Extended core 1 and core 2 branched O-glycans differentially modulate sialyl Lewis X-type L-selectin ligand activity

J Biol Chem. 2003 Mar 14;278(11):9953-61. doi: 10.1074/jbc.M212756200. Epub 2003 Jan 15.

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, NeuNAcalpha2-->3Galbeta1-->4(Fucalpha1-->3(sulfo-->6))GlcNAcbeta1--> 3Galbeta1-->3GalNAcalpha1-->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 beta1,3-N-acetylglucosaminyltransferase-3 (beta3GlcNAcT-3) is almost exclusively responsible for core 1 extension among seven different beta3GlcNAcTs and thus acts on core 1 O-glycans attached to PSGL-1. We found that transcripts encoding beta3GlcNAcT-3 were expressed in human neutrophils and lymphocytes but that their levels were lower than those of transcripts encoding core 2 beta1,6-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 beta3GlcNAcT-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.

Publication types

  • Research Support, U.S. Gov't, P.H.S.

MeSH terms

  • Animals
  • CHO Cells
  • Carbohydrate Sequence
  • Chromatography, High Pressure Liquid
  • Cloning, Molecular
  • Cricetinae
  • Dose-Response Relationship, Drug
  • Flow Cytometry
  • HeLa Cells
  • Humans
  • L-Selectin / chemistry*
  • L-Selectin / metabolism
  • Ligands
  • Lymphocytes / immunology
  • Lymphocytes / metabolism
  • Models, Biological
  • Molecular Sequence Data
  • N-Acetylglucosaminyltransferases / chemistry*
  • N-Acetylglucosaminyltransferases / immunology
  • Neutrophils / immunology
  • Neutrophils / metabolism
  • Oligosaccharides / chemistry*
  • Oligosaccharides / immunology
  • Polysaccharides / chemistry*
  • Protein Binding
  • Protein Structure, Tertiary
  • RNA, Messenger / metabolism
  • Reverse Transcriptase Polymerase Chain Reaction
  • Sialyl Lewis X Antigen
  • Transfection

Substances

  • Ligands
  • Oligosaccharides
  • Polysaccharides
  • RNA, Messenger
  • Sialyl Lewis X Antigen
  • L-Selectin
  • N-Acetylglucosaminyltransferases
  • beta-1,3-N--acetylglucosaminyltransferase T3