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Original Research ARTICLE Provisionally accepted The full-text will be published soon. Notify me

Front. Immunol. | doi: 10.3389/fimmu.2019.01967

Specific sialoforms required for the immunosuppressive activity of human soluble CD52

  • 1Department of Molecular Sciences, Macquarie University, Australia
  • 2Centre of Excellence for Nanoscale BioPhotonics, Australian Research Council, Australia
  • 3Al-Rayan Research and Innovation Center, Al-Rayan Colleges, Saudi Arabia
  • 4Walter and Eliza Hall Institute of Medical Research, Australia
  • 5Department of Medical Biology, The University of Melbourne, Australia
  • 6Institute for Glycomics, Griffith University, Australia
  • 7Commonwealth Scientific and Industrial Research Organisation (CSIRO), Australia

Human CD52 is a small glycopeptide (12 amino acid residues) with one N-linked glycosylation site at Asn3 and several possible O-glycosylation serine/threonine sites. Soluble CD52 is released from the surface of activated T cells and initiates immunosuppression by first sequestering pro-inflammatory HMGB1 followed by binding to the inhibitory sialic acid-binding immunoglobulin-like lectin-10 (Siglec-10) receptor on activated T cells. Our analysis of native CD52 purified from human spleen confirmed extensive heterogeneity in N-glycosylation and the presence of multi-antennary sialylated N-glycans with abundant polyLacNAc extensions, together with mainly di-sialylated O-glycosylation type structures. We then aimed to define the glycan structures on recombinant soluble human CD52- immunoglobulin Fc fusion protein that correlate with its immune suppressive activity. Glycomics (porous graphitised carbon-ESI-MS/MS) and glycopeptide (C8-LC-ESI-MS) analysis after factor Xa-based Fc removal revealed that bioactive CD52 was characterised by a higher abundance of tetra-antennary α-2,3/6 sialylated N-glycans compared to less bioactive CD52. Moreover, the relative abundance of the α-2,3 sialic acid linkage correlated with higher bioactivity. Removal of α-2,3 sialylation abolished bioactivity, which was restored by re-sialylation with α-2,3 sialyltransferases. Bioactive glycoforms of CD52-Fc were isolated by fractionation on an anion exchange MonoQ-GL column. The CD52 component of fractionated bioactive CD52-Fc displayed mainly tetra-antennary α-2,3 sialylated N-glycan structures and decreased relative abundance of bisecting GlcNAc structures compared to non-bioactive adjacent fractions. In addition, O-glycan core type-2 di-sialylated structures at Ser12 were more abundant in bioactive CD52 fractions. These features describe the active glycoforms of CD52 that confer immunosuppressive activity of soluble CD52.

Keywords: α-2,3 sialic acid , tetra-antennary type oligosaccharide, Glycosylation, Immunosuppression, CD52

Received: 14 Dec 2018; Accepted: 05 Aug 2019.

Copyright: © 2019 Shathili, Sanchez, John, Goddard-Borger, Thaysen-Andersen, Everest-Dass, Adams, Harrison and Packer. This is an open-access article distributed under the terms of the Creative Commons Attribution License (CC BY). The use, distribution or reproduction in other forums is permitted, provided the original author(s) and the copyright owner(s) are credited and that the original publication in this journal is cited, in accordance with accepted academic practice. No use, distribution or reproduction is permitted which does not comply with these terms.

* Correspondence: Prof. Nicolle H. Packer, Macquarie University, Department of Molecular Sciences, Sydney, 2109, New South Wales, Australia, nicki.packer@mq.edu.au