Biology subjects

Cummings, R. D.

Publications and source records attributed to Cummings, R. D..

At least 19 recordsLinked to original sources

bioRxiv2026

Disrupted O-GalNAc glycosylation as a mechanism and biomarker of SLC35A2-associated epilepsy

Rare germline and somatic variants in SLC35A2 cause a spectrum of severe glycosylation disorders that commonly present with epilepsy. SLC35A2 encodes the Golgi transporter for UDP-galactose, but how its deficiency leads to severe neurodevelopmental disorders is unknown. Using a mouse model deficient for Slc35a2 in the forebrain, we identified a specific defect in O-GalNAc glycan synthesis, while other galactose-containing glycoconjugates remained intact. O-GalNAc glycans were absent from their normal location within neuronal tracts of the corpus callosum, and truncated precursors accumulated in the cortex on critical extracellular matrix molecules. Cultured primary neurons lacking Slc35a2 showed impaired development, hyperexcitability, and impaired O-GalNAc glycosylation. Finally, human brain tissue from cases of SLC35A2-associated intractable epilepsy displayed a strong correlation between variant burden and truncated O-GalNAc glycans. These findings provide a mechanistic link between genetic causes of SLC35A2-associated epilepsy and protein O-glycosylation that can be targeted for biomarker and therapeutic development.

neuroscience↗

bioRxiv2025

N-Glycopedia: Libraries for Native N-glycan Structural Analysis

Studies on glycans of glycoproteins are hampered by the lack of standards that reflect the wide diversity in structure typically observed. To this end we have exploited a large library of N-glycan standards comprised of a unique collection of 226 N-glycans including oligomannose, hybrid, and complex-type. We generated a method employing porous graphitized carbon (PGC) and liquid chromatography mass spectrometry (PGC-LC-MS), which can provide a high degree of resolution of underivatized N-glycan structures. Chromatogram libraries arising from these studies include retention time data, diagnostic fragments, and validated structural assignments, providing a robust platform for both targeted and discovery-based glycomics. We refer to this as an N-glycopedia, the first type of resource in which researchers can compare this collective data to N-glycans under study and overcome the limitations of only having compositional data and predicted structures. The technology is easily expandable to include additional N-glycans as new standards become available.

biochemistry↗

bioRxiv2025

A glycan atlas of the mammalian intestine through ontogeny and inflammation

The muco-epithelial interface in the mammalian gut is composed of a mucus and epithelial lining fundamental to barrier function, microbe-host interactions, and intestinal homeostasis. This barrier is heavily glycosylated by O-linked sugars covalently linked to mucin glycoproteins, and N-linked sugars that coat epithelial surface proteins. Gut O- and N-glycans are thought to play central roles in barrier function, host defense, nutrition and attachment for commensals and pathogens, immunoregulation and cell-cell interactions. However, the precise nature of the glycans and how glycan composition changes through development, as a function of diet, and during inflammation, remains incompletely understood. Here, we apply O- and N-glycomic platforms to profile glycans on mucus and intestinal epithelium. By mapping individual glycan species spatially and temporally we identify 57 O- and 18 N-glycans in the mouse intestine, and observe that fucosylation and sialylation varies according to intestinal region and developmental stage. We identify a subset of glycans regulated by the gut microbiome, and observe a constriction of the glycan repertoire during inflammation in both mice and humans. Together, these results provide an atlas of individual intestinal glycans and their dynamic range through ontogeny and inflammation, and represent a significant resource for our understanding of the role of intestinal glycans in health and disease and glycan-focused therapies for intestinal inflammation and shaping the gut microbiome. Highlights- Individual glycans vary across gut region and developmental stage - Terminal fucose and sialic acid residues vary across space and time - The microbiome influences gut glycan composition early in life - Gut inflammation in mice and humans converge on a restricted glycan repertoire eTOC blurbMicrobes colonizing the mammalian intestines encounter mucus and an epithelial layer highly decorated by glycans. Siegel et al. use glycomics to map these sugars in high resolution across gut region, microbial colonization, development and inflammation in both humans and mice.

