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It has been suggested that the prevalence of obesity in black women is high partly because self-image in black women is not strongly dependent on body size. To determine associations between self-image, body size, and dieting behavior among black women, the authors assessed an Appearance Evaluation Subscale (AES) score (range, 1-5), a Body Image Satisfaction (BIS) score (range, 2-11), and reported dieting behavior in a population-based sample of 1,143 black women aged 24-42 years from the fourth follow-up examination (1992-1993) of the Coronary Artery Risk Development in Young Adults (CARDIA) Study. Lower AES and BIS scores indicate poorer self-image and lower body size satisfaction, respectively. After adjustment for age, education, smoking, and physical activity, women in the lowest, middle, and highest tertiles of body mass index (weight (kg)/height (m)2) had mean AES scores of 3.7, 3.3, and 2.9, respectively (p < 0.001), and mean BIS scores of 7.8, 6.7, and 5.9, respectively (p < 0.001). After additional control for body mass index as a continuous variable, both AES and BIS scores were inversely related to ever dieting, current dieting, and previous weight loss of 10 pounds (4.5 kg) or more in all tertiles of body mass index. These results suggest that among black women, a higher body mass index is associated with poorer self-image and lower body size satisfaction and that these perceptions may be an avenue to promoting weight control. [ABSTRACT FROM AUTHOR]

Deamidation of asparagine and glutamine residues occurs spontaneously, is influenced by pH, temperature, and incubation time, and can be accelerated by adjacent amino acid residues. Incubation conditions used for proteolytic digestion in bottom-up proteomic studies can induce significant deamidation that affects results, either knowingly or unknowingly. This has prompted studies into modifications to common trypsin digestion protocols to minimize chemical deamidation, including shorter incubation times and specific lysis buffers. Prior work suggested ammonium acetate at pH 6 to minimize chemical deamidation, but this buffer has compatibility issues with trypsin digestion and common assays (e.g., bicinchoninic acid assays). Here, we re-evaluated former comparisons of Tris-HCl, ammonium bicarbonate, and triethylammonium bicarbonate buffers for the amount of artificial, chemically induced deamidation generated in a standard bottom-up proteomics workflow, and we added an evaluation of three commonly used and biologically compatible buffers, HEPES (4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid), EPPS (3-[4-(2-Hydroxyethyl)piperazin-1-yl]propane-1-sulfonic acid), and PBS (phosphate buffered saline). Our findings show that HEPES exhibited the least amount of artificial deamidation and is a reasonable choice for general proteomic experiments, especially for studies considering N-glycosylation.

A critical step in infections is the attachment of many microorganisms to host cells using lectins that bind surface glycans, making lectins promising antimicrobial targets. Upon binding mannosylated glycans, FimH, the most studied lectin adhesin of type 1 fimbriae in E. coli , undergoes an allosteric transition from an inactive to an active conformation that can act as a catch-bond. Monoclonal antibodies that alter FimH glycan binding in various ways are available, but the mechanisms of these antibodies remain unclear. Here, we use cryoEM, mass spectrometry, binding assays, and molecular dynamics simulations to determine the structure-function relationships underlying antibody-FimH binding. Our study reveals four distinct antibody mechanisms of action: ligand mimicry by an N-linked, high-mannose glycan; stabilization of the ligand pocket in the inactive state; conformational trapping of the active and inactive states; and locking of the ligand pocket through long-range allosteric effects. These structures reveal multiple mechanisms of antibody responses to an allosteric protein and provide blueprints for new antimicrobial that target adhesins., Competing Interests: Competing Interests Statement EVS and VT hold US10722580B2 patent ‘Compositions and methods for treatment and prevention of uropathogenic E. coli infection’ that include therapeutic use of mAb475 and mAb926. N.M.R. receives support from Thermo Fisher Scientific under a nondisclosure agreement and is a consultant for Tegmine Therapeutics, Cartography Biosciences, and Augment Biologics.

