Expression and functional characterization of the P-selectin glycoprotein ligand-1 in various cells.
We have examined the expression and function of P-selectin glycoprotein ligand-1 (PSGL-1), which is a high affinity ligand for P-selectin. Northern blot and flow cytometric analysis demonstrated that a variety of hematopoietic cells and cell lines expressed PSGL-1. However, P-selectin binding ability was dependent on the additional expression of a carbohydrate structure, sialyl Lewis x (sLex). All the peripheral lymphocytes expressed PSGL-1 and subpopulation expressed sLex. Two color analysis showed that the majority of the cells that bound P-selectin were sLex-negative I lymphocytes, and most of the sLex-positive cells were B lymphocytes that did not blind P-selectin, indicating that the carbohydrate on T lymphocytes recognized by P-selectin is not sLex, and that the sLex on B lymphocytes is not readily presented for P-selectin recognition. Transfected 293 cells detectably bound P-selectin only when the cells expressed both PSGL-1 and sLex. When cysteine 310 of PSGL-1 was mutated to alanine, P-selectin binding was markedly reduced, suggesting the importance of dimerization of PSGL-1. These findings indicate that a preferable conformation of both carbohydrate and protein structure is necessary for a functional P-selectin ligand.
Fujimoto
T T
TT
Department of Hematology and Oncology, Hiroshima University, Japan. fujimot@ipc.hiroshima-u.ac.jp
Noda
M
M
Takafuta
T
T
Shimomura
T
T
Fujimura
K
K
Kuramoto
A
A
D001402:B-Lymphocytes / Q000378:metabolism; D002236:Carbohydrate Conformation; D002478:Cells, Cultured; D006412:Hematopoietic Stem Cells / Q000378:metabolism*; D006801:Humans; D008562:Membrane Glycoproteins / Q000032:analysis* / Q000378:metabolism / Q000502:physiology; D009844:Oligosaccharides / Q000378:metabolism; D019007:P-Selectin / Q000378:metabolism; D011485:Protein Binding; D011487:Protein Conformation; D000080506:Sialyl Lewis X Antigen; D013601:T-Lymphocytes / Q000378:metabolism
D008562:Membrane Glycoproteins; D009844:Oligosaccharides; D019007:P-Selectin; C084882:P-selectin ligand protein; D000080506:Sialyl Lewis X Antigen