Structural basis for simultaneous recognition of anO-glycan and its attached peptide of mucin family by immune receptor PILRα

Abstract

Paired Ig-like type 2 receptor α (PILRα) recognizes a wide range of O-glycosylated mucin and related proteins to regulate broad immune responses. However, the molecular characteristics of these recognitions are largely unknown. Here we show that sialylated O-linked sugar T antigen (sTn) and its attached peptide region are both required for ligand recognition by PILRα. Furthermore, we determined the crystal structures of PILRα and its complex with an sTn and its attached peptide region. The structures show that PILRα exhibits large conformational change to recognize simultaneously both the sTn O-glycan and the compact peptide structure constrained by proline residues. Binding and functional assays support this binding mode. These findings provide significant insight into the binding motif and molecular mechanism (which is distinct from sugar-recognition receptors) by which O-glycosylated mucin proteins with sTn modifications are recognized in the immune system as well as during viral entry.ucin-type O-glycosylation is highly important in physiological events, but it is too complicated to be fully understood because the structures of modified O-linked sugars are basically heterogeneous and are extremely difficult to predict. However, this complexity is beneficial in discriminating cellular conditions. To date, a number of molecules have been shown to recognize O-glycans, which are important in many biological events, including tumorigenesis and immune evasion (1), as well as infectious diseases. Selectins recognize the O-glycan parts of mucin-type glycoproteins on target cell surfaces to regulate the rolling and attachment of lymphocytes in inflammation and in cancer metastasis. However, how O-glycosylated proteins are recognized by specific receptors has remained largely unclear.To regulate function, the immune system has a fine-tuning mechanism using paired receptors families in which members show opposite (activating and inhibitory) signaling even though their cognate ligand recognitions are essentially similar. It is believed that activating members exhibit lower affinity to ligands than inhibitory members, possibly preventing undesired overactivation of immune responses (2-4) [e.g., killer cell Ig-like receptors, CD94/NKG2 heterodimers, leukocyte Ig-like receptors, and CD28/CTLA-4 (5)]. Paired Ig-like type 2 receptor (PILR) is a member of a paired receptor family comprising activating and inhibitory members designated "PILRβ" and "PILRα," respectively. PILRs have one Ig-like domain in the extracellular region, which is responsible for the ligand recognition. Our previous reports showed that PILRs recognize the O-glycosylated mucin proteins, mouse CD99 (6), and PILR-associating neural protein (PANP) (7). A recent study identified two O-glycosylated ligands for PILRs, neuronal differentiation and proliferation factor-1 (NPDC1) and collectin-12 (COLEC12), expanding the PILRrecognizing O-glycosylated ligand family (8). All PILR ligands are mucin or have mucin-like domains in which the heavy O-glycosylation occurs...

O-glycans direct selectin ligands to lipid rafts on leukocytes

“…However, all three lectins have cytoplasmic immunoreceptor tyrosine-based inhibitory motifs that negatively regulate inflammation (43,44), whereas raft association of PSGL-1, CD43, and CD44 promotes proinflammatory signaling. CD33 and PIRLα prefer sialic acid linked α2-6 to N-acetylgalactosamine (45,46), not the sialic acid linked α2-3 to galactose that caps core 1-derived O-glycans. Alternatively, desialylation or truncation of O-glycans could indirectly affect the conformation of targeting signals on the protein backbone.…”

Rapid Screening by Cell-Based Fusion Assay for Identifying Novel Antivirals of Glycoprotein B-Mediated Herpes Simplex Virus Type 1 Infection

“…25) To evaluate the non-specific effects of O-glycosylated sTn peptide on cell fusion, we chemically synthesized a control peptide RIIPRHLQL (Fig. 1).…”

“…25) It is likely that the concentration of inhibitors in a cell-based assay is higher than that in a cell-free infection assay.…”

“…23) Akkarawongsa et al also reported the inhibition of HSV-1 entry by gB-derived peptides. 24) Recently, we identified the sialylated O-linked sugar T antigen (sTn) and its attached peptide region, derived from gB (O-glycosylated sTn peptide; corresponding to amino acid residues 50-56), that effectively inhibited HSV-1 infection by binding to PILRα in vitro, 25) thus, this O-glycosylated sTn peptide could be a promising lead molecule for the inhibition of gB-mediated HSV-1 infection.…”

Sialic Acids on Varicella-Zoster Virus Glycoprotein B Are Required for Cell-Cell Fusion

“…5, cells that expressed gBs with mutations of putative O-glycosylation sites exhibited comparable fusion efficiency as WT-gB-expressing cells. We performed cell-cell fusion assays using gBs where other Ser/Thr residues were substituted with Ala. We thought that the SAs on O-glycans might be involved in the recognition of gB by MAG in the same way that SAs on the O-glycans of HSV gB are required for PILR␣ to bind to HSV gB (26,44). We selected Ser/Thr residues in gB that were predicted to have enhancement value product values of Ͼ2.00 using the ISOGlyP server.…”