Biology subjects

Macauley, M. S.

Publications and source records attributed to Macauley, M. S..

At least 19 recordsLinked to original sources

bioRxiv2026

Biological recognition of mirror-image glycans

Recent synthesis of essential enzymes, such as DNA and RNA polymerases with opposite chirality, has boosted the feasibility of creating mirror-image life. Such life, if ever produced, will undoubtedly be coated by a dense display of glycans (glycoproteins, glycolipids, polysaccharides) built from enantiomers of common monosaccharides. Recognition of mirror-image glycans by extant glycan-binding proteins (GBPs) may be critical for colonization by or immune response to mirror life organisms. We evaluated recognition of enantiomers of common glycans by a diverse set of purified GBPs (plant and human derived), antibodies (including IgM from human plasma), mammalian cells (including immune cells), and organs in live animals. We found that GBP binding to enantiomers of naturally prevalent glycans is widespread. Notably, L-glucose and L-galactose interact with fucose-binding lectins, including DC-SIGN, a C-type lectin expressed on immune cells. These interactions can be inhibited by soluble "natural" glycan ligands and enantiomeric ones confirming specificity. Binding of L-glycans to diverse immune cell repertoires revealed preferences for specific glycan enantiomers. IgM antibodies from human serum showed donor-specific recognition of L-glycans. We propose that the recognition of L-glycans by extant GBPs arises from their co-evolution over millennia with the L-glycans that are present in the glycocalyx of many microorganisms.

biochemistry↗

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

Ratiometric Fluorescent Protein Biosensors Reveal Citrate Dynamics and Cellular Heterogeneity

Citrate is a central intermediate metabolite linking the tricarboxylic acid cycle and lipid biosynthesis. Tools for monitoring of spatiotemporal citrate dynamics are critical for getting a better understanding of cellular metabolism. Here, we develope genetically encoded excitation ratiometric biosensors for citrate, based on our previous intensiometric green fluorescence protein-based citrate biosensor, Citron1. We find that a single mutation in the Citron1 chromophore-forming tripeptide provided an excitation ratiometric response. Further rounds of directed evolution yield highly responsive variants, exhibiting citrate-dependent fluorescence changes between two excitation peaks. When expressed in mammalian cells, these biosensors enable citrate dynamics to be monitored in both the cytosol and mitochondria. Comparative analysis across multiple human breast cancer cell lines uncovers cell line-specific differences in citrate levels and their heterogeneity, which could be linked to their malignancy. Furthermore, flow cytometry-based measurements in mouse embryonic stem cells demonstrate the proteomics signatures underlying the population-level variability in citrate concentrations and citrate rewiring during stem cell differentiation. Together, these results show that these excitation ratiometric citrate biosensors enable quantitative, compartment-resolved, and population-scale analysis of cellular metabolism.

molecular biology↗

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↗

bioRxiv2024

Reshaping the tumor microenvironment by degrading glycoimmune checkpoints Siglec-7 and -9

Cancer treatment has been rapidly transformed by the development of immune checkpoint inhibitors targeting CTLA-4 and PD-1/PD-L1. However, many patients fail to respond, especially those with an immunosuppressive tumor microenvironment (TME), suggesting the existence of additional immune checkpoints that act through orthogonal mechanisms. Sialic acid-binding immunoglobulin-like lectin (Siglec)-7 and -9 are newly designated glycoimmune checkpoints that are abundantly expressed by tumor-infiltrating myeloid cells. We discovered that T cells express only basal levels of Siglec transcripts; instead, they acquire Siglec-7 and -9 from interacting myeloid cells in the TME via trogocytosis, which impairs their activation and effector function. Mechanistically, Siglec-7 and -9 suppress T cell activity by dephosphorylating T cell receptor (TCR)-related signaling cascades. Using sulfur fluoride exchange (SuFEx) click chemistry, we developed a ligand that binds to Siglec-7 and -9 with high-affinity and exclusive specificity. Using this ligand, we constructed a Siglec-7/9 degrader that targets membrane Siglec-7 and -9 to the lysosome for degradation. Administration of this degrader induced efficient Siglec degradation in both T cells and myeloid cells in the TME. We found that Siglec-7/9 degradation has a negligible effect on macrophage phagocytosis, but significantly enhances T cell anti-tumor immunity. The degrader, particularly when combined with anti-CTLA-4, enhanced macrophage antigen presentation, reshaped the TME, and resulted in long-lasting T cell memory and excellent tumor control in multiple murine tumor models. These findings underscore the need to consider exogenous checkpoints acquired by T cells in the TME when selecting specific checkpoint blockade therapy to enhance T cell immunity.