microbiology↗

bioRxiv2024

The restricted N-glycome of neurons is programmed during differentiation

The protein glycome of individual cell types in the brain is unexplored, despite the critical function of these modifications in development and disease. In aggregate, the most abundant asparagine (N-) linked glycans in the adult brain are high mannose structures, and specifically Man5GlcNAc2 (Man-5), which normally exits the ER for further processing in the Golgi. Mannose structures are uncommon in other organs and often overlooked or excluded in most studies. To understand cell-specific contributions to the unique brain N-glycome and its abundance of Man-5, we performed RNAseq and MALDI-MS TOF protein N-glycomics at several timepoints during differentiation of multiple cell types. To this end, homogeneous cultures of glutamatergic neurons, GABAergic neurons, and brain-specific endothelial cells were generated from monoclonal human inducible pluripotent stem cells (hiPSCs) through cellular reprogramming. Small molecule induction of stably integrated synthetic transcription units driving morphogen expression generated differentiated cells with distinct patterns mirroring intact tissue. Comparing uninduced hiPSCs for each cell type revealed identical transcriptomic and glycomic profiles before differentiation, with low quantities of Man-5. In differentiated glutamatergic and GABAergic neurons, the most abundant N-glycans became Man-5 and its immediate precursor Man-6, despite the presence of transcripts encoding enzymes for their subsequent modification. Differentiation to brain-specific endothelial cells showed an opposite effect, with the N-glycome displaying an abundance of complex N-glycans and terminal modifications of the late secretory pathway. These results confirm that the restricted N-glycome profile of brain is programmed into neuronal differentiation, with regulation independent of the transcriptome and under tight evolutionary constraint.

neuroscience↗

bioRxiv2024

Antibody-Fab and -Fc features promote Mycobacterium tuberculosis restriction.

Mycobacterium tuberculosis (Mtb), the causative agent of tuberculosis (TB), is a leading cause of death by an infectious disease globally, with no efficacious vaccine. Antibodies are implicated in Mtb control, but the mechanisms of antibody action remain poorly understood. We assembled a library of TB monoclonal antibodies (mAb) and screened for the ability to restrict Mtb in mice, identifying protective antibodies targeting known and novel antigens. To dissect the mechanism of mAb-mediated Mtb restriction, we optimized a protective lipoarabinomannan-specific mAb through Fc-swapping. In vivo analysis of these Fc-variants revealed a critical role for Fc-effector function in Mtb restriction. Restrictive Fc-variants altered distribution of Mtb across innate immune cells. Single-cell transcriptomics highlighted distinctly activated molecular circuitry within innate immune cell subpopulations, highlighting early activation of neutrophils as a key signature of mAb-mediated Mtb restriction. Therefore, improved antibody-mediated restriction of Mtb is associated with reorganization of the tissue-level immune response to infection and depends on the collaboration of antibody Fab and Fc.

immunology↗

bioRxiv2024

O-GalNAc glycans enrich in white matter tracts and regulate nodes of Ranvier

Protein O-glycosylation is a critical modification in the brain, as genetic variants in the pathway are associated with both common and severe neuropsychiatric phenotypes. However, little is known about the most abundant type of O-glycan in the mammalian brain, which are O-GalNAc linked. Here, we determined the spatial localization, protein carriers, and cellular function of O-GalNAc glycans in mouse brain. We observed striking spatial enrichment of O-GalNAc glycans in white matter tracts and at nodes of Ranvier. Glycoproteomic analysis revealed that more than half of the identified O-GalNAc glycans were present on chondroitin sulfate proteoglycans termed lecticans, and display both domain enrichment and site-specific heterogeneity. Though genetic ablation in a single cell type failed to replicate the severe phenotypes seen in congenital disorders, inhibition of O-GalNAc synthesis in neurons reduced binding of Siglec-4, a known regulator neurite growth, and shortened the length of nodes of Ranvier. This work highlights a new function of O-GalNAc glycans in the brain and will inform future studies on their role in development and disease.

neuroscience↗

bioRxiv2023

Recognition of highly branched N-glycans of the porcine whipworm by the immune system

Glycans are key to host-pathogen interactions, whereby recognition by the host and immunomodulation by the pathogen can be mediated by carbohydrate binding proteins, such as lectins of the innate immune system, and their glycoconjugate ligands. Previous studies have shown that excretory-secretory products of the porcine nematode parasite Trichuris suis exert immunomodulatory effects in a glycan-dependent manner. To better understand the mechanisms of these interactions, we prepared N-glycans from T. suis and both analyzed their structures and used them to generate a natural glycan microarray. With this array we explored the interactions of glycans with C-type lectins, C-reactive protein and sera from T. suis infected pigs. Glycans containing LacdiNAc and phosphorylcholine-modified glycans were associated with the highest binding by most of these proteins. In-depth analysis revealed not only fucosylated LacdiNAc motifs with and without phosphorylcholine moieties, but phosphorylcholine-modified mannose and N-acetylhexosamine-substituted fucose residues, in the context of maximally tetraantennary N-glycan scaffolds. Furthermore, O-glycans also contained fucosylated motifs. In summary, the glycans of T. suis are recognized by both the innate and adaptive immune systems, and also exhibit species-specific features distinguishing its glycome from those of other nematodes.

