Abstract
Interactions between the leukocyte adhesion receptor L-selectin and P-selectin glycoprotein ligand-1 play an important role in regulating the inflammatory response by mediating leukocyte tethering and rolling on adherent leukocytes. In this study, we have examined the effect of post-translational modifications of PSGL-1 including Tyr sulfation and presentation of sialylated and fucosylated O-glycans for L-selectin binding. The functional importance of these modifications was determined by analyzing soluble L-selectin binding and leukocyte rolling on CHO cells expressing various glycoforms of PSGL-1 or mutant PSGL-1 targeted at N-terminal Thr or Tyr residues. Simultaneous expression of core-2 beta1,6-N-acetylglucosaminyltransferase and fucosyltransferase VII was required for optimal L-selectin binding to PSGL-1. Substitution of Thr-57 by Ala but not of Thr-44, strongly decreased L-selectin binding and leukocyte rolling on PSGL-1. Substitution of Tyr by Phe revealed that PSGL-1 Tyr-51 plays a predominant role in mediating L-selectin binding and leukocyte rolling whereas Tyr-48 has a minor role, an observation that contrasts with the pattern seen for the interactions between PSGL-1 and P-selectin where Tyr-48 plays a key role. Molecular modeling analysis of L-selectin and P-selectin interactions with PSGL-1 further supported these observations. Additional experiments showed that core-2 O-glycans attached to Thr-57 were also of critical importance in regulating the velocity and stability of leukocyte rolling. These observations pinpoint the structural characteristics of PSGL-1 that are required for optimal interactions with L-selectin and may be responsible for the specific kinetic and mechanical bond properties of the L-selectin-PSGL-1 adhesion receptor-counterreceptor pair.
Highlights
Interactions between the leukocyte adhesion receptor L-selectin and P-selectin glycoprotein ligand-1 play an important role in regulating the inflammatory response by mediating leukocyte tethering and rolling on adherent leukocytes
Sialylated, Fucosylated, Core-2 O-Glycans Are Essential to Support Leukocyte Rolling on P-selectin glycoprotein ligand-1 (PSGL-1)—The requirement in sLex/cutaneous lymphocyte antigen (CLA) and core-2 O-glycans to support L-selectin-mediated interactions with PSGL-1 was examined by analyzing L-selectin/ chimera binding and leukocyte rolling on CHO cells cotransfected with PSGL-1 cDNA in pcDNA3.1 vector and/or FucT-VII cDNA in pZeo SV and/or C2GnT and FucT-VII cDNAs in pIRES Zeo SV vector
The crystal structure analysis of P-selectin co-complexed with the N-terminal peptide of PSGL-1 showed that O-glycans terminated by sLex/CLA as well as Tyr-48 and -51 play an essential role in supporting P-selectin binding
Summary
Antibodies and Chimeric Selectins—The anti-L-selectin monoclonal antibodies (mAbs) LAM1–3, LAM-14 [36], HECA– 452 (ATCC HB 11485), and CSLEX-1 (ATCC number: HB-10135) were purified from hybridoma culture medium. mAbs PL1 and PL2 were purchased from Coulter Immunotech (Marseille, France) and KPL1 from BD Bioscience PharMingen (Heidelberg, Germany). The expression cassette was constructed by inserting the C2GnT sequence followed by the IRES and the FucT-VII cDNA sequences in the multiple cloning site of the pZeo SV vector. CHO cells were co-transfected with FucT-VII cDNA subcloned in pZeoSV vector (Invitrogen) or in the pIRES Zeo SV expression vector, which allows the simultaneous translation of C2GnT and FucT-VII sequences. The determination of sLex and CLA expression indicated that CHO cells used for L-selectin/ binding studies and adhesion assays expressed comparable levels of FucT-VII activity. In experiments performed to evaluate the role of sialic acid residues in supporting neutrophil rolling on PSGL-1, CHOPSGL-1 cells, co-expressing C2GnT and FucT-VII, were cultured for 30 min at 37 °C in PBS containing 0.1 units/ml Vibrio cholerae neuraminidase (Roche Diagnostics). The Mann-Whitney test was used to compare the medians of two unpaired groups. p values of Ͻ0.05 were considered as significant
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