Biology subjects

Schmidt, E. N.

Publications and source records attributed to Schmidt, E. N..

9 recordsLinked to original sources

bioRxiv2026

Siglec-G on B cells restrains the germinal center response by controlling T cell help during positive selection

The germinal center (GC) reaction requires tight regulation of B cell and T follicular helper (Tfh) cell interactions to ensure B cell expansion and antibody affinity maturation, while preventing oncogenesis. However, regulatory mechanisms fine-tuning B-T cell interactions within the GC to prevent aberrant activation and proliferation remain incompletely understood. Here, we identify Siglec-G, the mouse ortholog of human Siglec-10, as an immune checkpoint that restrains the GC by dampening B-T cell interactions. Selective and temporal ablation of Siglec-G on B cells after immunization triggers GC hyperplasia and enhanced plasma cell and antibody output. While Siglec-G is dispensable in B cell receptor (BCR)-mediated processes, it acts as an intrinsic inhibitory receptor of B-T cell interactions in the GC, ultimately limiting Myc and mTORC activation within positively selected GC B cells. Trans interactions of Siglec-G and its glycan ligands on Tfh likely contribute in fine-tuning the strength of bidirectional signaling following contact between GC B cells and Tfh cells. This interaction is further reinforced by glycan remodeling that occurs in the GC, resulting in concurrent decreased in glycan ligands on GC B cells and increased in glycan ligands on Tfh. This augmented binding of Siglec-G/10 on Tfh is mainly due to the upregulation of 2-6 linked sialic acid ligands. Moreover, APEX2-based proximity labeling revealed several candidate Siglec-G/10 binding partners on T cells, including BTLA, CD6, and Slamf6, which are known negative regulators of Tfh cell activation. Taken together, our findings identified that Siglec-G acts as a GC checkpoint receptor, restricting B cell proliferation by tuning T cell help following B-T cell interactions.

immunology↗

bioRxiv2026

Siglec-7 and -15 recognize repeated clustered sulfo-sialo O-glycan motifs on select O-glycoproteins

Siglecs are key immunoreceptors in immune homeostasis and cancer immunosuppression. Diverse endogenous and exogenous sialoglycan ligands induce Siglec-mediated immunoregulation, but the nature of endogenous Siglec ligands and how seemingly ubiquitous sialoglycans achieve selectivity is unclear. Here, we employed a HEK293 cell-based array and glycoprotein reporters designed from human mucins and mucin-like proteins with O-glycans in natural clusters and repeat motifs to dissect specificities of Siglec-7 and -15. We first used precomplexed Siglec-Fc chimera to demonstrate that CHST1-mediated 6-O-sulfation markedly enhanced binding to select O-glycoproteins, namely MAdCAM1, CD43, and PSGL-1. Probing binding properties of Siglec-7 expressed exogenously on CHO cells or endogenously on human monocytes by reverse assays employing fluorophore-tagged O-glycoprotein reporters revealed specific binding to the sulfo-sialyl core1 O-glycoform of the same O-glycoproteins. Our study indicates that Siglec-7 on immune cells employs multivalent interactions with repeated motifs of clustered sulfo-sialyl O-glycans to drive selectivity in ligand interactions and Siglec-induced immunosuppression.

molecular biology↗

bioRxiv2025

Non-Carbohydrate Inhibitors of Sialic Acid-binding Immunomodulatory-type Lectin-7 (Siglec-7) Discovered from Genetically Encoded Bicyclic Peptide Libraries

Glycan-binding proteins (GBP) are among the most difficult to drug targets. This deficiency delays clinical progress for therapeutically important GBPs. We employed bicyclic genetically encoded libraries (BiGELs), produced by chemical modification of phage-displayed libraries of peptides with two-fold symmetric linchpins, to discover inhibitors of therapeutically relevant Siglec-7:GD3 interactions. Next-generation sequencing (NGS) analysis of panning of BiGEL against Siglec-7 yielded 815 candidates from which 23 hits yielded KD = 1-100 {micro}M as determined by surface plasmon resonance (SPR). Competitive enzyme-linked immunosorbent assays (ELISA) identified a subset of leads that disrupted the Siglec-7:GD3 interaction with IC50= 3-300 {micro}M. Machine learning models trained on NGS datasets identified additional inhibitors with equivalent potency. Alanine scans of 8c (SWCRPATVNC, IC50 = 3.8 {micro}M) and 12c (SFCHYPTHVC, IC50= 11 {micro}M), identified key residues as crucial for activity. Ring reshaping studies of compound 8c highlighted the critical role of bicyclic topology produced by analogue 46e (SAAAAAWCRPATVNC, IC50= 9.5 {micro}M). Multivalent display of the lead bicycles alongside [~]100 glycans in Liquid glycan Array (LiGA), made it possible to compare the binding of bicycles and glycans to Siglec-7 expressed on CHO, Jurkat, and Raji cells. LiGA assays confirmed binding of the bicycles to Siglec-7 but revealed considerable non-specific interactions with receptor-negative cells. Saturation transfer difference nuclear magnetic resonance (STD-NMR) revealed 46e binds to Siglec-7 at a site distinct from the V-Ig domain, suggesting it might inhibit binding of glycans to the glycan-binding site of Siglec-7 via an allosteric site. Together these results demonstrate that BiGEL enables the discovery of bicyclic peptides for undruggable Siglec targets but highlights future challenges in molecular discoveries that aim to identify small, non-carbohydrate inhibitors of GBPs.

