Core2 beta-1,6-N-acetylglucosaminyltransferase enzyme activity is critical for P-selectin glycoprotein ligand-1 binding to P-selectin.
P-selectin glycoprotein ligand-1 (PSGL-1) is a high-affinity counterreceptor for P-selectin on myeloid cells and activated T-cells. In addition, PSGL-1 can serve, both in vitro and in vivo, as an E-selectin ligand. Appropriate glycosylation of PSGL-1 is crucial for binding to P-selectin. Functional PSGL-1 is known to bear sialyl lewis X (SLex) or a closely related oligosaccharide. In this study, we show that Chinese hamster ovary (CHO) cells expressing PSGL-1 and fucosyltransferase show a dramatic increase in binding to P-selectin when transfected with "core2" transferase, the enzyme that initiates branching of O-linked glycans. Moreover, only PSGL-1 from core2 transfectant CHO cells can be affinity-captured with P-selectin, suggesting that branched O-linked glycans are required for high-affinity binding to P-selectin. Analysis of PSGL-1-derived O-linked oligosaccharides produced in core2 transfected cells shows the presence of more elaborated glycans. Interestingly, transfection of core2 in these cells does not alter binding to E-selectin.
Small Molecule Drug Discovery, Genetics Institute, Cambridge, MA 02140, USA.
R
R
Kumar
R T
RT
Camphausen
F X
FX
Sullivan
D A
DA
Cumming
D000818:Animals; D016466:CHO Cells; D002236:Carbohydrate Conformation; D006224:Cricetinae; D003412:Cricetulus; D019040:E-Selectin / Q000378:metabolism; D005647:Fucosyltransferases / Q000378:metabolism; D005732:Gangliosides / Q000378:metabolism; D006031:Glycosylation; D006801:Humans; D008024:Ligands; D008562:Membrane Glycoproteins / Q000737:chemistry / Q000378:metabolism*; D017351:N-Acetylglucosaminyltransferases / Q000235:genetics / Q000502:physiology*; D009844:Oligosaccharides / Q000378:metabolism; D019007:P-Selectin / Q000378:metabolism*; D011485:Protein Binding; D011499:Protein Processing, Post-Translational; D011993:Recombinant Fusion Proteins / Q000378:metabolism; D000080506:Sialyl Lewis X Antigen; D014162:Transfection
D019040:E-Selectin; D005732:Gangliosides; D008024:Ligands; D008562:Membrane Glycoproteins; D009844:Oligosaccharides; D019007:P-Selectin; C084882:P-selectin ligand protein; D011993:Recombinant Fusion Proteins; D000080506:Sialyl Lewis X Antigen; C094688:sialyl Lewis(x) ganglioside; D005647:Fucosyltransferases; D017351:N-Acetylglucosaminyltransferases; C053024:beta-1,3-galactosyl-O-glycosyl-glycoprotein beta-1,6-acetylglucosaminyl transferase; C019818:3-galactosyl-N-acetylglucosaminide 4-alpha-L-fucosyltransferase