Poster Presentation 29th Annual Lorne Proteomics Symposium 2024

An in-depth multi-dimensional analysis of recombinant CD52 glycopeptide (#172)

Nicholas J DeBono 1 , Silvia D'Andrea 2 , Muhammad A Zenaidee 3 , Esther Bandala-Sanchez 4 , Elisa Fadda 2 , Leonard C Harrison 4 , Nicolle Packer 1 5
  1. ARC Centre of Excellence in Synthetic Biology, Macquarie University, Sydney, NSW, Australia
  2. Department of Chemistry, Maynooth University, Maynooth, County Kildare, Ireland
  3. Australian Proteome Analysis Facility, Macquarie University, Sydney, NSW, Australia
  4. The Walter and Eliza Hall Institute of Medical Research, Parkville, VIC, Australia
  5. Australian Proteome Analysis Facility, Macquarie University, Sydney, NSW, Australia

Soluble CD52 is an immune regulator which initially sequesters pro-inflammatory high mobility group box protein 1 (HMGB1) and inhibits immune responses (1). 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 on serine/threonine residues. Previously, we demonstrated that specific glycosylation of CD52 is essential for its immunosuppressive function (1), 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 (1, 2). However, production of recombinant proteins in different host cell lines for therapeutic use will result in expression of different glycoforms compared to the native protein, which can reduce its biological activity. 

Using top-down and traditional high-resolution mass spectrometry, we were able to confidently identify large numbers of glycoforms on recombinant CD52 produced in HEK293 cells. Combining this glycopeptide analysis with information on PNGase F released N-glycans using porous graphitised carbon LC-MS/MS in negative ion mode, we characterised the glycan structures comprising the various glycoforms on both native and recombinant CD52. Additionally, analysis of the glycopeptide after release of the N-glycans allowed the glycosylation site and monosaccharide composition of the O-glycosylation to be determined. As glycosylation of CD52 is essential to its function, 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 cell function. Proceedings of the National Academy of Sciences. 2018;115(30):7783-8.
  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(2):392-405.