Tandem mass spectrometry (MS/MS) is the gold standard for intact glycopeptide identification, enabling peptide sequence elucidation and site-specific localization of glycan compositions. Beam-type collisional activation is generally sufficient for N- glycopeptides, while electron-driven dissociation is crucial for site localization in O- glycopeptides. Modern glycoproteomic methods often employ multiple dissociation techniques within a single LC-MS/MS analysis, but this approach frequently sacrifices sensitivity when analyzing multiple glycopeptide classes simultaneously. Here we explore the utility of intelligent data acquisition for glycoproteomics through real-time library searching (RTLS) to match oxonium ion patterns for on-the-fly selection of the appropriate dissociation method. By matching dissociation method with glycopeptide class, this autonomous dissociation-type selection (ADS) generates equivalent numbers of N- glycopeptide identifications relative to traditional beam-type collisional activation methods while also yielding comparable numbers of site-localized O- glycopeptide identifications relative to conventional electron transfer dissociation-based methods. The ADS approach represents a step forward in glycoproteomics throughput by enabling site-specific characterization of both N- and O- glycopeptides within the same LC-MS/MS acquisition.

Prostate cancer is the second leading cause of male cancer death in the U.S. Current immune checkpoint inhibitor-based immunotherapies have improved survival for many malignancies; however, they have failed to prolong survival for prostate cancer. Siglecs (sialic acid-binding immunoglobulin-like lectins) are expressed on immune cells and regulate their function. Siglec-7 and Siglec-9 contribute to immune evasion in cancer by interacting with sialic acid-containing glycoprotein ligands on cancer cells. However, the role of Siglec-7/9 receptors and their ligands in prostate cancer remains poorly understood. Here, we find that Siglec-7 and Siglec-9 are associated with poor prognosis in patients with prostate cancer and are highly expressed in myeloid cells, including macrophages, in prostate tumor tissues. Siglec-7 and -9 ligands were expressed in prostate cancer cells and human prostate tumor tissues. Blocking the interactions between Siglec-7/9 and sialic acids inhibited prostate cancer xenograft growth and increased immune cell infiltration in humanized mice in vivo. Using a CRISPRi screen and mass spectrometry, we identified CD59 as a candidate Siglec-9 ligand in prostate cancer. The identification of Siglec-7 and -9 as potential therapeutic targets, including the CD59/Siglec-9 axis, opens up opportunities for immune-based interventions in prostate cancer.

RNA binding proteins ( RBPs ) control varied processes, including RNA splicing, stability, transport, and translation 1-3 . Dysfunctional RNA-RBP interactions contribute to the pathogenesis of human disease 1,4,5 , however, characterizing the nature and dynamics of multiprotein assemblies on RNA has been challenging. To address this, non-isotopic ligation-based ultraviolet crosslinking immunoprecipitation 6 was combined with mass spectrometry ( irCLIP-RNP ) to identify RNA-dependent associated proteins ( RDAPs ) co-bound to RNA with any RBP of interest. irCLIP-RNP defined landscapes of multimeric protein assemblies on RNA, uncovering previously unknown patterns of RBP-RNA associations, including cell-type-selective combinatorial relationships between RDAPs and primary RBPs. irCLIP-RNP also defined dynamic RDAP remodeling in response to epidermal growth factor ( EGF ), uncovering EGF-induced recruitment of UPF1 adjacent to HNRNPC to effect splicing surveillance of cell proliferation mRNAs. To identify the RNAs simultaneously co-bound by multiple studied RBPs, a sequential immunoprecipitation irCLIP ( Re-CLIP ) method was also developed. Re-CLIP confirmed binding relationships seen in irCLIP-RNP and detected simultaneous HNRNPC and UPF1 co-binding on RND3 and DDX3X mRNAs. irCLIP-RNP and Re-CLIP provide a framework to identify and characterize dynamic RNA-protein assemblies in living cells.