biochemistry↗

bioRxiv2025

Glycolipid recognition and binding by Siglec-6 hinges on interactions with the cell membrane

Sialic acid-binding immunoglobulin-type lectins (Siglecs) regulate immune response through interactions with sialylated glycans on glycoproteins and glycolipids. Human Siglecs count 14 unique proteins and in all of those the recognition and binding of the sialic acid on the glycan target involves a conserved, or canonical, Arg residue. For a subset of human Siglecs, namely MAG, Siglec-6, and Siglec-11, this Arg appears not to be essential, suggesting that a different binding mechanism may be at play. In this work, we used all-atom molecular dynamics (MD) simulations, binding assays, and mutagenesis to investigate the structural, mechanistic and energetic details of the binding of Siglec-6 to monosialylated gangliosides. Our results show that Siglec-6 relies only partially on its conserved Arg122 for recognition of membrane-bound gangliosides and that it supplements its binding free energy through interactions with the phospholipids in the membrane surrounding the target epitope. We confirmed by mutagenesis assays that the loss of the key residues (Lys 126 and Trp 127) for membrane interaction abrogates binding. These results provide a step-change in our understanding of the diversification of human Siglecs as molecular precision tools to bind specific sialosides by adapting their structure to the biological environment where these are found.

biochemistry↗

bioRxiv2025

An ultrasensitive and modular platform to detect Siglec ligands and control immune cell function

Siglecs are immunomodulatory receptors that regulate immune cell function. A fundamental challenge in studying Siglec-ligand interactions is the low affinity of Siglecs for their ligands. Inspired by how nature uses multivalency, we developed Siglec-liposomes as a highly multivalent and versatile platform for detecting Siglec glycan ligands in which recombinant Siglecs were conjugated to liposomes using the SpyCatcher-SpyTag system. Siglec-liposomes offer tunable multivalency and a modular assembly, enabling presentation of different Siglecs on the same liposome. Using Siglec-liposomes, we profiled Siglec ligands on human leukocytes, revealing new insights into Siglec ligands. Moreover, Siglec-liposomes are in vivo compatible, where we demonstrated that Siglec-7-liposomes bind to the brain vasculature in a mucin-dependent manner. Given the abundance of Siglec ligands on T cells, we investigated whether Siglec-liposomes modulate T cell function and find that Siglec-7-liposomes increase T cell proliferation in a ST3Gal1-dependent and CD43-independent manner. Taken together, Siglec-liposomes are a versatile and sensitive tool for detecting Siglec ligands and immunomodulation.

biochemistry↗

bioRxiv2025

The unique molecular recognition features of Siglec-10: structural insights into sialoglycan and antibody interactions

Siglec-10 is a sialic acid-binding immunoglobulin-like lectin implicated in immune regulation, yet the molecular basis for ligand recognition and how this is functionally linked to immune modulation remains poorly defined. Herein, we present a multidisciplinary study encompassing structural, biochemical, and cellular approaches to elucidate Siglec-10-carbohydrate interactions and their functional consequences. The crystal structure of the extracellular domain of Siglec-10 in complex with 2-6 sialyllactose revealed the presence of two key arginine residues within the Siglec-10 binding site that interact with the carboxyl group of sialic acid, the canonical R119 and R127, suggesting potential dual contributions to ligand engagement. Saturation Transfer Difference (STD)-Nuclear Magnetic Resonance (NMR) confirmed that R119 is essential for sialoglycan binding in solution, whereas R127 appears dispensable for interactions with glycans under these conditions. In contrast, cell-based binding assays using primary human T cells and engineered monocytic lines demonstrated that both arginine residues (R119 and R127) are critical for cellular recognition, revealing a context-dependent interaction. By obtaining direct images at a molecular resolution of 6-7 nm, super-resolution microscopy further revealed glycan-independent dimerization of the Siglec-10 receptor on the surface of human monocytes. Ligand blockade mediated by anti-Siglec-10 mAb (clone S10A) restores CAR-T cell cytotoxicity in vitro, supporting its role as an immune checkpoint receptor. Finally, although CD24 was not identified as a Siglec-10 ligand on T cells, proximity labeling and mass spectrometry uncovered other sialylated glycoproteins that may mediate this interaction. Together, these results identify Siglec-10 as a modulatory receptor with structural and functional features distinct from other Siglec family members and highlight its potential for therapeutic targeting in cancer immunotherapy.

