Evolutionarily conserved paired immunoglobulin-like receptor α (PILRα) domain mediates its interaction with diverse sialylated ligands

Abstract

Paired immunoglobulin-like receptor (PILR) α is an inhibitory receptor that recognizes several ligands, including mouse CD99, PILR-associating neural protein, and Herpes simplex virus-1 glycoprotein B. The physiological function(s) of interactions between PILRα and its cellular ligands are not well understood, as are the molecular determinants of PILRα/ligand interactions. To address these uncertainties, we sought to identify additional PILRα ligands and further define the molecular basis for PILRα/ligand interactions. Here, we identify two novel PILRα binding partners, neuronal differentiation and proliferation factor-1 (NPDC1), and collectin-12 (COLEC12). We find that sialylated O-glycans on these novel PILRα ligands, and on known PILRα ligands, are compulsory for PILRα binding. Sialylation-dependent ligand recognition is also a property of SIGLEC1, a member of the sialic acid-binding Ig-like lectins. SIGLEC1 Ig domain shares ∼22% sequence identity with PILRα, an identity that includes a conserved arginine localized to position 97 in mouse and human SIGLEC1, position 133 in mouse PILRα and position 126 in human PILRα. We observe that PILRα/ligand interactions require conserved PILRα Arg-133 (mouse) and Arg-126 (human), in correspondence with a previously reported requirement for SIGLEC1 Arg-197 in SIGLEC1/ligand interactions. Homology modeling identifies striking similarities between PILRα and SIGLEC1 ligand binding pockets as well as at least one set of distinctive interactions in the galactoxyl-binding site. Binding studies suggest that PILRα recognizes a complex ligand domain involving both sialic acid and protein motif(s). Thus, PILRα is evolved to engage multiple ligands with common molecular determinants to modulate myeloid cell functions in anatomical settings where PILRα ligands are expressed.

FIGURE 1.

NPDC1 and COLEC12 are novel ligands of PILRα. A, COS7 cells were transfected with hNPDC1 and hCOLEC12 expression vectors and then stained with hPILRα-AP, mPILRα-AP, and control supernatants followed with alkaline phosphatase substrate. B, 293T cells were transfected with mCD99, hNPDC1, and hCOLEC12, and the transfectants were stained with isotype control, hPILRα-Fc, or mPILRα-Fc (black line). Binding to mock transfectants (gray area) represents background binding. Transfected ligand expressing cells were gated, and their binding to PILRα-Fc is shown. C, 293T cells were transfected with human and mouse PILRα, and the transfectants were stained with hNPDC1-Fc or hCOLEC12-His (black line). Binding to mock transfectants (gray area) represents background binding. PILRα-positive cells were gated, and ligand fusion staining was shown. D, radioligand assays were used to determine the equilibrium binding affinity of hPILRα-Fc to hNPDC1 transiently expressed on 293T cells. ¹²⁵I-Labeled hPILRα-Fc was allowed to bind to cells in the presence of increasing amounts of unlabeled hPRILα-Fc. The average equilibrium K_D value from two replicate assays was 49 nm. E, SPR equilibrium binding analysis of hPILRα-Fc binding to immobilized hCOLEC12 is shown. The equilibrium K_D value for hPILRα/hCOLEC12 was 1.1 μm.

FIGURE 2.

Human NPDC1 and COLEC12 are glycosylated with sialylated glycans. Comparison of MALDI-TOF MS spectra of permethylated O-glycans, released by reductive β-elimination, from human NPDC1-Fc and human COLEC12-His is shown. Molecular ions of permethylated glycans (glycan alditols) were detected in positive ion mode as sodium adducts (M + Na)⁺.

FIGURE 3.

Sialylated glycans on NPDC1 or COLEC12 are required for their binding to PILRα. The binding of selected proteins to hPILRα was determined by SPR. Human PILRα-Fc (25 μg/ml) was immobilized to a CM5 sensor chip resulting in 11,551 resonance units (RU) bound to chip. Fusion proteins with and without sialidase A treatment were used as analytes (1 μm). Sensograms were corrected for response difference between active and reference flow cell.

