Role of O-linked carbohydrate chains on leukocyte cell membranes in platelet-induced leukocyte activation.
We previously reported that activated platelets stimulated neutrophils and monocytes to produce superoxide anion (O2-) through the interaction between P-selectin and its carbohydrate ligand, sialyl Lewis X (sLeX) (Nagata, K., Tsuji, T., Todoroki, N., Katagiri, Y., Tanoue, K., Yamazaki, H., Hanai N., and Irimura, T. (1993) J. Immunol. 151, 3267-3273). In the present study, we investigated the role of cell surface carbohydrate chains of leukocytes in this process. Glycoconjugate-specific hydrolytic enzymes and inhibitors of glycosylation processing were applied. Granulocyte-like differentiated HL-60 (gHL-60) cells released an increased amount of O2- in response to activated platelets in a P-selectin-dependent manner. When HL-60 cells were differentiated in the presence of benzyl-alpha-N-acetylgalactosaminide (Bzl-alpha-GalNAc), an inhibitor of chain elongation of O-linked carbohydrates, the enhanced generation of O2- was abrogated in parallel with decrease in the expression of sLex structure and in the adhesion capacity to activated platelets. In contrast, treatment with swainsonine or 1-deoxymannojirimycin, inhibitors of processing of N-linked carbohydrate chains, did not show such effects. O-Sialoglycoprotease treatment of gHL-60 cells decreased the activated platelet-induced O2- production with concomitant reduction of cell surface sLex expression. Treatment of these cells with N-glycanase did not affect the O2- production. These results strongly suggested that serine/threonine-linked carbohydrate chains containing sLex played an essential role in the P-selectin-mediated leukocyte activation. By Western blotting analysis of gHL-60 cell lysates, we identified two glycoproteins which carried sLex structures and were sensitive to Bzl-alpha-GalNAc treatment.
Nagata
K
K
Division of Chemical Toxicology and Immunochemistry, Faculty of Pharmaceutical Sciences, University of Tokyo, Japan.
Tsuji
T
T
Hanai
N
N
Irimura
T
T
D001792:Blood Platelets / Q000502:physiology*; D002241:Carbohydrates / Q000502:physiology*; D002462:Cell Membrane / Q000502:physiology; D006023:Glycoproteins / Q000378:metabolism; D006031:Glycosylation; D006801:Humans; D007962:Leukocytes / Q000276:immunology*; D009844:Oligosaccharides / Q000378:metabolism; D019007:P-Selectin; D015539:Platelet Activation; D010980:Platelet Membrane Glycoproteins / Q000378:metabolism; D000080506:Sialyl Lewis X Antigen; D013481:Superoxides / Q000378:metabolism
D002241:Carbohydrates; D006023:Glycoproteins; D009844:Oligosaccharides; D019007:P-Selectin; D010980:Platelet Membrane Glycoproteins; D000080506:Sialyl Lewis X Antigen; D013481:Superoxides