Reconstitution of functional L-selectin ligands on a cultured human endothelial cell line by cotransfection of alpha1-->3 fucosyltransferase VII and newly cloned GlcNAcbeta:6-sulfotransferase cDNA

Proc Natl Acad Sci U S A. 1999 Apr 13;96(8):4530-5. doi: 10.1073/pnas.96.8.4530.

Abstract

Recently, we proposed sialyl 6-sulfo Lewis X as a major carbohydrate-capping group of the L-selectin ligands on high endothelial venules in human lymph nodes. In this study we succeeded in reconstituting functional L-selectin ligands on a cultured human endothelial cell line, ECV304, by transfecting the alpha1-->3fucosyltranseferase VII (Fuc-T VII) and newly cloned GlcNAcbeta:6-sulfotransferase (6-Sul-T) cDNAs. The ECV304 cells transfected with Fuc-T VII cDNA expressed conventional sialyl Lewis X detected with specific antibodies including 2H5, whereas the cells transfected with 6-Sul-T cDNA expressed sialyl 6-sulfo lactosamine as well as MECA-79-defined carbohydrate determinants, but these singly transfected cells failed to express sialyl 6-sulfo Lewis X, as detected with the antisialyl 6-sulfo Lewis X mAb G152. Sialyl 6-sulfo Lewis X appeared only on the cells that were cotransfected with both 6-Sul-T and Fuc-T VII cDNAs. Significant adhesion of L-selectin-expressing cells was seen only to the doubly transfected ECV304 cells and was inhibited by G152. No adhesion was observed to the cells transfected either with 6-Sul-T or with Fuc-T VII cDNA alone. The mRNAs of the two enzymes were expressed or were inducible upon interleukin 1 stimulation in human endothelial cells. These results indicate that a set of carbohydrate determinants synthesized by the concerted action of the two enzymes, as typically represented by the sialyl 6-sulfo Lewis X-capping group, serves as an essential component of the ligand for L-selectin and that the reagents 2H5 and MECA-79, utilized in earlier studies to detect L-selectin ligand on high endothelial venules, recognize two different aspects of the same set of synthetic products.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Antibodies, Monoclonal
  • Carbohydrate Sequence
  • Cell Line
  • Cloning, Molecular
  • DNA, Complementary
  • Endothelium, Vascular / cytology
  • Endothelium, Vascular / physiology*
  • Fucosyltransferases / genetics
  • Fucosyltransferases / metabolism*
  • Genetic Variation
  • Humans
  • L-Selectin / physiology*
  • Lymph Nodes / immunology
  • Molecular Sequence Data
  • Oligosaccharides / biosynthesis*
  • Oligosaccharides / chemistry
  • Oligosaccharides / genetics
  • Sialyl Lewis X Antigen
  • Sulfotransferases / genetics
  • Sulfotransferases / metabolism*
  • Transfection
  • Umbilical Veins

Substances

  • Antibodies, Monoclonal
  • DNA, Complementary
  • Oligosaccharides
  • Sialyl Lewis X Antigen
  • L-Selectin
  • Fucosyltransferases
  • galactoside 3-fucosyltransferase
  • Sulfotransferases
  • carbohydrate sulfotransferases