Structural insights into Siglec-15 reveal glycosylation dependency for its interaction with T cells through integrin CD11b

Abstract

Sialic acid-binding Ig-like lectin 15 (Siglec-15) is an immune modulator and emerging cancer immunotherapy target. However, limited understanding of its structure and mechanism of action restrains the development of drug candidates that unleash its full therapeutic potential. In this study, we elucidate the crystal structure of Siglec-15 and its binding epitope via co-crystallization with an anti-Siglec-15 blocking antibody. Using saturation transfer-difference nuclear magnetic resonance (STD-NMR) spectroscopy and molecular dynamics simulations, we reveal Siglec-15 binding mode to α(2,3)- and α(2,6)-linked sialic acids and the cancer-associated sialyl-Tn (STn) glycoform. We demonstrate that binding of Siglec-15 to T cells, which lack STn expression, depends on the presence of α(2,3)- and α(2,6)-linked sialoglycans. Furthermore, we identify the leukocyte integrin CD11b as a Siglec-15 binding partner on human T cells. Collectively, our findings provide an integrated understanding of the structural features of Siglec-15 and emphasize glycosylation as a crucial factor in controlling T cell responses.

Fig. 1. Overall structure of Siglec-15 in complex with anti-Siglec-15 Fab.

a Cartoon and surface representation of two views of the crystal structure of Siglec-15 in complex with anti-Siglec-15 5G12 Fab. 5G12 is composed of a heavy chain (HC) (in orange) and a light chain (LC) (in yellow). The variable (VH and VL) region of 5G12 Fab binds to the V-set domain of Siglec-15 (in cyan). The conserved R143 residue on Siglec-15 ligand-binding site that forms the salt bridge with the carboxylate C1 of sialic acid moiety is represented with a red sphere. b Zoom-in views of the interaction between Siglec-15 and VH (top) and VL (bottom) domains of 5G12 Fab. The heavy-chain CDRs 1 and 3 (HCDR1 and HCRD3) (in orange) of 5G12 interact with C–C′ and G-G′ loops and the F strand of Siglec-15 (cyan). The light-chain CDR3 (LCDR3) from 5G12 (in yellow) interacts with the G strand and G-G´ loop (in blue). Polar contacts (H-bonds and salt bridges) are represented with yellow dashed lines. c Cartoon representation of the two β-sheets forming the V-set Ig domain of Siglec-15. The internal disulfide linkages (between C64 and C142; and between C95 and C104) are represented as sticks. d Topology diagram of V type Ig-like domain in Siglec-15. The β-sheets of are formed by strands AA´BED and C′′C′CFGG′. C64-C142 and C95-C104 intra-disulfide linkages are represented with dashed yellow lines.

Fig. 2. Binding of Siglec-15 to human T cells depends on α(2,3) and α(2,6) sialylation.

a Representative flow histograms showing the expression of STn on leukemia cell lines (K562 and Jurkat), compared to unstimulated (unstim) or activated human T cells. b Representative histograms of SNA and MAL II lectin binding to the surface of unstimulated or activated T cells. The general glycan structure recognized by SNA and MAL II lectins is drawed using the Symbol Nomenclature for Glycans (SNFG). Sialic acid with magenta rhomboid, galactose (Gal) with yellow circle and N-acetylglucosamine (GalNAc) with blue square. c The binding of lectins (SNA or MAL II) to CD8⁺ or CD4⁺ T cells before and after activation was quantified by flow cytometry (SNA CD8⁺: p < 0.0001, SNA CD4⁺: p < 0.0001,MAL II CD8⁺: p < 0.0001, MAL II CD4⁺: p < 0.0001, n = 4 donors). d Bar graphs representing the binding of SNA and MAL II lectins to activated T cells after preincubation with Siglec-15 or IgG-Fc control (SNA CD8⁺: p = 0.0047, SNA CD4⁺: p = 0.0002,MAL II CD8⁺: p = 0.0006, MAL II CD4⁺: p = 0.002, n = 4 donors). e Representative flow cytometric histograms (left) and pooled data (right) of Siglec-15-Fc binding after pan-deglycosylation (De-glyco) treatment of human CD8⁺ and CD4⁺ T cells (CD4⁺: p = 0.0005, CD8⁺: 0.0007, n = 4 donors). f Representative histograms of Siglec-15-Fc binding after desialylation with Neuraminidase A (Neur A) of human CD8⁺ and CD4⁺ T cells measured by flow cytometry (n = 4 donors). g Representative flow cytometric histograms (left) and pooled data (right) showing the binding of Siglec-15-Fc to human CD8⁺ and CD4⁺ T cells treated with Neuraminidase S (Neur S) (CD4⁺: 0.007, CD8⁺: 0.011, n = 7 donors). h Representative histograms (top) and pooled data (bottom) of Siglec-15-Fc R143A binding to activated CD8⁺ and CD4⁺ T cells (CD4⁺: 0.0002, CD8⁺: 0.0003, n = 4 healthy donors). Secondary control means that only an anti-Fc detector antibody, but not recombinant Fc-chimera protein was added to the sample. Error bars denote SEM. *p < 0.05, **p < 0.01; ***p < 0.001; ****p < 0.0001) as determined by two-tailed, unpaired Student’s t test. Source data are provided as a Source Data file.

