Post-translational modifications of recombinant P-selectin glycoprotein ligand-1 required for binding to P- and E-selectin

P-selectin glycoprotein ligand-1 (PSGL-1) is a mucin-like ligand for P- and E-selectin on human leukocytes. PSGL-1 requires sialylated, fucosylated O-linked glycans and tyrosine sulfate to bind P-selectin. Less is known about the determinants that PSGL-1 requires to bind E-selectin. To further define the modifications required for PSGL-1 to bind P- and E-selectin, we transfected Chinese hamster ovary (CHO) cells with cDNAs for PSGL-1 and specific glycosyltransferases. CHO cells synthesize only core 1 O-linked glycans (Gal beta 1-3Gal-NAc alpha 1-Ser/Thr); they lack core 2 O-linked glycans (Gal beta 1-3(Gal beta 1-4GlcNAc beta 1-6)GalNAc alpha 1- Ser/Thr) because they do not express the core 2 beta 1-6-N-acetylglucosaminyltransferase (C2GnT), CHO cells also lack alpha 1-3 fucosyltransferase activity. PSGL-1 expressed on transfected CHO cells bound P- and E-selectin only when it was co-expressed with both C2GnT and an alpha 1-3 fucosyl-transferase (Fuc-TIII, Fuc-TIV, or Fuc-TVII). Chromatography of beta-eliminated O-linked glycans from PSGL-1 co-expressed with C2GnT confirmed synthesis of core 2 structures. Tyrosine residues on PSGL-1 expressed in CHO cells were shown to be sulfated. Phenylalanine replacement of three tyrosines within a consensus sequence for tyrosine sulfation abolished binding to P-selectin but not to E-selectin, These results demonstrate that PSGL-1 requires core 2 O-linked glycans that are sialylated and fucosylated to bind P- and E-selectin, PSGL-1 also requires tyrosine sulfate to bind P-selectin but not E-selectin.