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.
Highlights
CD52 is a glycoprotein composed of only 12 amino acid extensively modified by both N-linked and possible O-linked glycosylation, anchored by glycosylphosphatidylinositol (GPI) to the surface of leukocytic, and male reproductive cells [1, 2]
This glycan heterogeneity raises the question whether particular bioactive glycoforms of CD52 exist and whether such heterogeneity is reflected in the recombinant form of human CD52
We engineered human CD52 as a recombinant fusion protein conjugated with an IgG1 Fc fragment as described [3]
Summary
CD52 is a glycoprotein composed of only 12 amino acid extensively modified by both N-linked and possible O-linked glycosylation, anchored by glycosylphosphatidylinositol (GPI) to the surface of leukocytic, and male reproductive cells [1, 2]. The conserved CD52 peptide backbone probably functions only as a scaffold for presentation of the large N-linked glycan, which masks the small GPI-anchored peptide and acts as the prime feature of the CD52 antigen with respect to cell-cell contacts [1, 2]. Activated human T cells with high expression of CD52 were found to exhibit immune suppressive activity via phospholipase C-mediated release of soluble CD52, which was shown to bind to the inhibitory sialic acid-binding immunoglobulin (Ig)-like lectin-10 (Siglec-10) receptor on neighboring T cell populations [3] This sialic acid interaction was subsequently shown to require initial binding of soluble CD52 glycan to the damage-associated molecular pattern (DAMP) protein, high-mobility group box 1 (HMGB1). Complexing of soluble CD52 with HMGB1 promoted binding of the CD52 N-glycan, preferentially in α-2,3 sialic acid linkage, to Siglec-10 [4]
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