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.
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.
Kimura
N
N
Program of Experimental Pathology, Aichi Cancer Center, Nagoya 464-8681, Japan.
Mitsuoka
C
C
Kanamori
A
A
Hiraiwa
N
N
Uchimura
K
K
Muramatsu
T
T
Tamatani
T
T
Kansas
G S
GS
Kannagi
R
R
D000911:Antibodies, Monoclonal; D002240:Carbohydrate Sequence; D002460:Cell Line; D003001:Cloning, Molecular; D018076:DNA, Complementary; D004730:Endothelium, Vascular / Q000166:cytology / Q000502:physiology*; D005647:Fucosyltransferases / Q000235:genetics / Q000378:metabolism*; D014644:Genetic Variation; D006801:Humans; D019041:L-Selectin / Q000502:physiology*; D008198:Lymph Nodes / Q000276:immunology; D008969:Molecular Sequence Data; D009844:Oligosaccharides / Q000096:biosynthesis* / Q000737:chemistry / Q000235:genetics; D000080506:Sialyl Lewis X Antigen; D015238:Sulfotransferases / Q000235:genetics / Q000378:metabolism*; D014162:Transfection; D014471:Umbilical Veins; D000097811:Carbohydrate Sulfotransferases
D000911:Antibodies, Monoclonal; D018076:DNA, Complementary; D005647:Fucosyltransferases; D019041:L-Selectin; D009844:Oligosaccharides; D000080506:Sialyl Lewis X Antigen; D015238:Sulfotransferases; D000097811:Carbohydrate Sulfotransferases; C031692:galactoside 3-fucosyltransferase