TY - JOUR
T1 - 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
AU - Kimura, N.
AU - Mitsuoka, C.
AU - Kanamori, A.
AU - Hiraiwa, N.
AU - Uchimura, K.
AU - Muramatsu, T.
AU - Tamatani, T.
AU - Kansas, G. S.
AU - Kannagi, R.
PY - 1999/4/13
Y1 - 1999/4/13
N2 - 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.
AB - 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.
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U2 - 10.1073/pnas.96.8.4530
DO - 10.1073/pnas.96.8.4530
M3 - Article
C2 - 10200296
AN - SCOPUS:0033551049
SN - 0027-8424
VL - 96
SP - 4530
EP - 4535
JO - Proceedings of the National Academy of Sciences of the United States of America
JF - Proceedings of the National Academy of Sciences of the United States of America
IS - 8
ER -