molecular biology↗

bioRxiv2025

CD33 Epitope Editing Unlocks UM171-Expanded Cord Blood Grafts for AML Immunotherapy

Immunotherapies in acute myeloid leukemia (AML) are limited by shared antigen expression between leukemic and healthy hematopoietic cells, leading to on-target toxicity. Here we developed a clinically scalable strategy to engineer cord blood (CB)-derived hematopoietic stem and progenitor cell (HSPC) grafts resistant to CD33-directed therapies. Leveraging UM171-mediated expansion and adenine base editing, we precisely disrupted a critical epitope in CD33 required for gemtuzumab ozogamicin (GO) binding, centered on phenylalanine 21, while preserving CD33 expression and its sialic acid binding function. Ex vivo edited HSPCs maintained robust multilineage engraftment, T-cell output, and conferred protection from GO-induced myelotoxicity in xenograft models, without impairing anti-leukemic efficacy. Editing was efficient across multiple donors, enriched in primitive subsets, and exhibited minimal off-target activity by ultra-deep exome sequencing. Our work establishes base editor-driven epitope engineering as an improved approach to CD33-targeted immunotherapy-compatible HSPC grafts, enabling safe integration of currently available agents into post-transplant care.

immunology↗

bioRxiv2023

Measuring carbohydrate recognition profile of lectins on live cells using liquid glycan array (LiGA)

Glycans constitute a significant fraction of biomolecular diversity on the surface of cells across all the species in all kingdoms of life. As the structure of glycans is not encoded by the DNA of the host organisms, it is impossible to use cutting-edge DNA technology to study the role of cellular glycosylation or to understand how cell-surface glycome is recognized by glycan-binding proteins (GBPs). To address this gap, we recently described a genetically-encoded liquid glycan array (LiGA) platform that allows profiling of glycan:GBP interactions on the surface of live cells in vitro and in vivo using next-generation sequencing (NGS). LiGA is a library of DNA-barcoded bacteriophages coated with 5-1500 copies of a glycan; the DNA barcode inside each bacteriophage encodes the structure and density of the displayed glycans. Deep sequencing of the glycophages associated with live cells yields a glycan-binding profile of GBPs displayed on the surface of such cells. This protocol provides detailed instructions of using LiGA to probe cell surface receptors and includes information on the preparation of glycophages, analysis by MALDI-TOF MS, the assembly of a LiGA library, and its deep-sequencing. Using the protocol detailed in this report, we measure a glycan-binding profile of the immunomodulatory SiglecLJ1, -2, -6, -7, and -9 expressed on the surface of different cell types and uncover previously unknown environment-dependent recognition of glycans by Siglec-receptors on the surface of live cells. Protocols similar to the one described in this report will make it possible to measure the precise glycan-binding profile of any GPBs displayed on the surface of any cell types.

biochemistry↗

bioRxiv2023

ST3Gal1 synthesis of Siglec ligands mediates anti-tumour immunity in prostate cancer

Immune checkpoint blockade trials have yet to produce a robust anti-cancer response in prostate cancer patients as a monotherapy due to the immunosuppressed prostate cancer tumour immune microenvironment. ST3Gal1 and other sialyltransferases are implicated in cancer and immune suppression by synthesizing sialoglycans, which act as ligands for Siglec receptors. These checkpoints are important for the immune response. However, its unclear how the synthesis of Siglec ligands is regulated, and little is known about the role of sialoglycan-Siglec-axis in prostate cancers evasion of anti-tumour immunity. We report that ST3Gal1 levels negatively correlate with androgen signalling in prostate tumours. Utilising syngeneic mouse models, we demonstrate that ST3Gal1 plays an important role in modulating tumour immune evasion. Using mouse models, patient samples and in vitro models we show that ST3Gal1 synthesises sialoglycans with the capacity to engage the Siglec-7 and Siglec-9 immunoreceptors preventing immune clearance of cancer cells. For the first time we provide evidence of the expression of Siglec-7/9 ligands and their respective immunoreceptors in prostate tumours. Importantly, we show that these interactions can be modulated by enzalutamide and may maintain immune suppression in enzalutamide treated tumours. We conclude that the activity of ST3Gal1 is critical to prostate cancer anti-tumour immunity and provide rationale for the use of glyco-immune checkpoint targeting therapies in advanced prostate cancer.

cancer biology↗