In Vivo Specificity of Human α1,3/4-Fucosyltransferases III-VII in the Biosynthesis of LewisX and Sialyl LewisX Motifs on Complex-type N-Glycans

Grabenhorst, Eckart; Nimtz, Manfred; Costa, Júlia; Conradt, Harald S.

doi:10.1074/jbc.273.47.30985

Cited by 72 publications

(77 citation statements)

References 56 publications

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“…FT VI has been previously reported to attach peripheral fucose moieties on sialylated as well as nonsialylated antenna of biantennary N-glycans with similar efficiency [20]. Preferred attachment sites in the multiantennary substrates used in this study, which offer both potential acceptor epitopes, were however, unknown.…”

Section: Tetraantennary N-glycan Containing Three Sialic Acids and Onmentioning

confidence: 84%

“…In vitro ␣1-3fucosylation was performed as described previously [20] using GDP-fucose along with a soluble form of recombinant human ␣1-3fucosyltransferase VI which has been shown to add peripheral fucose to asialo as well as to ␣2-3sialylated N-glycans thus forming both the Le x as well the sLe x motifs [21].…”

Section: In Vitro ␣1-3fucosylation Of the Trisialylated N-glycansmentioning

confidence: 99%

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Sequencing of tri- and tetraantennary N-glycans containing sialic acid by negative mode ESI QTOF tandem MS

Sagi¹,

Peter‐Katalinić²,

Conradt

et al. 2002

J. Am. Soc. Mass Spectrom.

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Application of the negative mode electrospray ionization-quadrupole time-of-flight mass spectrometry (ESI QTOF) tandem MS for determination of substitution patterns by sialic acid and/or fucose and extention by additional LacNAc disaccharide units in single branches of multiantennary N-glycans from biological samples is described. Fragmentation patterns which can be obtained by low energy collision-induced dissociation (CID) using the QTOF instrument include cleavage ions, diagnostic for determination of antennarity and for specific structural features of single antennae. Systematic fragmentation studies in the negative ion mode were focussed toward formation of the D diagnostic ion relevant for assignment of 3-and 6-antennae in complex N-glycans carrying three and four antennae in combination with epitope-relevant B-and C-type ions. For validation of this approach ESI QTOF fragmentation of the permethylated analogues was carried out in the positive ion mode. Using this strategy, products of in vitro glycosylation reactions were investigated in order to clarify some general aspects of N-glycan acceptor specificity during biosynthesis. ␣1-3fucosylation using GDPfucose along with a soluble form of the recombinant human ␣1-3fucosyltransferase VI was carried out on tri-and tetraantennary precursors to test structural requirements for formation of Le A number of biologically relevant glycoproteins contain large N-linked complex carbohydrate moieties, proposed to be involved in specific interactions with other molecules and/or cell surfaces. Highly regulated biosynthetic pathways in which Nacetylglucosaminyltransferases are incorporating GlcNAc residues on the mannotriose core are determining factors for the formation of a number of antennae in complex-type N-glycans [1]. In multiantennary glycans different antennae can show different extent of elongation by N-acetyllactosaminyl repeats and number and site of modifications by sialic acid and fucose moieties, contributing to the microheterogeneity on the single glycosylation site. Besides, the expression of a certain oligosaccharide epitope may be restricted to just one of the antennae present. In order to carry on a complete characterization of complex type N-glycan mixtures obtained from glycoproteins after release by Nglycanase, combined chromatographic, mass spectrometric and nuclear magnetic resonance spectroscopic approach can be mandatory, as demonstrated in the case of human erythropoietin (hEPO), a glycoprotein containing bi-, tri-and tetraantennary N-glycan structures [2]. Its biological activity in terms of the clearance rate was positively correlated with the ratio of tetra-to biantennary N-glycans, where the hEPO with wellbranched tetraantennary glycans remained at higher levels in the plasma [3]. Particular attention is to be paid to identification of glycosylation patterns in terms of antennarity in glycoproteins from different organs, since they may express identical protein cores, carrying glycoforms of different antennarity and therefore different ant...

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Section: Tetraantennary N-glycan Containing Three Sialic Acids and Onmentioning

confidence: 84%

Section: In Vitro ␣1-3fucosylation Of the Trisialylated N-glycansmentioning

confidence: 99%

Sequencing of tri- and tetraantennary N-glycans containing sialic acid by negative mode ESI QTOF tandem MS

Sagi¹,

Peter‐Katalinić²,

Conradt

et al. 2002

J. Am. Soc. Mass Spectrom.

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“…Transfection of Chinese hamster ovary (CHO) cells of the DHFR Ϫ strain with human Fuc-TIV led to the expression of CSLEX-1-reactive sLe x ,although no sLe x could be generated by this enzyme in CHO-Pro Ϫ 5 cells (10). In BHK-21 cells, human Fuc-TIV generates Le x seven times more efficiently than sLe x (14). In COS cells, human as well as mouse Fuc-TIV could not generate sLe x -epitopes on the cell surface as defined by the mAb CSLEX-1 (15).…”

