Poster Presentation 23rd Annual Lorne Proteomics Symposium 2018

Human CD52 initiates its immunosuppressive activity via specific sialoforms  (#106)

Abdulrahman Shathili 1 2 , Esther Bandala-Sanchez 3 , Ethan Goddard-Borger 3 , Alan John 3 , Arun Evervest-Dass 4 , Leonard Harrison 3 , Nicolle Packer 1 2 4
  1. MSc, Macquarie University , Sydney, NSW, Australia
  2. Macquarie node, Centre of Excellence for nanoscale biophotonics , Sydney, nsw, Australia
  3. walter and hall institute , Melbourne
  4. Institute for Glycomics, Grifith University, Gold-Coast, QLD, Australia

Regulation of T cells is necessary to limit their proliferation and to prevent autoimmune diseases. High expression of the cluster of differentiation protein 52 (CD52) results in suppression of other T cells by interacting with sialic acid-binding immunoglobulin-like lectin receptor-10 (Siglec-10) (1). CD52 is a low molecular weight (1208 Da) glycopeptide with a single N-linked, and several potential O-linked, glycosylation sites (2). We aimed to perform a comprehensive glycomics/glycoproteomics characterisation of several CD52-Fc recombinant proteins to determine the bioactive glycoforms of CD52. CD52 was expressed as a recombinant protein in fusion with carrier immunoglobulin Fc glycoprotein in host cells (HEK293 or CHO), and was functionally tested for suppression of T-cell proliferation and interferon-γ secretion. N-glycans were released after PNGase F treatment and reduced glycans were analysed using porous graphitised carbon-liquid chromatography-MS/MS in (-) mode on an ion trap mass spectrometer. Intact-mass of CD52 analysis was performed by (+) mode reversed phase C8-ESI-MS/MS on a Q-TOF mass spectrometer. The N-glycosylation profile of CD52 with the carrier Fc N297 glycosylation site mutated showed the presence of several bi-, tri- and tetra-antennary sialylated N-glycan structures. This glycan profile was also observed on CD52 after Factor X cleavage from the Fc carrier. O-glycan structures were observed by intact-mass analysis of the CD52 glycopeptide after PNGase F treatment, and showed a low abundance of core type-2 di-silaylated structures. Interestingly, the relative abundance of tri- and tetra- antennary sialylated structures, as well as the specific α-2, 3 sialic acid linkage, correlated with two variants of CD52-Fc that showed different immunosuppressive activity.  Furthermore, anion exchange chromatography on a Mono Q GL column separated CD52-Fc into glycoforms that varied in their capacity to suppress T-cell proliferation, strongly supporting different sialylation as a determinant of active CD52. These findings define glycan structures responsible for the bioactivity of CD52.

  1. 1. Bandala-Sanchez E, Zhang Y, Reinwald S, Dromey JA, Lee B, Qian J, Böhmer RM and Harrison LC (2013) T cell regulation mediated by interaction of soluble CD52 with the inhibitory receptor Siglec-10. Nature Immunol 14: 741-48.
  2. 2. Treumann A, Lifely MR, Schneider P and Ferguson MAJ (1995) Primary structure of CD52. J Biol Chem 270: 6088-99.