg : mL 21 ). Catalytic activity and kinetic parameters of sfb4gal-T1mut produced in E. coli were shown not to differ to any significant extent from those of the native enzyme.Keywords: b1,4-galactosyltransferase; heterologous expression; site-directed mutagenesis; protein solubility.Glycosylation reactions are of great biological importance to both prokaryotes and eukaryotes, and require the combined action of glycosidases and glycosyltransferases that are differentially expressed throughout tissues. As many as a few hundred different glycosyltransferases and their corresponding genes account for the synthesis of the large variety of oligosaccharide structures observed at the surface of glycoproteins and glycolipids in mammalian species. The first glycosyltransferase to be cloned was the UDPGal:GlcNAc-R b-1,4-galactosyltransferase (b4gal-T1) [1][2][3], and this glycosyltransferase has been characterized thoroughly. At present, . 118 different human glycosyltransferase genes have been cloned and classified [4,6] http://afmb.cnrs-mrs.fr/~pedro/CAZY/gtf.html. Although they have very different amino-acid sequences, these type II endoplasmic reticulum and Golgi resident membrane-bound glycoprotein enzymes have a similar domain organization [7]. In general, the eukaryotic glycosyltransferases cloned to date consist of a short N-terminal cytoplasmic tail, and a noncleavable signal-anchor/transmembrane domain followed by a stem region and a large C-terminal catalytic domain both exposed to the Golgi lumen. Secreted forms of glycosyltransferases are produced by proteolysis at multiple protease-sensitive positions within the stem region of the protein. The resulting soluble enzymes are responsible for the glycosyltransferase activities detected in milk, serum and saliva [8,9].The human b4gal-T1 is a type II membrane-bound glycoprotein that, in the pres...

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“…To achieve elongation and termination of complex glycans, additional glycosyltransferases and nucleotide sugar transporters are required. Surprisingly, heterologous expression of human b1,4galac-tosyltransferase led to the incorporation of galactose into several yeast proteins [103], suggesting the presence of the metabolic pathways to synthesize UDP-gal and to transport it across the Golgi membranes. This has in fact been confirmed recently by a direct demonstration of a UDPgal/UMP antiporter in S. cerevisiae Golgi membranes [133].…”

Section: Cell Engineeringmentioning

confidence: 99%

The yeast expression system for recombinant glycosyltransferases

Malissard¹,

Zeng²,

Berger³

1999

Glycotechnology

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Glycosyltransferases are increasingly being used for in vitro synthesis of oligosaccharides. Since these enzymes are difficult to purify from natural sources, expression systems for soluble forms of the recombinant enzymes have been developed. This review focuses on the current state of development of yeast expression systems. Two yeast species have mainly been used, i.e. Saccharomyces cerevisiae and Pichia pastoris. Safety and ease of fermentation are well recognized for S. cerevisiae as a biotechnological expression system; however, even soluble forms of recombinant glycosyltransferases are not secreted. In some cases, hyperglycosylation may occur. P. pastoris, by contrast, secrete soluble orthoglycosylated forms to the supernatant where they can be recovered in a highly purified form.The review also covers some basic features of yeast fermentation and describes in some detail those glycosyltransferases that have successfully been expressed in yeasts. These include b1,4galactosyltransferase, a2,6sialyltransferase, a2,3sialyltransferase, a1,3fucosyltransferase III and VI and a1,2mannosyltransferase. Current efforts in introducing glycosylation systems of higher eukaryotes into yeasts are briefly addressed.

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Recombinant Soluble β‐1,4‐Galactosyltransferases Expressed in Saccharomyces cerevisiae

Cited by 48 publications

References 61 publications

Genomic Cloning and Expression of Three Murine UDP-galactose: β-N-Acetylglucosamine β1,3-Galactosyltransferase Genes

Genomic Cloning and Expression of Three Murine UDP-galactose: β-N-Acetylglucosamine β1,3-Galactosyltransferase Genes

Improving solubility of catalytic domain of human β‐1,4‐galactosyltransferase 1 through rationally designed amino acid replacements

The yeast expression system for recombinant glycosyltransferases

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