Reconstitution of functional L-selectin ligands on a cultured human endothelial cell line by cotransfection of α1→3 fucosyltransferase VII and newly cloned GlcNAcβ:6-sulfotransferase cDNA

N. Kimura, C. Mitsuoka, A. Kanamori, N. Hiraiwa, K. Uchimura, T. Muramatsu, T. Tamatani, G. S. Kansas, R. Kannagi*

*Corresponding author for this work

Research output: Contribution to journalArticlepeer-review

114 Scopus citations

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 α1→3fucosyltranseferase VII (Fuc-T VII) and newly cloned GlcNAcβ: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.

Original languageEnglish (US)
Pages (from-to)4530-4535
Number of pages6
JournalProceedings of the National Academy of Sciences of the United States of America
Volume96
Issue number8
DOIs
StatePublished - Apr 13 1999

ASJC Scopus subject areas

  • General

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