Distinct sulfation requirements of selectins disclosed using cells that support rolling mediated by all three selectins under shear flow. L-selectin prefers carbohydrate 6-sulfation totyrosine sulfation, whereas p-selectin does not.
l- and P-selectin are known to require sulfation in their ligand molecules. We investigated the significance of carbohydrate 6-sulfation and tyrosine sulfation in selectin-mediated cell adhesion. COS-7 cells were genetically engineered to express P-selectin glycoprotein ligand-1 (PSGL-1) or its mutant in various combinations with 6-O-sulfotransferase (6-Sul-T) and/or alpha1-->3fucosyltransferase VII (Fuc-T VII). The cells transfected with PSGL-1, 6-Sul-T, and Fuc-T VII cDNAs supported rolling mediated by all three selectins and provided the best experimental system so far to estimate kinetic parameters in selectin-mediated cell adhesion for all three selectins using the identical rolling substrate and to compare the ligand specificity of each selectin. L-selectin-mediated rolling was drastically impaired if the cells lacked carbohydrate 6-sulfation elaborated by 6-Sul-T, but not affected when PSGL-1 was replaced with a mutant lacking three tyrosine residues at its NH(2) terminus. L-selectin-mediated adhesion was also hardly affected by mocarhagin treatment of the cells, which cleaved a short peptide containing sulfated tyrosine residues from PSGL-1. In contrast, P-selectin-mediated rolling was abolished when PSGL-1 was either mutated or cleaved by mocarhagin at its NH(2) terminus, whereas the cells expressing PSGL-1 and Fuc-T VII but not 6-Sul-T showed only a modest decrease in P-selectin-mediated adhesion. These results indicate that L-selectin prefers carbohydrate 6-sulfation much more than tyrosine sulfation, whereas P-selectin favors tyrosine sulfation in the PSGL-1 molecule far more than carbohydrate 6-sulfation. E-selectin-mediated adhesion was sulfation-independent requiring only Fuc-T VII, and thus the three members of the selectin family have distinct requirements for ligand sulfation.
Kanamori
Akiko
A
Program of Molecular Pathology, Aichi Cancer Center, Research Institute, Nagoya 464-8681, Japan.
Kojima
Naoya
N
Uchimura
Kenji
K
Muramatsu
Takashi
T
Tamatani
Takuya
T
Berndt
Michael C
MC
Kansas
Geoffrey S
GS
Kannagi
Reiji
R
D000818:Animals; D000911:Antibodies, Monoclonal / Q000378:metabolism; D018952:Antigens, CD34 / Q000096:biosynthesis; D019556:COS Cells; D050260:Carbohydrate Metabolism; D002448:Cell Adhesion; D004546:Elapid Venoms / Q000494:pharmacology; D005434:Flow Cytometry; D005647:Fucosyltransferases / Q000378:metabolism; D017353:Gene Deletion; D005822:Genetic Vectors; D006801:Humans; D019041:L-Selectin / Q000378:metabolism*; D008024:Ligands; D008666:Metalloendopeptidases / Q000494:pharmacology; D019007:P-Selectin / Q000378:metabolism*; D017434:Protein Structure, Tertiary; D013379:Substrate Specificity; D015238:Sulfotransferases / Q000378:metabolism; D013455:Sulfur / Q000378:metabolism*; D014162:Transfection; D014443:Tyrosine / Q000031:analogs & derivatives / Q000737:chemistry / Q000378:metabolism*
D000911:Antibodies, Monoclonal; D018952:Antigens, CD34; D004546:Elapid Venoms; D008024:Ligands; D019007:P-Selectin; D019041:L-Selectin; C005662:tyrosine O-sulfate; D014443:Tyrosine; D013455:Sulfur; D005647:Fucosyltransferases; C031692:galactoside 3-fucosyltransferase; D015238:Sulfotransferases; D008666:Metalloendopeptidases; C099092:mocarhagin