It’s all about the glycans: activity of the CD52 immunoglycopeptide — ASN Events
  1. Schedule
  2. PROTEOMICS SATELLITE SESSION 3 - THE ROLE OF GLYCOSYLATION
  3. It’s all about the glycans: activity of the CD52 immunoglycopeptide

It’s all about the glycans: activity of the CD52 immunoglycopeptide (#Omics8)

Nicolle Packer 1 2 3 , Nicholas J DeBono 1 2 4 , Muhammad A Zenaidee 3 , Esther Bandala-Sanchez 5 , Edward S.X. Moh 1 2 , Leonard C Harrison 5
  1. School of Natural Sciences, Macquarie University, Sydney, NSW, Australia
  2. ARC Centre of Excellence in Synthetic Biology, Macquarie University, Sydney, NSW, Australia
  3. Australian Proteome Analysis Facility, Macquarie University, Sydney, NSW, Australia
  4. CSIRO Synthetic Biology Future Science Platform, Canberra, ACT, Australia
  5. The Walter and Eliza Hall Institute of Medical Research, Melbourne, VIC, Australia

Soluble CD52 is an immune regulator glycopeptide which sequesters pro-inflammatory high mobility group box protein 1 (HMGB1) and inhibits immune responses. Recombinant CD52-Fc has been shown to act as a broad anti-inflammatory agent, dampening both adaptive (1) and innate (2) immune responses. Human soluble CD52 is a short glycopeptide comprising 12 amino acids (GQNDTSQTSSPS) carrying an N-linked sialylated complex glycan at Asn3 and with potential O-linked glycosylation sites possible on four serine/threonine residues.

Previously (3), we demonstrated that specific glycosylation of CD52 is essential for its immunosuppressive function, with terminal α-2,3-linked sialic acids playing a key role in binding to the inhibitory Siglec-10 receptor leading to T-cell suppression.

Using top-down and traditional high-resolution mass spectrometry, we were able to identify large numbers of glycoforms of recombinant CD52 produced in HEK293 cells. Combining glycopeptide analysis with glycomics, using porous graphitised carbon LC-MS/MS in negative ion mode, we characterised the glycosylation of both native and recombinant CD52. Additionally, analysis of the glycopeptide after release of the N-glycans allowed the O- glycosylation site(s) to be determined.

As glycosylation of CD52 is essential to its function and production of recombinant proteins in different host cell lines results in expression of different glycoforms compared to the native protein, accurate identification of its glycoforms will inform the development of CD52 as a therapeutic agent.

  1. Bandala-Sanchez E, G. Bediaga N, Goddard-Borger ED, Ngui K, Naselli G, Stone NL, et al. CD52 glycan binds the proinflammatory B box of HMGB1 to engage the Siglec-10 receptor and suppress human T
  2. Rashidi M, Bandala-Sanchez E, Lawlor KE, Zhang Y, Neale AM, Vijayaraj SL, et al. CD52 inhibits Toll-like receptor activation of NF-κB and triggers apoptosis to suppress inflammation. Cell Death & Differentiation. 2018: 25:392-405.
  3. Shathili AM, Bandala-Sanchez E, John A, Goddard-Borger ED, Thaysen-Andersen M, Everest-Dass AV, Adams TE, Harrison LC, Packer NH. Specific Sialoforms Required for the Immune Suppressive Activity of Human Soluble CD52. Front Immunol. 2019: 10:1967.
#AusPeptide2023