An Essential Role of Sialylated O-Linked Sugar Chains in the Recognition of Mouse CD99 by Paired Ig-Like Type 2 Receptor (PILR)

Abstract

The paired Ig-like type 2 receptor (PILR), which comprises both inhibitory and activating isoforms, is well conserved among most mammalians. The inhibitory PILRα possesses an ITIM in its cytoplasmic domain, whereas the activating PILRβ does not have an ITIM but transduces activating signals by associating with the ITAM-bearing DAP12 adapter molecule. Both mouse PILRα and PILRβ recognize mouse CD99, which is broadly expressed on various cells, including lymphocytes, and is involved in the regulation of immune responses. We herein report that sialylated O-linked sugar chains on CD99 are essential for the recognition by PILR. Mutations of one of two O-glycosylation sites on CD99 significantly reduced recognition of CD99 by the activating PILRβ, whereas recognition by the inhibitory PILRα was not affected. In contrast, mutations of both O-glycosylation sites on CD99 completely abrogated the recognition by both PILRα and PILRβ. PILR did not recognize CD99 treated with neuraminidase, and CD99 expressed on cells transfected with core 2 β-1,6-N-acetylglucosaminyltransferase was not recognized by PILR. NK cells expressing endogenous activating PILRβ receptors mediated cytotoxicity against cells expressing wild-type CD99 but not cells expressing mutant CD99 that lacked O-glycosylation sites. These findings indicate that sialylated O-linked sugar structures on CD99 play an important role in the recognition of PILR.

Evolutionarily Conserved Paired Immunoglobulin-like Receptor α (PILRα) Domain Mediates Its Interaction with Diverse Sialylated Ligands

“…S2) and/or the differences in glycosylation (type and/or number of glycans). It has been shown that sialylated O-linked glycans on mCD99 play an essential role in PILR␣ binding (13). hCD99 has been reported to be O-glycosylated (35), and like mCD99, it does not bear any potential N-glycosylation sites.…”

“…LC-MS analysis of reduced fusion proteins (with and without PNGase F and sialidase digestion) showed that hCD99-Fc carried two O-glycans and mCD99-Fc carried three O-glycans with the composition NeuAc 1-2 Hex 1 HexNAc 1 . In the case of mCD99, two adjacent O-glycosylation sites, Thr-45 and Thr-50 (NMKPT 45 PKAPT 50 PKKPS) are relevant for PILR␣ recognition (13). Sequence alignment of human and mouse CD99 showed that Thr-41 of hCD99 corresponds to the Thr-45 O-glycosylation site of mCD99 and that hCD99 lacks the second potential O-glycosylation site corresponding to Thr-50 of mCD99 (supplemental Fig.…”

“…O-Glycosylation of Human NPDC1 and COLEC12 Is Required for Their Binding to PILR␣-The presence of sialylated O-glycans is required for PILR␣ to bind its known ligands, including mCD99, HSV-1 gB, and PANP (8,13,17). Human NPDC1 and COLEC12 have multiple potential O-glycosylation sites when analyzed using the NetOGlyc 3.1 prediction server (34).…”

“…Ligands-Sialylated glycan modifications appear to be a general feature of all known PILR␣ ligands and coincidently a few other ligands of receptors such as SIGLECs (8,12,13,17,19). A conserved arginine in the ECD of SIGLECs plays a critical role in their binding to sialic acid (20,39,40).…”

“…In mice, CD99 binds to PILR␣ and -␤ with 2.2 and 85 M affinities, respectively (12). Two sialylated O-linked glycans present on mCD99 (Thr-45 and Thr-50) are crucial for inter-actions with PILR proteins (13). Sialic acid-containing glycans are abundantly expressed on cell surfaces, secreted glycoproteins, and in the extracellular matrix, where they are usually found at the exposed, nonreducing termini of oligosaccharide chains and are therefore well suited to function as ligands in cellular recognition events (14).…”