Specific Sialoforms Required for the Immune Suppressive Activity of Human Soluble CD52

Abstract

Human CD52 is a small glycopeptide (12 amino acid residues) with one N-linked glycosylation site at asparagine 3 (Asn3) and several potential O-glycosylation serine/threonine sites. Soluble CD52 is released from the surface of activated T cells and mediates immune suppression via its glycan moiety. In suppressing activated T cells, it first sequesters the pro-inflammatory high mobility group Box 1 (HMGB1) protein, which facilitates its binding to the inhibitory sialic acid-binding immunoglobulin-like lectin-10 (Siglec-10) receptor. We aimed to identify the features of CD52 glycan that underlie its bioactivity. Analysis of native CD52 purified from human spleen revealed extensive heterogeneity in N-glycosylation and multi-antennary sialylated N-glycans with abundant polyLacNAc extensions, together with mainly di-sialylated O-glycosylation type structures. Glycomic (porous graphitized carbon-ESI-MS/MS) and glycopeptide (C8-LC-ESI-MS) analysis of recombinant soluble human CD52-immunoglobulin Fc fusion proteins revealed that CD52 bioactivity was correlated with a high abundance of tetra-antennary α-2,3/6 sialylated N-glycans. Removal of α-2,3 sialylation abolished bioactivity, which was restored by re-sialylation with α-2,3 sialyltransferases. When glycoforms of CD52-Fc were fractionated by anion exchange MonoQ-GL chromatography, bioactive fractions displayed mainly tetra-antennary, α-2,3 sialylated N-glycan structures and a lower relative abundance of bisecting GlcNAc structures compared to non-bioactive fractions. In addition, O-glycan core type-2 di-sialylated structures at Ser12 were more abundant in bioactive CD52 fractions. Understanding the structural features of CD52 glycan required for its bioactivity will aid its development as an immunotherapeutic agent.

Keywords: CD52; analysis; glycan structure; immune suppression; tetra-antennary; α-2,3 sialylation.

Figure 1

Glycosylation analysis of human spleen CD52. (A) Summed MS profile of released N-glycans from CD52 purified from human spleen tissue. (B) Distribution of O-linked glycans released from human spleen CD52. CD52 was purified from one healthy donor spleen.

Figure 2

Comparative N-glycoprofiling of recombinant human IgG Fc and CD52. (A) Proliferation of human PBMCs (³H thymidine uptake) followed 5 days incubation with tetanus toxoid (10 LfU), histograms show mean ± SD of within-assay triplicates, in the presence of different concentration of proteins (CD52-Fc 5, 10, 50 μg/ml; Cleaved CD52-Fc 50 μg/ml and Fc control 50 μg/ml). The Fc component was cleaved from CD52-Fc with Factor Xa. (B) Factor Xa treated-CD52 was analyzed by Western blotting with anti-CD52-HRP antibody (Campath-H1). (C) Summed MS profile of N-glycans released from the Fc (I) and CD52 (II); the latter variant was generated by introducing a point mutation (A297N) into the conventional Fc N-glycosylation site.

Figure 3

Comparison of recombinant human CD52-Fc variants (I, II, and III) with different immunosuppressive activities. (A) IFN–γ production measured by ELISpot assay from human PBMCs (2 × 10⁶) in 200 μL/well. Samples were incubated with no antigen or tetanus toxoid in the presence of two different preparations of CD52-Fc (CD52 I or CD52 II; 5, 25, and 50 μg/ml). (B) N-linked glycans released from cleaved CD52 I and CD52 II. The abundance of each N-glycan class is the sum of all EICs measured for all glycans in that class relative to the total of all EICs observed for all N-glycans. (C) EIC of m/z 1140.4²⁻ (GlcNAc₅Man₃Gal₂NeuAc₁) demonstrating the PGC-based separation of sialo-glycan isomers observed in CD52 I and CD52 II. (D) Binding of CD52-Fc I and CD52-Fc II (5 and 20 μg/ml) to the α-2,3 sialic acid recognizing lectin MAL-1. (E) ELISpot assay showing activity of CD52-Fc III reconstituted with sialic acid in α2-6, α2-3, and α2-8 linkages with galactose. The data points in (A,D,E) are plotted as mean ± SEM of three independent replicate experiments. Data in B and C are mean ± SDs (n = 3). ANOVA, post-hoc comparisons of pairs and Bonferroni correction were used to test for significant difference between group means.

Figure 4

CD52-Fc after fractionation by anion-exchange chromatography. (A) Anion exchange chromatography on a MonoQ-GL column fractionated the recombinant human CD52-Fc III into a gradient of anionic glycoforms displaying a spectrum of pI (see Supplementary Figure 2). (B) IFN–γ ELISpot assay with 2 × 10⁶ PBMCs in 200 μL/well incubated with no antigen or with anti-CD3/CD28 antibody Dynabeads in the presence of recombinant human CD52-Fc fractions (F29–F52; 5 μg/ml).

Figure 5

Sialic linkage analysis of active monoQ active fractions. (A) EICs of the di-sialylated N-glycan m/z 1111.4²⁻ after sequential α-2,3 sialidase treatment of bovine fetuin, known to carry tri-antennary α-2,3-sialylated N-glycans. The EICs assess the removal of each of the sialic acid residues. (B) Summed MS of all N-glycans observed for the active CD52 fractions F48 and F49 before (i) and after treatment with α-2,3-specific sialidase (ii). (C) Summary of the degree of α-2,3 sialylation and bisecting GlcNAcylation of late-eluting MonoQ fractions of particular interest. (D) High-resolution intact mass analysis of the immune suppressive CD52 fractions (F48/F49).

Figure 6

Mapping the O-glycosylation of recombinant human CD52. (A) N- and O-glycan occupancy of CD52 I, CD52 II, and selected MonoQ fractions (F31 and F46–F51) measured at the protein level after de-N-glycosylation. (B) PGC resolution of O-glycosylated isomers from active fractions m/z 665.2²⁻ (GalNAc₁GlcNAc₁Gal₂NeuAc₂) and m/z 1040.4¹⁻ (GalNAc₁GlcNAc₁Gal₂NeuAc₁). (C) EThcD-MS/MS based site localization analysis showing the peptide backbone fragments and the ions diagnostic of the amino acid site for both aforementioned O-glycans. Fragmentation to c and z ions are shown that indicate that (i) di-sialylated O-glycans were conjugated to Ser12, and possibly Ser10, whereas (ii) the mono-sialylated O-glycans are found on Thr8.