biochemistry↗

bioRxiv2023

Platelet-Localized ST6Gal1 Does Not Impact IgG Sialylation

The IgG antibody class forms an important basis of the humoral immune response, conferring reciprocal protection from both pathogens and autoimmunity. IgG function is determined by the IgG subclass, as defined by the heavy chain, as well as the glycan composition at N297, the conserved site of N-glycosylation within the Fc domain. For example, lack of core fucose promotes increased antibody-dependent cellular cytotoxicity, whereas 2,6-linked sialylation by the enzyme ST6Gal1 helps to drive immune quiescence. Despite the immunological significance of these carbohydrates, little is known about how IgG glycan composition is regulated. We previously reported that mice with ST6Gal1-deficient B cells have unaltered IgG sialylation. Likewise, ST6Gal1 released into the plasma by hepatocytes does not significantly impact overall IgG sialylation. Since IgG and ST6Gal1 have independently been shown to exist in platelet granules, it was possible that platelet granules could serve as a B cell-extrinsic site for IgG sialylation. To address this hypothesis, we used a platelet factor 4 (Pf4)-Cre mouse to delete ST6Gal1 in megakaryocytes and platelets alone or in combination with an albumin-Cre mouse to also remove it from hepatocytes and the plasma. The resulting mouse strains were viable and had no overt pathological phenotype. We also found that despite targeted ablation of ST6Gal1, no change in IgG sialylation was apparent. Together with our prior findings, we can conclude that in mice, neither B cells, the plasma, nor platelets have a substantial role in homeostatic IgG sialylation.

immunology↗

bioRxiv2023

N-glycans show distinct spatial distribution in mouse brain

Protein N-linked glycosylation is a ubiquitous modification in the secretory pathway that plays a critical role in the development and function of the brain. N-glycans have a distinct composition and undergo tight regulation in the brain, but the spatial distribution of these structures remains relatively unexplored. Here, we systematically employed carbohydrate binding lectins with differing specificities to various classes of N-glycans and appropriate controls to identify multiple regions of the mouse brain. Lectins binding high-mannose-type N-glycans, the most abundant class of brain N-glycans, showed diffuse staining with some punctate structures observed on high magnification. Lectins binding specific motifs of complex N-glycans, including fucose and bisecting GlcNAc, showed more partitioned labeling, including to the synapse-rich molecular layer of the cerebellum. Understanding the distribution of N-glycans across the brain will aid future studies of these critical protein modifications in development and disease of the brain.

neuroscience↗

bioRxiv2022

Plasma ST6Gal1 is Dispensable for IgG Sialylation

The glycosylation of IgG has attracted increased attention due to the impact of N-glycan modifications at N297 on IgG function, acting primarily through modulation of Fc domain conformation and Fc{gamma} receptor binding affinities and signaling. However, the mechanisms regulating IgG glycosylation and especially 2,6-sialylation of its N-glycan remain poorly understood. We observed previously that IgG is normally sialylated in mice with B cells lacking the sialyltransferase ST6Gal1. This supported the hypothesis that IgG may be sialylated outside of B cells, perhaps through the action of hepatocyte-released plasma ST6Gal1. Here we demonstrate that this model is incorrect. Animals lacking hepatocyte expressed ST6Gal1 retain normal IgG 2,6-sialylation, despite the lack of detectable ST6Gal1 in plasma. Moreover, we confirmed that B cells were not a redundant source of IgG sialylation. Thus, while 2,6-sialylation is lacking in IgG from mice with germline ablation of ST6Gal1, IgG 2,6-sialylation is normal in mice lacking ST6Gal1 in either hepatocytes or B cells. These results indicate that IgG 2,6-sialylation arises after release from a B cell, but is not dependent on plasma-localized ST6Gal1 activity.

immunology↗

bioRxiv2021

A Useful Guide to Lectin Binding: Machine-Learning Directed Annotation of 57 Unique Lectin Specificities

