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 PILRalpha possesses an ITIM in its cytoplasmic domain, whereas the activating PILRbeta does not have an ITIM but transduces activating signals by associating with the ITAM-bearing DAP12 adapter molecule. Both mouse PILRalpha and PILRbeta 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 PILRbeta, whereas recognition by the inhibitory PILRalpha was not affected. In contrast, mutations of both O-glycosylation sites on CD99 completely abrogated the recognition by both PILRalpha and PILRbeta. PILR did not recognize CD99 treated with neuraminidase, and CD99 expressed on cells transfected with core 2 beta-1,6-N-acetylglucosaminyltransferase was not recognized by PILR. NK cells expressing endogenous activating PILRbeta 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.

FIGURE 1

Posttranscriptional modification of CD99 affects the recognition by PILR. A, Mouse CD99- or mock-transfected Ba/F3, WEHI-231, and IIA1.6 cells stained with control Ig (dotted line), PILRα-Ig (solid line) or PILRβ-Ig (solid line). B, Mouse CD99- or mock-transfected Ba/F3, WEHI-231, and IIA1.6 cells stained with anti-CD99 mAb (5–2, solid line) or control mAb (dotted line). C, Western blot analysis of CD99 expressed on several cell lines. CD99 was transfected into Ba/F3, WEHI-231, and IIA1.6 cells, and cell-surface molecules were biotinylated. CD99 was precipitated from the lysates of CD99-transfected cells (CD99) or parental cells (P) by using anti-CD99 mAb. The precipitates were analyzed by SDS-PAGE, and biotinylated proteins were detected by using peroxidase-conjugated streptavidin.

FIGURE 2

Involvement of O-glycosylation of CD99 in the recognition by PILR. A, Effect of GalNAc-α-O-benzyl on the recognition of CD99 by PILR. CD99- or mock-transfected Ba/F3 cells were incubated with GalNAc-α-O-benzyl at the indicated concentrations for 2 days and were stained with PILR-Ig or anti-CD99 mAb. Mean fluorescence intensities of the stained cells are shown. B, Western blot analysis of CD99 expressed on Ba/F3 cells treated with GalNAc-α-O-benzyl. CD99- or mock-transfected Ba/F3 cells were cultured in the presence (+) or absence (−) of GalNAc-α-O-benzyl (5 mM) for 2 days, and cell lysates were analyzed by SDS-PAGE. CD99 was detected by Western blotting using anti-CD99 mAb.

FIGURE 3

Identification of O-glycosylation sites on CD99. A, Possible O-glycosylation sites were predicted by using an O-glycosylation prediction program (www.cbs.dtu.dk/services/netoglyc). Serine and threonine residues that were predicted to be possible O-linked glycosylation sites are double underlined. Serine and threonine residues that were not predicted to be O-glycosylation sites are underlined. Threonines at 45 and 50 residues are indicated as boldface characters. B, Ba/F3 cells were transfected with various CD99 mutants and the transfectants were stained with PILRα-Ig (solid line), PILRβ-Ig (solid line), or control Ig (dotted line). Expression of CD99 was analyzed by staining with anti-CD99 mAb (solid line) or control mAb (dotted line). C, Western blot analysis of various CD99 mutants. Lysates of 293T cells transfected with wild-type CD99 and CD99 mutants were separated by SDS-PAGE and were blotted with anti-CD99 mAb.

FIGURE 4

Sialic acid is involved in the recognition of CD99 by PILR. A, Mock-, CD99-, or CD99W-T45A-T50A-transfected Ba/F3 cells were incubated in the presence (solid line) or absence (dotted line) of neuraminidase and were stained with PILRα-Ig, PILRβ-Ig, or control-Ig fusion protein. Expression of CD99 was analyzed by staining with anti-CD99 mAb. B, Western blot analysis of neuraminidase-treated cells. Mock-, CD99-, or CD99W-T45A-T50A-transfected Ba/F3 cells were incubated in the presence (+) or absence (−) with neuraminidase. Cell-surface molecules were biotinylated, and lysates of cells were precipitated with anti-CD99 mAb. The precipitates were separated by SDS-PAGE, and biotinylated proteins were detected by using peroxidase-conjugated streptavidin.

FIGURE 5

Expression of core 2-branching enzyme abrogates PILR recognition. A, Mock- or CD99-transfected Ba/F3 cells were further transfected with core 2-branching enzyme (solid line) or control vector (dotted line) and were stained with control-Ig, PILRα-Ig, PILRβ-Ig, or anti-CD99 mAb. B, Western blot analysis of CD99 modified with core 2-branching enzyme. CD99 was precipitated from cell lysates of mock- or CD99-transfected Ba/F3 cells that were further transfected with core 2-branching enzyme (C2) or control vector (M) by anti-CD99 mAb. The precipitants were blotted with PILR-Ig (right) or anti-CD99 mAb (left), respectively.

FIGURE 6

Recognition of activated CD4⁺ T cells, but not B cells, by PILRα. Freshly isolated splenocytes or splenocytes activated with anti-CD3 mAb or anti-IgM Ab were stained with PE-conjugated anti-CD4, PE-conjugated anti-CD8, or PE-conjugated anti-B220 mAb in combination with biotinlylated PILRα-Ig, PILRβ-Ig, or anti-CD99 mAb, followed by APC-conjugated streptavidin (solid line). Cells were also stained with control-Ig or control mAb (dotted line). Staining patterns of PILRα-Ig, PILRβ-Ig, and anti-CD99 mAb on CD4⁺, CD8⁺, or B220⁺ cells are shown.

FIGURE 7

Cytotoxicity of NK cells against Ba/F3 cells transfected with wild-type CD99 or mutant CD99. Cytotoxicity of IL-2-expanded NK cells against wild-type CD99-transfected Ba/F3 cells (●), CD99W-T45A-T50A-transfected Ba/F3 cells (□), CD99W-T45A (▲), CD99W-T50A (○), and parental Ba/F3 cells (■) is shown. Means (±SD) of triplicate assays are shown. Data are representative of three independent experiments.