immunology↗

bioRxiv2024

A Unified Atlas of T cell Glycophysiology

Glycans are emerging as important regulators of T cell function but remain poorly characterized across the functionally distinct populations that exist in vivo. Here, we couple single-cell analysis technologies with soluble lectins and chemical probes to interrogate glycosylation patterns on major T cell populations across multiple mouse and human tissues. Our analysis focused on terminal glycan epitopes with immunomodulatory functions, including sialoglycan ligands for Siglecs. We demonstrate that glycosylation patterns are diverse across the resting murine T cell repertoire and dynamically remodelled in response to antigen-specific stimulation. Surprisingly, we find that human T cell populations do not share the same glycoprofiles or glycan remodelling dynamics as their murine counterparts. We show that these differences can be explained by divergent regulation of glycan biosynthesis pathways between the species. These results highlight fundamental glycophysiological differences between mouse and human T cells and reveal features that are critical to consider for glycan-targeted therapies.

immunology↗

bioRxiv2023

Multivalent DNA-encoded lectins on phage enable detecting compositional glycocalyx differences

Selective detection of disease-associated changes in the cellular glycocalyx is a foundation of modern targeted therapies. Detecting minor changes in the density and identity of glycans on the cell surface is a technological challenge exacerbated by lack of 1:1 correspondence between cellular DNA/RNA and glycan structures on cell surface. We demonstrate that multivalent displays of up to 300 lectins on DNA-barcoded M13 phage on a liquid lectin array (LiLA), detects subtle differences in composition and density of glycans on cells ex vivo and in immune cells or organs in animals. For example, constructs displaying 73 copies of diCBM40 lectin per 700x5 nm virion ({varphi}-CBM73) exhibit non-linear ON/OFF-like recognition of sialoglycans on the surface of normal and cancer cells. In contrast, a high-valency {varphi}-CBM290 display, or soluble diCBM40, exhibit canonical progressive scaling in binding with increased epitope density; these constructs cannot amplify the subtle differences detected by {varphi}-CBM73. Similarly, multivalent displays of diCBM40 and Siglec-7 detect differences in the glycocalyx between stem-like and non-stem populations in cancer cells that are not detected with soluble lectins. Multivalent display of lectins on M13 scaffold with protected DNA inside the phage offer non-destructive detection of minor differences in glycocalyx in cells in vitro and in vivo not feasible to currently available technologies.

biochemistry↗

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

Alzheimer's disease associated isoforms of human CD33 distinctively modulate microglial cell responses in 5XFAD mice

Microglia play diverse pathophysiological roles in Alzheimers disease (AD), with genetic susceptibility factors skewing microglial cell function to influence AD risk. CD33 is an immunomodulatory receptor associated with AD susceptibility through a single nucleotide polymorphism that modulates mRNA splicing, skewing protein expression from a long protein isoform (CD33M) to a short isoform (CD33m). Understanding how human CD33 isoforms differentially impact microglial cell function in vivo has been challenging due to functional divergence of CD33 between mice and humans. We address this challenge by studying transgenic mice expressing either of the human CD33 isoforms crossed with the 5XFAD mouse model of amyloidosis and find that human CD33 isoforms have opposing effects on the response of microglia to amyloid-{beta} (A{beta}) deposition. Mice expressing CD33M have increased A{beta} levels, mo7re diffuse plaques, fewer disease-associated microglia, and more dystrophic neurites compared to control 5XFAD mice. Conversely, CD33m promotes plaque compaction and microglia-plaque contacts, and minimizes neuritic plaque pathology, highlighting an AD protective role for this isoform. Protective phenotypes driven by CD33m are detected at an earlier timepoint compared to the more aggressive pathology in CD33M mice that appears at a later timepoint, suggesting that CD33m has a more prominent impact on microglia cell function at earlier stages of disease progression. In addition to divergent roles in modulating phagocytosis, scRNAseq and proteomics analyses demonstrate that CD33m+ microglia upregulate nestin, an intermediate filament involved in cell migration, at plaque contact sites. Overall, our work provides new functional insights into how CD33, as a top genetic susceptibility factor for AD, modulates microglial cell function.

neuroscience↗

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↗

bioRxiv2022

Genetically-Encoded Discovery of Perfluoroaryl-Macrocycles that Bind to Albumin and Exhibit Extended Circulation in-vivo