Fig. 3. Anti-Siglec-15 blocking mAb 5G12 competes for the sialic acid-binding site of Siglec-15.

a Representative flow cytometry histograms of activated CD8+ and CD4+ human T cells showing the binding of recombinant Siglec-15-Fc in the presence or absence of anti-Siglec-15 5G12 Fab. Here, secondary control means that only an anti-Fc detector antibody, but not recombinant Fc-chimera protein was added to the sample. b Bar graphs show Siglec-15-Fc binding to human T cells in the presence or absence of 5G12 Fab (CD4⁺: 0.0001, CD8⁺: 0.0002, n = 4 donors). Errors bars denote SEM. ***p < 0.001 as determined by two-tailed, unpaired Student’s t test. c Competition of 3′SL (left)/6′SL (right) and 5G12 mAb for the same binding site of Siglec-15. Source data are provided as a Source Data file.

Fig. 4. Molecular recognition of 3′SL, 6′SL and STn-Ser by Siglec-15.

a STD-based epitope mapping for 3′SL, 6′SL and STn-Ser in the presence of Siglec-15. The relative STD response is coded according to the legend. b Representative frames derived from 0.5 µs molecular dynamics (MD) simulations of 3′SL (left), 6’SL (middle) and STn-Ser (right) (sticks, purple for Neu5Ac, yellow for GalNAc and Gal, blue for Glc, and gray for serine) in complex with Siglec-15 V-set domain (cartoon and sticks, light cyan). The residues of the protein involved in interactions with the ligands are identified and represented in sticks. Besides the essential salt bridge between Neu5Ac carboxylate group and R143 guanidinium group, four hydrogen bonds are maintained throughout the simulation: OH9-Neu5Ac with CO-E155; O9-Neu5Ac with OH-Y87; OH4-Neu5Ac with CO-V150; and NHAc-Neu5Ac with CO-R153. In addition, CH–π interactions are established between C9-Neu5Ac and Y154; NHAc-Neu5Ac and W44. Source data are provided as a Source Data file.

Fig. 5. Siglec-15 binds to CD11b in a sialic acid-dependent manner.

a OD values corresponding to an ELISA performed with serially diluted IgG1-Fc (gray), Siglec-15-Fc WT (red) or Siglec-15-Fc R143A (blue) against plate-coated CD11b/CD18. Averages of triplicates are shown. b Co-immunoprecipitation showing the interaction of Siglec-15-Fc or Siglec-15-Fc R143A with CD11b. Assay repeated twice with similar results. c Bar graph representing the absolute STD-NMR intensities corresponding to proton signals of STn-Ser + Siglec-15 before the addition of CD11b/CD18 (gray), and after (orange). d Representative contour plots showing the expression of CD11b on activated CD4⁺ and CD8⁺ T cells by flow cytometry. e Quantitation of Siglec-15-Fc binding to CD4⁺ and CD8⁺ T cells in the presence or absence of anti-CD11b blocking mAb (clone M1/70), measured by flow cytometry (CD4+: p = 0.0052, CD8+: 0.0098, n = 4 donors). f Representative flow cytometric histograms (gMFI) and pooled data showing the fold change in the binding of Siglec-15–Fc to T cells transfected with indicated siRNAs (CD4+: p = 0.046, CD8+: p = 0.036, n = 5 donors). g Representative flow cytometric histograms (gMFI) and pooled data of the fold change in the binding of Siglec-15-Fc to T cells overexpressing CD11b/CD18 compared to untransduced cells (CD4+: 0.0007, CD8+: 0.036, n = 6 donors). Error bars denote SEM. *p < 0.05, **p < 0.01; ***p < 0.001; ****p < 0.0001) as determined by two-tailed, unpaired Student’s t test. Source data are provided as a Source Data file.

Fig. 6. Glycosylation pattern of CD11b expressed on T cells.

a Western blot of CD11b on T cell lysates treated with PNGase F, O-glycosidases or Neuraminidase A for 4 h as indicated. b Lectin blot analysis of purified CD11b-flag from transduced human CD3⁺ T cells. Complete membrane blots are included in Supplementary Fig. 18. a, b One representative western blot from n = 2 biologically independent experiments. c Model of the interaction between Siglec-15 present on the surface of interacting cells and CD11b/CD18 integrin from the cell surface of T cells. The full extracellular domain of Siglec-15 (cyan) was manually built using the crystal structure of the V-set domain and the C2-type Ig-like domain of CD22 (PDB ID:5VKJ). The full extracellular domain of CD11b (blue)/CD18 (wheat) (PDB ID 7USM) heterodimer (from the cell surface of T cells) is represented. The I domain (taken from PDB ID 3K72) and the thigh region of CD11b are colored in gray and pink, respectively. The N-linked glycans present in CD11b are represented as blue spheres. In this model, Siglec-15 binding pocket at V domain is interacting with the sialylated N692-linked glycan of CD11b.