Glycans are critical to every facet of biology and medicine, from viral infections to embryogenesis. Tools to study glycans are rapidly evolving, however the majority of our knowledge is deeply dependent on binding by glycan binding proteins (e.g., lectins). The specificities of lectins, which are often naturally isolated proteins, have not been well- defined, making it difficult to leverage their full potential for glycan analysis. Herein, we use glycan microarray analysis of 116 commercially available lectins, including different preparations of the same lectin, to extract the specific glycan features required for lectin binding. Data was obtained using the Consortium for Functional Glycomics microarray (CFG v5.0) containing 611 glycans. We use a combination of machine learning algorithms to define lectin specificity, mapping inputs (glycan sequences) to outputs (lectin-glycan binding) for a large-scale evaluation of lectin-glycan binding behaviours. Our motif analysis was performed by integrating 68 manually defined glycan features with systematic probing of computational rules for significant binding motifs using mono- and disaccharides- and linkages. Using a combination of machine learning and manual annotation of the data, we created a detailed interpretation of glycan-binding specificity for 57 unique lectins, categorized by their major binding motifs: mannose, complex-type N-glycan, O-glycan, fucose, sialic acid and sulfate, GlcNAc and chitin, Gal and LacNAc, and GalNAc. Our work provides fresh insights into the complex binding features of commercially available lectins in current use, providing a critical guide to these important reagents.

biochemistry↗

bioRxiv2020

The schizophrenia-associated variant in SLC39A8 alters N-glycosylation in the mouse brain

A missense mutation (A391T) in the manganese transporter SLC39A8 is strongly associated with schizophrenia in genomic studies, though the molecular connection to the brain remains hypothetical. Human carriers of A391T have reduced serum manganese, altered plasma glycosylation, and brain MRI changes consistent with altered metal transport. Here, using a knock-in mouse model homozygous for A391T, we show that the schizophrenia-associated variant changes protein glycosylation in the brain. N-linked glycosylation was most significantly impaired, with effects differing between regions. RNAseq analysis showed negligible regional variation, consistent with changes in the activity of glycosylation enzymes rather than gene expression. Finally, nearly one third of detected glycoproteins were differentially N-glycosylated in the cortex, including members of several pathways previously implicated in schizophrenia such as cell adhesion molecules and neurotransmitter receptors. These findings provide a mechanistic link between a risk allele and biochemical changes in the brain, furthering our molecular understanding of the pathophysiology of schizophrenia.

neuroscience↗

bioRxiv2020

Endogenous Galectin-3 is Required for Skeletal Muscle Repair

Skeletal muscle has the intrinsic ability to self-repair through a multifactorial process, but many aspects of its cellular and molecular mechanisms are not fully understood. There is increasing evidence that some members of the mammalian {beta}-galactoside-binding protein family (galectins) are involved in the muscular repair process (MRP), including galectin-3 (Gal-3). However, there are many questions about the role of this protein on muscle self-repair. Here, we demonstrate that endogenous Gal-3 is required for: i) muscle repair in vivo using a chloride-barium myolesion mouse model, and ii) mouse primary myoblasts myogenic programming. Injured muscle from Gal-3 knockout mice (GAL3KO) showed persistent inflammation associated with compromised muscle repair and the formation of fibrotic tissue on the lesion site. In GAL3KO mice, osteopontin expression remained high even after 7 and 14 days of the myolesion, while MyoD and myogenin had decreased their expression. In GAL3KO mouse primary myoblast cell culture, Pax7 detection seems to sustain even when cells are stimulated to differentiation and MyoD expression is drastically reduced. These findings suggest that the detection and temporal expression levels of these transcriptional factors appear to be altered in Gal-3-deficient myoblast cell culture compared to Wild Type (WT) cells. We observed Gal-3 expression in WT states, both in vivo and in vitro, in sarcoplasm/cytoplasm and myonuclei; as differentiation proceeds, Gal-3 expression is drastically reduced, and its location is confined to the sarcolemma/plasma cell membrane. We also observed a change in the temporal-spatial profile of Gal-3 expression and muscle transcription factors levels during the myolesion. Overall, these results demonstrate that endogenous Gal-3 is required for the skeletal muscle repair process.

cell biology↗

bioRxiv2020

The restricted nature of protein glycosylation in the mammalian brain

Glycosylation is essential to brain development and function, though prior studies have often been limited to a single analytical technique. Using several methodologies, we analyzed Asn-linked (N-glycans) and Ser/Thr/Tyr-linked (O-glycans) protein glycosylation between brain regions and sexes in mice. Brain N-glycans were surprisingly less complex in sequence and variety compared to other tissues, consisting predominantly of high-mannose precursors and fucosylated/bisected structures. Most brain O-glycans were unbranched, sialylated O-GalNAc and O-mannose structures. A consistent pattern was observed between regions, and sex differences were minimal compared to those observed in plasma. Brain glycans correlate with RNA expression of their synthetic enzymes, and analysis of all glycosylation genes in humans showed a global downregulation in the brain compared to other tissues. We hypothesize that the restricted repertoire of protein glycans arises from their tight regulation in the brain. These results provide a roadmap for future studies of glycosylation in neurodevelopment and disease.