FIGURE 4.

PILRα binds to mouse but not human CD99 and transfer of the mouse CD99 PKAPT motif to human CD99 restores PILRα binding. A, 293T cells were transfected with mouse and human CD99 expression vectors, and the transfectants were stained with hPILRα-Fc (black line), mouse PILRα-Fc (black line), or control Ig (gray area). CD99-expressing cells were gated, and their binding to PILRα-Fc is shown. B, comparison of profiles of O-glycans from human and mouse CD99-Fc fusion proteins. O-Glycans were released by reductive β-elimination, permethylated, and then analyzed by MALDI-TOF MS. C, 293T cells were transfected with either mouse or human CD99 or human CD99 with the mouse PKAPT motif inserted after Thr-41. The transfectants were stained with hPILRα-Fc (black line), mouse PILRα-Fc (black line), or control Ig (gray area). The amino acid sequences of regions surrounding the O-glycosylated threonines (underlined and bold) in mouse and human CD99 are shown.

FIGURE 5.

A conserved arginine in PILRα is required for ligand binding. A, amino acid sequence alignment of PILRα from various species, PILRβ and the N terminus of SIGLEC1. The positions of Ig fold residues are shown in yellow based on comparison with Igκ/λ and TCRβ V set Ig domains. Conserved non-Ig PILRα residues are shown in purple. SIGLEC1 residues involved in the sialic acid-binding are in green. SIGLEC residues conserved across the family are shown in red. Asterisks represent PILRα amino acids that are important for sialic acid interaction. Blue arrows denote the positions corresponding to active sites of the SIGLEC1 crystal structure and PILRα homology model. The underlined segments designate β-strands within PILRα. Black circles represent PILRα residues that were mutated to screen for HSV-1/gB binding by others (16). The pairwise percentage residue identity between PILRα and SIGLEC1 was 23%. B, 293T cells were transfected with WT human and mouse PILRα (blue line), or human PILRαR126A and mouse PILRαR133A (red line) expression constructs, and the transfectants were stained with mCD99-Fc, hNPDC1-Fc, or hCOLEC12-His. Background binding to mock transfectants is shown in gray. PILRα-positive cells were gated, and ligand fusion staining is shown. C, 293T cells were transfected with mouse CD99, human NPDC1 and COLEC12, or HSV-1 gB expression vectors, and transfectants were stained with hPILRα-Fc or mPILRα-Fc (blue line), hPILRαR126A-Fc, or mPILRαR133A-Fc (red line) or control IgG (gray area). Ligand-expressing cells were gated, and PILRα-Fc staining was shown. D, 293T cells were infected with HSV-1. Left panel, 24 h later, gB expression in HSV-1 (black line) or mock (gray area) infected cells is shown; right panel, hPILRα-Fc or mPILRα-Fc (blue line), hPILRαR126A-Fc, or mPILRαR133A-Fc (red line) binding to HSV-1 infected cells is shown, and their binding to mock transfectants (gray area) is shown as background binding. E, binding of ligand fusion proteins to wild type and arginine mutated human PILRα. The binding of selected proteins to hPILRα was determined by SPR. Human PILRα-Fc and PILRαR126A-Fc was immobilized to a CM5 sensor chip resulting in 10,540.0 response units (RU) bound to the chip. Fusion proteins were used as analytes (1 μm). Sensograms were corrected for response difference between active and reference flow cell. F, binding of hPILRα-Fc or mPILRα-Fc (blue line) and hPILRαR126A-Fc or mPILRαR133A-Fc (red line) to total human PBMC and T cells (left panels), mouse thymocytes, and CD8⁺ T cells (right panels) is shown, and the binding of isotype control to these cells is shown in gray.

FIGURE 5.