In this paper, we report selection of albumin-binding macrocyclic peptides from genetically encoded libraries of peptides modified by perfluoroaryl-cysteine SNAr chemistry. Modification of phage-displayed libraries SXCXnC-phage, n=3-5, where X is any amino acid except for cysteine by decafluoro-diphenylsulfone (DFS), yields genetically-encoded library of octafluoro-diphen-ylsulfone-crosslinked macrocycles (OFS-SXCXnC-phage). Selection from these libraries using albumin as a bait identified a family of significantly enriched perfluoroaryl-macrocycles. Synthesis of perfluoroaryl-macrocycles predicted by phage display and testing their binding properties by 19F NMR and fluorescent polarization identified OFS-macrocycle with SICRFFC sequence as the most potent albumin binder. We observed that OFS-macrocycles slowly react with biological nucleophiles such as glutathione. Replacing decafluoro-diphenylsulfone by nearly isosteric pentafluorophenyl sulfide yielded perfluorophenylsulfide (PFS)-crosslinked macrocycles devoid of undesired reactivity. The augmented lead PFS-macrocycle with SICRFFC sequence exhibited KD = 4-6 M towards human serum albumin and similar affinities towards rat and mouse albumins. When injected in mouse, the PFS-SICRFFCGGG compound was significantly retained in circulation in vivo when compared to control PFS-macrocyclic peptide. The perfluoroaryl-macrocycles with SICRFFC motif are the smallest known peptide macrocycle with significant affinity for human albumin and they are a productive starting point for future development of compact macrocycles with predictable circulation half-life in vivo. O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=67 SRC="FIGDIR/small/504611v1_ufig1.gif" ALT="Figure 1"> View larger version (17K): org.highwire.dtl.DTLVardef@cd54d2org.highwire.dtl.DTLVardef@7bd9aborg.highwire.dtl.DTLVardef@10f7515org.highwire.dtl.DTLVardef@1cfc49a_HPS_FORMAT_FIGEXP M_FIG C_FIG

biochemistry↗

bioRxiv2022

Chemoenzymatic Synthesis of Genetically-Encoded Multivalent Liquid N-glycan Arrays

A hallmark of cellular glycosylation is its chemical complexity and heterogeneity, which can be challenging to capture synthetically. Using chemoenzymatic synthesis on M13 phage, we produce a genetically-encoded liquid glycan array (LiGA) of biantennary complex type N-glycans. Ligation of azido-functionalized sialylglycosyl-asparagine derived from egg yolk to phage functionalized with 50-1000 copies of dibenzocyclooctyne produced divergent intermediate that can be trimmed by glycosidases and extended by glycosyltransferases to yield a library of phages with different N-glycans. Post-reaction analysis by MALDI-TOF MS provided a rigorous approach to confirm N-glycan structure and density, both of which were encoded in the bacteriophage DNA. The binding of this N-glycan LiGA by ten lectins, including CD22 or DC-SIGN expressed on live cells, uncovered an optimal structure/density combination for recognition. Injection of the LiGA into mice identified glycoconjugates with structures and avidity necessary for enrichment in specific organs. This work provides an unprecedented quantitative evaluation of the interaction of complex N-glycans with GBPs in vitro and in vivo.

biochemistry↗

bioRxiv2021

Carbohydrate sulfation as a mechanism for fine-tuning Siglec ligands

The immunomodulatory family of Siglecs recognize sialic acid-containing glycans as self, which is exploited in cancer for immune-evasion. The biochemical nature of Siglec ligands remains incompletely understood with emerging evidence suggesting the importance of carbohydrate sulfation. Here, we investigate how specific sulfate modifications affect Siglec ligands by overexpressing eight carbohydrate sulfotransferases (CHSTs) in five cell lines. Overexpression of three CHSTs (CHST1, CHST2, or CHST4) significantly enhances the binding of numerous Siglecs. Unexpectedly, two other CHSTs (Gal3ST2 and Gal3ST3) diminish Siglec binding, suggesting a new mode to modulate Siglec ligands via sulfation. Results are cell type dependent, indicating that the context in which sulfated glycans are presented is important. Moreover, pharmacological blockade of N- and O-glycan maturation reveals a cell type-specific pattern of importance for either class of glycan. Production of a highly homogenous CD33 (Siglec-3) fragment enabled a mass spectrometry-based binding assay to determine 10-fold and 3-fold enhanced affinity for Neu5Ac2-3(6-O-sulfo)Gal{beta}1-4GlcNAc and Neu5Ac2-3Gal{beta}1-4(6-O- sulfo)GlcNAc, respectively, over Neu5Ac2-3Gal{beta}1-4GlcNAc. CD33 showed significant additivity in affinity (36-fold) for the disulfated ligand, Neu5Ac2-3(6-O-sulfo)Gal{beta}1-4(6-O-sulfo)GlcNAc. Moreover, overexpression of both CHST1 and CHST2 in cells greatly enhanced the binding of several Siglecs, including CD33. Finally, we reveal that CHST1 is upregulated in numerous cancers, correlating with poorer survival rates and sodium chlorate sensitivity for the binding of Siglecs to cancer cell lines. These results provide new insights into carbohydrate sulfation as a modification that is a general mechanism for tuning Siglec ligands on cells, including in cancer.

immunology↗