neuroscience↗

bioRxiv2020

Galectin-1-deficient mice are protected against Trypanosoma cruzi infection through altered neutrophil migration and production of reactive oxygen species and nitric oxide

Trypanosoma cruzi is an intracellular parasite that causes Chagas disease that affects millions of people worldwide. Many cellular and molecular aspects of this neglected disease are not fully understood. Prior studies have shown that galectin-1 (Gal-1), a {beta}-galactoside-binding protein that regulates leukocyte recruitment to the inflammatory site, and promotes T. cruzi infection, but the mechanism is unclear. Here, we report that C57BL/6 mice lacking Gal-1 (Lgals1-/-) exhibited lower parasitemia and higher survival rates than their wildtype (WT) counterparts when infected with T. cruzi Y strain. Two weeks after infection, Lgals1-/- mice displayed greater neutrophil accumulation in infection site and heart tissue than WT mice. In T. cruzi-infected Lgals1-/- mice, infiltrated neutrophils produced increased levels of reactive oxygen species (ROS), while macrophages and neutrophils produced increased levels of nitric oxide (NO), which reduced replication and viability of parasites in vitro and downregulated IL-1{beta} production. Pharmacological inhibition of NADPH oxidase and NO synthase during early in vivo infection reversed the protective effect of Gal-1 deficiency in Lgals1-/- mice. Together, our findings demonstrate that lacking Gal-1 favors neutrophil migration to the infection site and increases production of ROS and NO, thereby controlling the early steps of T. cruzi infection by reducing parasitemia and prolonging survival of infected mice.

immunology↗

bioRxiv2020

SARS-CoV-2 Spike Protein Interacts with Multiple Innate Immune Receptors

The spike (S) glycoprotein in the envelope of SARS-CoV-2 is densely glycosylated but the functions of its glycosylation are unknown. Here we demonstrate that S is recognized in a glycan-dependent manner by multiple innate immune receptors including the mannose receptor MR/CD206, DC-SIGN/CD209, L-SIGN/CD209L, and MGL/CLEC10A/CD301. Single-cell RNA sequencing analyses indicate that such receptors are highly expressed in innate immune cells in tissues susceptible to SARS-CoV-2 infection. Binding of the above receptors to S is characterized by affinities in the picomolar range and consistent with S glycosylation analysis demonstrating a variety of N- and O-glycans as receptor ligands. These results indicate multiple routes for SARS-CoV-2 to interact with human cells and suggest alternative strategies for therapeutic intervention.

biochemistry↗

bioRxiv2020

Mucin glycans signal through the sensor kinase RetS to inhibit virulence-associated traits in Pseudomonas aeruginosa

Mucus is a densely populated ecological niche that coats all epithelia, and plays a critical role in protecting the human body from infections. Although traditionally viewed as a physical barrier, emerging evidence suggests that mucus can directly suppress virulence traits in opportunistic pathogens like Pseudomonas aeruginosa. However, the molecular mechanisms by which mucus affords this protection are unclear. Here, we show that mucins, and particularly their associated glycans, activate the sensor kinase RetS via its Dismed2 domain in P. aeruginosa. We find that this RetS-dependent signaling leads to the direct inhibition of the GacS-GacA two-component system, the activity of which is associated with a chronic infection state. This signaling includes the downregulation of the type VI secretion system (T6SS), and prevents T6SS-dependent bacterial killing by P. aeruginosa. Overall, these results shed light on how mucus impacts P. aeruginosa behavior in the human host, and may inspire novel approaches for controlling P. aeruginosa infections.

microbiology↗

bioRxiv2020

Resident and elicited macrophages differ in expression of their glycomes and lectins

The pleiotropic functions of macrophages in immune defense, tissue repair, and maintenance of tissue homeostasis are supported by the heterogeneity in macrophage sub-populations that differ both in ontogeny and polarization. Although glycans and lectins are integral to macrophage function, little is known about the factors governing their expression. Here we show that the cellular glycome of murine peritoneal macrophages primarily reflects developmental origin and to a lesser degree, cellular polarization. Resident macrophages were characterized by a simple glycome, predominantly consisting of core 1 O-glycans, while elicited macrophages also expressed core 2 O-glycans, along with highly branched and extended complex-type N-glycans, that exhibited a higher N-acetylneuraminic acid:N-glycolylneuraminic acid ratio. Strikingly, our analysis revealed that resident and elicited macrophages express 139 lectin genes, with differential expression of 49 lectin genes, including galectins, Siglecs, and C-type lectins. These results suggest that regulation of self-glycan-protein complexes may be central to macrophage residence and recruitment.

biochemistry↗