Polylactosamine on glycoproteins influences basal levels of lymphocyte and macrophage activation

Abstract

beta1,3-N-acetylglucosaminyltransferase 2 (beta3GnT2) is a polylactosamine synthase that synthesizes a backbone structure of carbohydrate structures onto glycoproteins. Here we generated beta3GnT2-deficient (beta3GnT2(-/-)) mice and showed that polylactosamine on N-glycans was markedly reduced in their immunological tissues. In WT mice, polylactosamine was present on CD28 and CD19, both known immune costimulatory molecules. However, polylactosamine levels on these molecules were reduced in beta3GnT2(-/-) mice. beta3GnT2(-/-) T cells lacking polylactosamine were more sensitive to the induction of intracellular calcium flux on stimulation with anti-CD3epsilon/CD28 and proliferated more strongly than T cells from WT mice. beta3GnT2(-/-) B cells also showed hyperproliferation on BCR stimulation. Macrophages from beta3GnT2(-/-) mice had higher cell surface CD14 levels and enhanced responses to endotoxin. These results indicate that polylactosamine on N-glycans is a putative immune regulatory factor presumably suppressing excessive responses during immune reactions.

Fig. 1.

Decreased numbers of polylactosamine repeating units on N-glycan oligosaccharides in β3GnT2^−/− mice. (A) Predicted carbohydrate structures possibly absent in β3GnT2^−/− mice are indicated by rectangles. The bar indicates the LEL-binding epitope consisting of more than three repeated lactosamine units. The dotted rectangle indicates the DSA-binding structure that represents tri- and tetraantenna N-glycans. (B) Lysates of thymus, spleen, and macrophages were incubated in the absence or presence of N-glycanase F (PNGase F) overnight and subjected to LEL blots for polylactosamine. +/+, WT; +/−, heterozygous; −/−, homozygous null. The asterisks indicate nonspecific bands recognized by HRP-streptavidin. The positions of molecular mass markers are indicated. (C) Bio-Gel P-4 column chromatography of desialylated N-glycans derived from stimulated T cells of WT (bold line), β3GnT2^+/− (dotted line), and β3GnT2^−/− mice (solid line). Arrows with the numbers 9–24 indicate the elution positions of glucose oligomers, and the numbers indicate glucose units. Arrows with M_5–9 and S_2–4 indicate the elution positions of standard oligosaccharides, Man_5–9·GlcNAc·GlcNAc_OT and (Gal·GlcNAc)_2–4·Man₃·GlcNAc·GlcNAc_OT, respectively. Arrows with R indicate the elution range of oligosaccharides bearing putative polylactosamine repeating units.

Fig. 2.

T cells from β3GnT2^−/− mice are hypersensitive to stimulation by TCR/CD28. (A) Isolated splenic T cells stained with FITC-LEL. The shaded peak indicates the no lectin control. Mean fluorescence intensity (MFI) on LEL staining was 32 for WT (solid line) and 11 for β3GnT2^−/− (bold line) cells. (B) Glycan analysis of immunoprecipitated CD28 protein by lectin microarraying. The signals of LEL (reactive to polylactosamine) and DSA (reactive to tri- and tetraantennary N-glycan structures) are shown. (C) CD28 proteins detected on Western blot with anti-mouse CD28 (M-20; Santa Cruz Biotechnology, Santa Cruz, CA). Immunoprecipitation with anti-CD28 mAb (Left) or LEL-agarose (Right). The same volume of each extract from WT and β3GnT2^−/− splenic T cells was subjected to Western blot analysis. (D) The calcium response was induced by anti-CD3ε/anti-CD28 antibodies. Splenic T cells were loaded with Fluo-3 and Fura-Red dyes. Arrows indicate the time of addition of streptavidin for cross-linking of receptors. (E) Splenic T cells cultured with the indicated doses of immobilized anti-CD3ε and 750 ng/ml of soluble anti-CD28 antibodies (Left), or reciprocally with immobilized CD3ε (1 μg/ml) and the indicated doses of soluble anti-CD28 antibodies (Right). Proliferative responses are given as [³H]TdR incorporation for the final 6 h of the 42 h culture period. Each assay was performed in triplicate, and all data are representative of three to five experiments.

Fig. 3.

Resting B cells from β3GnT2^−/− mice are hypersensitive to BCR-mediated stimulation. (A) Isolated B cells stained with FITC-LEL. The shaded peak indicates the no lectin control. MFI on LEL staining was 1,267 for WT (solid line) and 717 for β3GnT2^−/− (bold line) cells. (B) Glycan analysis of immunoprecipitated CD19 protein by lectin microarraying. The signals of LEL (reactive to polylactosamine) and DSA (reactive to tri- and tetraantenna N-glycan structures) are shown. (C) CD19 proteins detected on Western blot with anti-mouse CD19 (Cell Signaling Technology). Immunoprecipitation with anti-CD19 antibody or LEL agarose. The same volume of each extract was subjected to Western blot. (D) Resting B cells (ρ = 1.079) cultured with the indicated dose of F(ab′)₂ anti-IgM. Proliferative responses are given as [³H]TdR incorporation for the final 6 h of the 42 h culture period. Each assay was performed in triplicate, and all data are representative of two to four experiments.

Fig. 4.

Peritoneal macrophages from β3GnT2^−/− mice are hypersensitive to LPS stimulation. (A) Peritoneal macrophages stained with LEL. The shaded peak indicates the no lectin control. MFI on LEL staining was 767 for WT (solid line) and 188 for β3GnT2^−/− (bold line) cells. (B) Peritoneal macrophages stimulated with LPS for 6 h. Cytokine production measured by ELISA. Data are given as means and standard deviation of triplicate determinations. Stimulated β3GnT2^−/− macrophages produced significantly greater amounts of cytokine than WT (TNF-α, P = 0.037; IL-1β, P = 0.0077; IL-6, P = 0.0048). (C) Peritoneal cells stained with FITC-F4/80 and PE-CD14. CD14 expression was elevated ≈3-fold (MFI, 679) compared with WT mice (MFI, 216). (D) Peritoneal macrophages stimulated with LPS for 6 h. CD14 proteins detected on Western blot with anti-mouse CD14. (E) Peritoneal macrophages stimulated with LPS for the indicated time. Phosphorylation of ERK and p38 analyzed by immunoblots.