Equine PSGL-1 modifications required for P-selectin binding.

Area of Science:

• Immunology
• Glycobiology
• Molecular Biology

Background:

• Equine P-selectin glycoprotein ligand-1 (ePSGL-1) is expressed on peripheral blood mononuclear cells (PBMCs) as a sialylated homodimer.
• Sialylation contributes to P-selectin binding affinity, but other post-translational modifications may also be involved.

Purpose of the Study:

• To investigate the specific post-translational modifications required for high-affinity P-selectin binding to ePSGL-1.
• To identify the functional binding domain of ePSGL-1.

Main Methods:

• Transfection of Chinese Hamster Ovary (CHO) cells with ePSGL-1 and equine FucT-VII and/or C2GnT enzymes.
• Binding assays using P-selectin-IgG chimera.
• Enzymatic removal and site-directed mutagenesis of N-glycans.
• Cleavage with O-sialoglycoprotein endopeptidase (OSGP).
• Point-mutation deletion techniques to map the binding domain.

Main Results:

• High-affinity P-selectin binding occurred only when both FucT-VII and C2GnT were co-expressed, indicating the necessity of fucosylation and core-2 branching.
• N-glycosylation was not required for P-selectin binding.
• OSGP cleavage confirmed the presence of clustered, sialylated O-glycans within the binding domain.
• The ePSGL-1 binding domain was localized between residues 48 and 100.
• Dimerization was essential, while sulfation was not required for binding.

Conclusions:

• Fucosylation and core-2 branching on O-glycans are critical post-translational modifications for high-affinity ePSGL-1 binding to P-selectin.
• N-glycosylation is dispensable for this interaction.
• The species-specific glycosylation requirements of ePSGL-1 offer insights into mononuclear cell trafficking mechanisms.