Immunosuppressive activity of human CD52 via specific sialoforms

Shathili AM1, Bandala-Sanchez E2, Goddard-Borger ED2, John A2, Thaysen-Andersen M1, Everest-Dass A3, Harrison LC2 and Packer NH1,3

  1. Dept. of Molecular Sciences and ARC Centre of Excellence in Nanoscale BioPhotonics, Macquarie University, Sydney, New South Wales, Australia.
  2. Walter & Eliza Hall institute of Medical Research and University of Melbourne, Parkville, Victoria, Australia.
  3. Institute for Glycomics, Griffith University, Gold Coast, Queensland, Australia.

Homeostatic mechanisms are required to limit immune T-cell proliferation and prevent autoimmune diseases. Human CD52 is a small glycoprotein (12 amino acid residues), with an N-linked glycan at Asn3 and possible O-glycosylation. Glycosylated and soluble CD52 contributes to immune homeostasis by ligating the inhibitory sialic acid-binding immunoglobulin-like lectin-10 (Siglec-10) receptor on the surface of activated T-cells. We aimed to define the bioactive glycoforms of CD52. Our initial analysis of purified human spleen CD52 confirmed the presence of multi-antennary sialylated N-glycans with abundant polyLacNAc extensions, together with some O-glycan structures. To gain insight into the molecular basis of CD52 binding mechanism, recombinant soluble CD52-Fc expressed from HEK293 or Expi293 cells, but not the Fc alone, suppressed T-cell function and was used to relate bioactivity with the CD52 glycoform structure. Glycomics (porous graphitised carbon-liquid chromatography-ESI-MS) and intact glycopeptide (high resolution C8-ESI-MS) analyses of recombinant CD52 revealed that only specific type of N-glycans contributed to the bioactivity of CD52. Interestingly, the relative abundance of a specific sialic acid linkage correlated with higher bioactivity, which was verified by de- then re-sialylation experiments. Anion exchange chromatography on a MonoQ-GL column fractionated CD52-Fc into glycoforms with increased capacity to suppress T-cell function. Fractions with suppressive activity confirmed previous results. O-glycans were assigned their site localisation in the active fractions. These findings define glycans involved in the immune suppressive bioactivity of CD52 and resulted in CD52 fractions with higher suppressive activity than the biologically isolated CD52.