Extended Core 1 and Core 2 Branched O-Glycans Differentially Modulate Sialyl Lewis x-type L-selectin Ligand Activity

Abstract

It has been established that sialyl Lewis x in core 2 branched O-glycans serves as an E-and P-selectin ligand. Recently, it was discovered that 6-sulfosialyl Lewis x in extended core 1 O-glycans, NeuNAc␣233Gal-␤134(Fuc␣133(sulfo36))GlcNAc␤13 3Gal␤133Gal-NAc␣13 Ser/Thr, functions as an L-selectin ligand in high endothelial venules. Extended core 1 O-glycans can be synthesized when a core 1 extension enzyme is present. In this study, we first show that ␤1,3-N-acetylglucosaminyltransferase-3 (␤3GlcNAcT-3) is almost exclusively responsible for core 1 extension among seven different ␤3GlcNAcTs and thus acts on core 1 O-glycans attached to PSGL-1. We found that transcripts encoding ␤3GlcNAcT-3 were expressed in human neutrophils and lymphocytes but that their levels were lower than those of transcripts encoding core 2 ␤1,6-N-acetylglucosaminyltransferase I (Core2GlcNAcT-I). Neutrophils also expressed transcripts encoding fucosyltransferase VII (FucT-VII) and Core2GlcNAcT-I, whereas lymphocytes expressed only small amounts of transcripts encoding FucT-VII. To determine the roles of sialyl Lewis x in extended core 1 O-glycans, Chinese hamster ovary (CHO) cells were stably transfected to express PSGL-1, FucT-VII, and either ␤3GlcNAcT-3 or Core2GlcNAcT-I. Glycan structural analyses disclosed that PSGL-1 expressed in these transfected cells carried comparable amounts of sialyl Lewis x in extended core 1 and core 2 branched O-glycans. In a rolling assay, CHO cells expressing sialyl Lewis x in extended core 1 O-glycans supported a significant degree of shear-dependent tethering and rolling of neutrophils and lymphocytes, although less than CHO cells expressing sialyl Lewis x in core 2 branched O-glycans. These results indicate that sialyl Lewis x in extended core 1 O-glycans can function as an L-selectin ligand and is potentially involved in neutrophil adhesion on neutrophils bound to activated endothelial cells.Mucin-type O-glycans are unique in having clusters of large numbers of O-glycans. These O-glycans contain N-acetylgalactosamine residues at reducing ends, which are linked to serine or threonine residues in a polypeptide (1). These attached Oglycans can be classified into several different groups according to the core structure (2). In many cells, a structure called core 1, Gal␤133GalNAc, is the major constituent of O-glycans. Core 1 oligosaccharides are converted to core 2 oligosaccharides, Gal␤133(GlcNAc␤136)GalNAc, when core 2 ␤1,6-N-acetylglucosaminyltransferase (Core2GlcNAcT) 1 is present (3, 4). Various ligand carbohydrates can be formed in core 2 branched oligosaccharides. For example, sialyl Lewis x in mucin-type glycoproteins of blood cells can be found in core 2 branched oligosaccharides such as NeuNAc␣233Gal␤134(Fuc␣133)GlcNAc␤136(NeuNAc␣23 3Gal␤133)GalNAc␣13 Ser/Thr (see Fig.

Introducing α(1,2)‐linked fucose into hepatocarcinoma cells inhibits vasculogenesis and tumor growth

“…13,37 It is proposed that glycosyltransferases are organised in the same order in which they act; such that those acting early in glycan biosynthesis are localised to the cis and medial compartments, whereas enzymes acting later tend to localize to the transGolgi cisternae and the trans-Golgi network (reviewed in 38 ). Accordingly, the sLe x -specific a(2,3)-sialyltransferases (denoted as ST3GalTs) have been proposed to localize to the late Golgi compartments 39,40 and our earlier studies have shown that FUT1 overlaps very well with the a-mannosidase-II and the core2 b(1,6)-N-acetyglucosaminyltransferase (C2GnT-I) in the cis/ medial Golgi compartments.…”

“…11 HepG2 cells infected with particles of pRRLpgk-GFP.sin18 to express EGFP alone are denoted as parental cells throughout the manuscript. The construct for E-selectin conjugated to the human IgM portion 13 (denoted as E-selectin-IgM chimera) was kindly provided by Dr Minoru Fukuda (The Burnham Institute, La Jolla, CA). The E-selectin-IgM chimera was produced by transiently transfecting the human 293-T cells as described.…”

Core3 O-Glycan Synthase Suppresses Tumor Formation and Metastasis of Prostate Carcinoma PC3 and LNCaP Cells through Down-regulation of α2β1 Integrin Complex

“…For this assay, PC3 and LNCaP cells in cloning plates were dissociated into monodispersed cells using an enzyme-free dissociation solution (Hanks' balanced saline solution-based) purchased from Cell and Molecular Technologies. Dissociated cells were incubated with fluorescein isothiocyanate-conjugated PNA and subjected to fluorescence-activated cell sorting analysis using FACScan flow cytometry (BD Biosciences) as described previously (26). As controls, PC3 and LNCaP cells were transfected with empty pcDNA 3.1(N) and pcDNA 3 and selected in Geneticin.…”

“…cDNA encoding core3 synthase (␤3GlcNAcT-6) (15) was amplified by reverse transcription (RT)-PCR and cloned into pcDNA 3.1(N) as described previously (26). pcDNA 3.1(N) was prepared by deleting the Zeocin resistance gene and f1 origin from pcDNA 3.1/Zeo as described previously (26). PC3 and LNCaP cells were transfected with pcDNA 3.1(N) harboring core3-synthase cDNA and pcDNA 3 harboring the neomycin resistance gene using Lipofectamine.…”

Endoglycan, a Member of the CD34 Family, Functions as an L-selectin Ligand through Modification with Tyrosine Sulfation and Sialyl Lewis x

“…Mounting evidence indicates these features are accommodated in the capping group 6-sulfo sLex in which the sLex tetrasaccharide (Sia␣2,3Gal␤1,4-[Fuc␣1,3] GlcNAc) is modified with sulfation on the C-6 position of GlcNAc (7)(8)(9). This structure can be presented as the terminus of both a core-2 branch and an extended core-1 branch of an O-glycan, and contributes to ligand activity in both positions (9,10). A potential role for Gal-6-sulfate has also been suggested for ligands in the mouse, but the structural context for this modification is presently unknown (7,11).…”