A conserved arginine in PILRα is required for ligand binding. A, amino acid sequence alignment of PILRα from various species, PILRβ and the N terminus of SIGLEC1. The positions of Ig fold residues are shown in yellow based on comparison with Igκ/λ and TCRβ V set Ig domains. Conserved non-Ig PILRα residues are shown in purple. SIGLEC1 residues involved in the sialic acid-binding are in green. SIGLEC residues conserved across the family are shown in red. Asterisks represent PILRα amino acids that are important for sialic acid interaction. Blue arrows denote the positions corresponding to active sites of the SIGLEC1 crystal structure and PILRα homology model. The underlined segments designate β-strands within PILRα. Black circles represent PILRα residues that were mutated to screen for HSV-1/gB binding by others (16). The pairwise percentage residue identity between PILRα and SIGLEC1 was 23%. B, 293T cells were transfected with WT human and mouse PILRα (blue line), or human PILRαR126A and mouse PILRαR133A (red line) expression constructs, and the transfectants were stained with mCD99-Fc, hNPDC1-Fc, or hCOLEC12-His. Background binding to mock transfectants is shown in gray. PILRα-positive cells were gated, and ligand fusion staining is shown. C, 293T cells were transfected with mouse CD99, human NPDC1 and COLEC12, or HSV-1 gB expression vectors, and transfectants were stained with hPILRα-Fc or mPILRα-Fc (blue line), hPILRαR126A-Fc, or mPILRαR133A-Fc (red line) or control IgG (gray area). Ligand-expressing cells were gated, and PILRα-Fc staining was shown. D, 293T cells were infected with HSV-1. Left panel, 24 h later, gB expression in HSV-1 (black line) or mock (gray area) infected cells is shown; right panel, hPILRα-Fc or mPILRα-Fc (blue line), hPILRαR126A-Fc, or mPILRαR133A-Fc (red line) binding to HSV-1 infected cells is shown, and their binding to mock transfectants (gray area) is shown as background binding. E, binding of ligand fusion proteins to wild type and arginine mutated human PILRα. The binding of selected proteins to hPILRα was determined by SPR. Human PILRα-Fc and PILRαR126A-Fc was immobilized to a CM5 sensor chip resulting in 10,540.0 response units (RU) bound to the chip. Fusion proteins were used as analytes (1 μm). Sensograms were corrected for response difference between active and reference flow cell. F, binding of hPILRα-Fc or mPILRα-Fc (blue line) and hPILRαR126A-Fc or mPILRαR133A-Fc (red line) to total human PBMC and T cells (left panels), mouse thymocytes, and CD8⁺ T cells (right panels) is shown, and the binding of isotype control to these cells is shown in gray.

FIGURE 6.

Role of sialic acid in mediating PILRα/ligand interactions. A, binding of hPILRα-Fc or mPILRα-Fc to sialidase A-treated (red line) and -untreated (blue line) total human PBMC and T cells (left panels) and mouse thymocyte and CD8⁺ T cells (right panels) is shown, and the binding of isotype control to these cells is shown in gray. B, binding of hPILRα and the glycans to human NPDC1 was determined by SPR. Human NPDC1 (15 μg/ml) was immobilized to a CM5 sensor chip resulting in 4866.7 response units for the 6′-sialyllactose figure and 3787.3 response units for the 3′-sialyllactose and lactose figures. Human PILRa (1 μm) was used as a positive control. Lactose was used as a negative control for the glycans. For the competition experiment, human PILRα was incubated with various concentrations of the glycans at room temperature for 30 min before running as analyte over the chip. Sialylated carbohydrates containing sialic acid (Neu5Ac) in α2–3 (3′-siallylactose) and α2–6 linkage (6′-siallylactose) were used. Human PILRa was run between each glycan concentration to account for surface variability. Sensograms were corrected for response difference between active and reference flow cell.

FIGURE 7.

Model of PILRα/ligand interactions based on SIGLEC1 structure. Ligand interaction diagrams of SIGLEC1 (top, from Protein Data Bank structure 1QFO) and PILRα (bottom, from the homology model). Ligand contacts are numbered in bold, according to Table 1. Residues having hydrophobic contacts with the ligand are shown in green, and those having a polar or hydrogen bonding interactions are shown in purple. Blue shading denotes solvent-exposed atoms.