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confidence: 97%

The α(1,3)-Fucosyltransferase Fuc-TIV, but Not Fuc-TVII, Generates Sialyl Lewis X-like Epitopes Preferentially on Glycolipids

Huang

Laskowska²,

Vestweber

et al. 2002

Journal of Biological Chemistry

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Fuc-TIV and Fuc-TVII are the two ␣(1, 3)-fucosyltransferases in myeloid cells responsible for the biosynthesis of sialyl Lewis X (sLe x ), the minimal ligand structure for the selectins. We have compared the ability of Fuc-TIV and Fuc-TVII to generate sLe x -like epitopes in transfected Chinese hamster ovary (CHO)-Pro ؊ 5 cells expressing the P-selectin glycoprotein ligand-1 and the core-2 branching enzyme C2GnT. We found that mouse Fuc-TIV and Fuc-TVII can generate similar levels of cell surface sLe x . Surprisingly however, Fuc-TIV-generated sLe x was resistant to proteinase K and trypsin treatment and could be removed from cells by delipidation with chloroform/methanol, whereas 80 -90% of Fuc-TVIIgenerated sLe x was protease-sensitive, and most of it resistant to delipidation. Despite similar levels of sLe x on the cell surface, Fuc-TVII transfectants adhered to immobilized E-selectin-IgG under static and flow conditions better than Fuc-TIV transfectants. Binding was mainly protease sensitive, indicating that glycoproteins were more efficient ligands than glycolipids. In summary, we conclude that the two fucosyltransferases differ in their in vivo specificity for acceptor substrates with Fuc-TVII generating sLe x preferentially on glycoproteins, whereas most of the Fuc-TIV-generated sLe x is found on glycolipids. Interestingly, the non-catalytic portion of Fuc-TIV in a Fuc-TIV/VII chimeric enzyme mediated the specificity for glycolipid substrates.The three known selectins, L-, E-, and P-selectin are cell adhesion molecules that initiate interactions between leukocytes and endothelial cells during leukocyte extravasation (1). The minimal ligand structure for all three selectins is the tetrasaccharide sialyl Lewis X (sLe x ). 1

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“…Also, the maturation process of N-glycans in the Golgi apparatus involves elongation reactions that are performed by N-acetylglucosaminyl-transferase, galactosyl-, fucosyl-and sialyltransferases, resulting in biantennary, triantennary or tetraantennary oligosaccharides (Breton et al, 1998;Grabenhorst et al, 1998;Roth 2002). These types of glycosylation have been reported for KLKs, while the unbranched N-glycosaminglycan (GAG, mucopolysaccharide) trees have not yet been observed for them.…”

Section: Introductionmentioning

confidence: 99%

Sweetened kallikrein-related peptidases (KLKs): glycan trees as potential regulators of activation and activity

Guo

Skala

Magdolen

et al. 2014

Biological Chemistry

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Most kallikrein-related peptidases (KLKs) are N-glycosylated with N-acetylglucosamine 2 -mannose 9 units at Asn-Xaa-Ser/Thr sequons during protein synthesis and translocation into the endoplasmic reticulum. These N-glycans are modified in the Golgi machinery, where additional O-glycosylation at Ser and Thr takes place, before exocytotic release of the KLKs into the extracellular space. Sequons are present in all 15 members of the KLKs and comparative studies for KLKs from natural and recombinant sources elucidated some aspects of glycosylation. Although glycosylation of mammalian KLKs 1, 3, 4, 6, and 8 has been analyzed in great detail, e.g., by crystal structures, the respective function remains largely unclear. In some cases, altered enzymatic activity was observed for KLKs upon glycosylation. Remarkably, for KLK3/PSA, changes in the glycosylation pattern were observed in samples of benign prostatic hyperplasia and prostate cancer with respect to healthy individuals. Potential functions of KLK glycosylation in structural stabilization, protection against degradation, and activity modulation of substrate specificity can be deduced from a comparison with other glycosylated proteins and their regulation. According to the new concept of protein sectors, glycosylation distant from the active site might significantly influence the activity of proteases. Novel pharmacological approaches can exploit engineered glycans in the therapeutical context.

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In Vivo Specificity of Human α1,3/4-Fucosyltransferases III-VII in the Biosynthesis of LewisX and Sialyl LewisX Motifs on Complex-type N-Glycans

Cited by 72 publications

References 56 publications

Sequencing of tri- and tetraantennary N-glycans containing sialic acid by negative mode ESI QTOF tandem MS

Sequencing of tri- and tetraantennary N-glycans containing sialic acid by negative mode ESI QTOF tandem MS

The α(1,3)-Fucosyltransferase Fuc-TIV, but Not Fuc-TVII, Generates Sialyl Lewis X-like Epitopes Preferentially on Glycolipids

Sweetened kallikrein-related peptidases (KLKs): glycan trees as potential regulators of activation and activity

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