The molecular basis of immunosuppression by soluble CD52 is defined by interactions of N-linked and O-linked glycans with HMGB1 box B.

Human soluble CD52 is a short glycopeptide comprising 12 amino acids (GQNDTSQTSSPS) which functions as an immune regulator by sequestering the pro-inflammatory high mobility group box protein 1 (HMGB1) and suppressing immune responses. Recombinant CD52 has been shown to act as a broad anti-inflammatory agent, dampening both adaptive and innate immune responses. This short glycopeptide is heavily glycosylated, with a complex sialylated N-linked glycan at N3 and reported O-linked glycosylation possible on several serine and threonine residues. Previously we demonstrated that specific glycosylation features of CD52 are essential for its immunosuppressive function, with terminal α-2,3-linked sialic acids required for binding to the inhibitory SIGLEC-10 receptor leading to T-cell suppression. Using high resolution mass spectrometry, we have further characterized the N- and O-linked glycosylation of Expi293 recombinantly produced CD52 at a glycopeptide and released glycan level, accurately determining glycan heterogeneity of both N- and O-linked glycosylation, and localizing the site of O-glycosylation to T8 with high confidence and direct spectral evidence. This detailed knowledge of CD52 glycosylation informed the construction of a model system, which we analyzed by molecular dynamics simulations to understand the mechanism of recognition and define interactions between bioactive CD52, HMGB1 and the SIGLEC-10 receptor. Our results confirm the essential role of glycosylation, more specifically hyper-sialylation, in the function of CD52, and identify at the atomistic level specific interactions between CD52 glycans and the Box B domain of HMGB1 that determine recognition, and the stability of the CD52/HMGB1 complex. These insights will inform the development of synthetic CD52 as an immunotherapeutic agent.