Structural characterisation of high affinity Siglec-2 (CD22) ligands in complex with whole Burkitt’s lymphoma (BL) Daudi cells by NMR spectroscopy

Abstract

Siglec-2 undergoes constitutive endocytosis and is a drug target for autoimmune diseases and B cell-derived malignancies, including hairy cell leukaemia, marginal zone lymphoma, chronic lymphocytic leukaemia and non-Hodgkin’s lymphoma (NHL). An alternative to current antibody-based therapies is the use of liposomal nanoparticles loaded with cytotoxic drugs and decorated with Siglec-2 ligands. We have recently designed the first Siglec-2 ligands (9-biphenylcarboxamido-4-meta-nitrophenyl-carboxamido-Neu5Acα2Me, 9-BPC-4-mNPC-Neu5Acα2Me) with simultaneous modifications at C-4 and C-9 position. In the current study we have used Saturation Transfer Difference (STD) NMR spectroscopy to monitor the binding of 9-BPC-4-mNPC-Neu5Acα2Me to Siglec-2 present on intact Burkitt’s lymphoma Daudi cells. Pre-treatment of cells with periodate resulted in significantly higher STD NMR signal intensities for 9-BPC-4-mNPC-Neu5Acα2Me as the cells were more susceptible to ligand binding because cis-binding on the cell surface was removed. Quantification of STD NMR effects led to a cell-derived binding epitope of 9-BPC-4-mNPC-Neu5Acα2Me that facilitated the design and synthesis of C-2, C-3, C-4 and C-9 tetra-substituted Siglec-2 ligands showing an 88-fold higher affinity compared to 9-BPC-Neu5Acα2Me. This is the first time a NMR-based binding study of high affinity Siglec-2 (CD22) ligands in complex with whole Burkitt’s lymphoma Daudi cells has been described that might open new avenues in developing tailored therapeutics and personalised medicine.

Boronate affinity-based photoactivatable magnetic nanoparticles for the oriented and irreversible conjugation of Fc-fused lectins and antibodies

“…In contrast, nanoprobe-based affinity mass spectrometry (NBA-MS)50 provides an efficient and straightforward tool for the proteomic study (identification) of interacting glycoprotein ligands. Thus, to further evaluate the advantage afforded by the BA–Diaz immobilization strategy in NBA-MS, Siglec-2–BA–Diaz@MNP C was used to enrich interacting membrane proteins from the BJAB human Burkitt lymphoma cell line, which is known to express Siglec-2 ligands 51,52. To investigate the effect of the immobilization strategy on the resulting Siglec-2 binding activity for interacting protein enrichment, the side chain amines of lysine residues of Siglec-2 were randomly conjugated with activated esters of NHS@MNP to yield Siglec-2–R@MNP.…”

Characterisation of the Dynamic Interactions between Complex N‐Glycans and Human CD22

“…In conclusion, we provide detailed characterisation of complex N ‐glycan behaviour in interaction with h‐CD22. It is worth noting that various studies aiming to propose therapeutic strategies against autoimmune disorders and some types of tumours through targeting of Siglecs have previously been published . However, to the best of our knowledge, no ligand‐based NMR studies on the recognition of naturally occurring complex‐type N ‐glycans by Siglecs in solution have been performed so far.…”

“…These immunoregulatory functions made CD22 an attractive target for therapies against autoimmune diseases and various B‐cell‐derived malignancies including hairy cell leukaemia, marginal zone lymphoma, chronic lymphocytic leukaemia and non‐Hodgkin lymphoma . Thus, deeper molecular insights into the mechanisms that govern sensing of N ‐glycans by CD22 might provide valuable clues to assist the development of new mimetics targeting CD22–glycan interactions.…”

Integrative Analysis of Siglec-15 mRNA in Human Cancers Based on Data Mining

“…Although their structures are similar, their binding specificity differs, and they bind specific sialoglycoconjugates [20]. Most Siglec family members are observed on only hematopoietic cells, as they have very low expression in other tissues, but a recent study found that their expression level changed in some tumor cells [21][22][23]. Several lines of evidence have shown that interactions with sialic acid-binding receptors can influence cancer progression; for example, hypersialylation can induce changes in the physical properties of tumor cells and potentiate the evasion of apoptosis in cancer cells [24,25].…”