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12 p.-6 fig.-1 schem., CD22 (Siglec-2) is a B-cell surface inhibitory protein capable of selectively recognising sialylated glycans, thus dampening autoimmune responses against self-antigens. Here we have characterised the dynamic recognition of complex-type N-glycans by human CD22 by means of orthogonal approaches including NMR spectroscopy, computational methods and biophysical assays. We provide new molecular insights into the binding mode of sialoglycans in complex with h-CD22, highlighting the role of the sialic acid galactose moieties in the recognition process, elucidating the conformational behaviour of complex-type N-glycans bound to Siglec-2 and dissecting the formation of CD22 homo-oligomers on the B-cell surface. Our results could enable the development of additional therapeutics capable of modulating the activity of h-CD22 in autoimmune diseases and malignancies derived from B-cells., H2020 Marie Skłodowska-Curie Actions. Grant Numbers: 642157, 814102 Mizutani Foundation for Glycoscience. Grant Number: 2014-2015

Here, two conformationally constrained sialyl analogues were synthesized and characterized in their interaction with the inhibitory Siglec, human CD22 (h-CD22). An orthogonal approach, including biophysical assays (SPR and fluorescence), ligand-based NMR techniques, and molecular modelling, was employed to disentangle the interaction mechanisms at a molecular level. The results showed that the Sialyl-TnThr antigen analogue represents a promising scaffold for the design of novel h-CD22 inhibitors. Our findings also suggested that the introduction of a biphenyl moiety at position 9 of the sialic acid hampers the canonical accommodation of the ligand in the protein binding pocket, even though the affinity with respect to the natural ligand is increased. Our outcomes address the search of novel modifications of the Neu5Ac-a(2-6)-Gal epitope, outlining new hints for the design and synthesis of high-affinity h-CD22 ligands, offering new prospects for therapeutic intervention to prevent autoimmune diseases and B-cells malignancies.

Summary Siglecs (sialic acid binding immunoglobulin (Ig)-like lectins) constitute a group of 15 human and 9 murine cell-surface transmembrane receptors belonging to the I-type lectin family, mostly expressed on innate immune cells and characterized by broadly similar structural features. Here, the prominent inhibitory CD22 (Siglec-2), well known in maintaining tolerance and preventing autoimmune responses on B cells, is studied in its human and murine forms in complex with sialoglycans. In detail, the role of the N-glycolyl neuraminic acid (Neu5Gc) moiety in the interaction with both orthologues was explored. The analysis of the binding mode was carried out by the combination of NMR spectroscopy, computational approaches, and CORCEMA-ST calculations. Our findings provide a first model of Neu5Gc recognition by h-CD22 and show a comparable molecular recognition profile by h- and m-CD22. These data open the way to innovative diagnostic and/or therapeutic methodologies to be used in the modulation of the immune responses., Graphical abstract, Highlights • The structural basis of sialoglycans recognition by h/m CD22 has been investigated • The binding modes of Neu5Gc-/Neu5Ac-containing ligands to m/h-CD22 were compared • The bioactive conformation of sialoglycans has been derived • Our findings may help in the regulation of immune response and cancer prevention, Biochemistry; Immunology; Structural Biology

Summary Siglec-10 is an inhibitory I-type lectin selectively recognizing sialoglycans exposed on cell surfaces, involved in several patho-physiological processes. The key role Siglec-10 plays in the regulation of immune cell functions has made it a potential target for the development of immunotherapeutics against a broad range of diseases. However, the crystal structure of the protein has not been resolved for the time being and the atomic description of Siglec-10 interactions with complex glycans has not been previously unraveled. We present here the first insights of the molecular mechanisms regulating the interaction between Siglec-10 and naturally occurring sialoglycans. We used combined spectroscopic, computational and biophysical approaches to dissect glycans' epitope mapping and conformation upon binding in order to afford a description of the 3D complexes. Our outcomes provide a structural perspective for the rational design and development of high-affinity ligands to control the receptor functionality., Graphical Abstract, Highlights • We unveiled the molecular basis of sialoglycans recognition by Siglec-10 • The conformation of sialoglycans drives the interaction with the protein • Siglec-10 is able to recognize and bind complex N-glycans • Our outcomes may open the venue for the design and development of novel glycomimetics, Biochemistry; Biochemistry Methods; Structural Biology; Data Analysis in Structural Biology