Glycoproteomics is a rapidly developing field, and data analysis has been stimulated by several technological innovations. As a result, there are many software tools from which to choose; and each comes with unique features that can be difficult to compare. This work presents a head-to-head comparison of five modern analytical software: Byonic, Protein Prospector, MSFraggerGlyco, pGlyco3, and GlycoDecipher. To enable a meaningful comparison, parameter variables were minimized. One potential confounding variable is the glycan database that informs glycoproteomic searches. We performed glycomic profiling of the samples and used the output to construct matched glycan databases for each software. Up to 19,000 glycopeptide spectra were identified across three replicates of wild-type SH-SY5Y cells. There was substantial overlap among most software for glycoproteins identified, locations of glycosites, and glycans, although Byonic reported a suspiciously large number of glycoproteins and glycosites of questionable reliability. We show that Protein Prospector identified the most glycopeptide spectrum matches with high agreement to known glycosites in UniProt. Overall, our results indicate that glycoproteomic searches should involve more than one software to generate confidence. It may be useful to consider software with peptide-first approaches and with glycan-first approaches., Competing Interests: Competing Interests The authors have no conflicts of interest to declare.

Proteomics is the large scale study of protein structure and function from biological systems through protein identification and quantification. "Shotgun proteomics" or "bottom-up proteomics" is the prevailing strategy, in which proteins are hydrolyzed into peptides that are analyzed by mass spectrometry. Proteomics studies can be applied to diverse studies ranging from simple protein identification to studies of proteoforms, protein-protein interactions, protein structural alterations, absolute and relative protein quantification, post-translational modifications, and protein stability. To enable this range of different experiments, there are diverse strategies for proteome analysis. The nuances of how proteomic workflows differ may be challenging to understand for new practitioners. Here, we provide a comprehensive overview of different proteomics methods. We cover from biochemistry basics and protein extraction to biological interpretation and orthogonal validation. We expect this Review will serve as a handbook for researchers who are new to the field of bottom-up proteomics., Competing Interests: The authors declare no competing financial interest., (© 2024 The Authors. Published by American Chemical Society.)

Isolates of the citrus brown rot pathogens Phytophthora citrophthora and P. syringae from the Inland Empire (IE) and Ventura Co. (VE) regions of southern California were evaluated for their sensitivity to ethaboxam, fluopicolide, mandipropamid, and oxathiapiprolin, and the previously published baselines that were generated for Central Valley (CV) isolates of California were expanded. Fungicides were generally more toxic to CV isolates of both species for all four fungicides. Specific differences were found in the toxicity of ethaboxam to P. syringae where CV isolates on average were 6.8 or 8.2 times more sensitive than those from the VE or IE regions, respectively. Based on the grouping of isolates in an unweighted pair-group method with arithmetic mean (UPGMA) dendrogram, as well as fastStructure analyses and plotting of principal component analyses (PCAs), differences in ethaboxam sensitivity could be related to differences in genetic background of the isolates. Isolates of P. citrophthora from the IE and VE had slightly reduced (i.e., 1.5×) sensitivity to mandipropamid as compared with isolates from the CV and were found on distinct branches in the UPGMA dendrogram. Differences in genetic background of less sensitive isolates within each species indicate that these two phenotypes emerged multiple times independently. IE and VE isolates of both species were sensitive to mefenoxam. Moderate resistance to potassium phosphite (EC 50 values of 25 to 75 μg/ml) was present in IE and VE isolates of P. syringae , whereas some IE isolates of P. citrophthora were considered resistant with EC 50 values of up to 113.69 μg/ml. Resistance to potassium phosphite did not relate to distinct genotypes., Competing Interests: The author(s) declare no conflict of interest.

Brain metastatic breast cancer is particularly lethal largely due to therapeutic resistance. Almost half of the patients with metastatic HER2-positive breast cancer develop brain metastases, representing a major clinical challenge. We previously described that cancer-associated fibroblasts are an important source of resistance in primary tumors. Here, we report that breast cancer brain metastasis stromal cell interactions in 3D cocultures induce therapeutic resistance to HER2-targeting agents, particularly to the small molecule inhibitor of HER2/EGFR neratinib. We investigated the underlying mechanisms using a synthetic Notch reporter system enabling the sorting of cancer cells that directly interact with stromal cells. We identified mucins and bulky glycoprotein synthesis as top-up-regulated genes and pathways by comparing the gene expression and chromatin profiles of stroma-contact and no-contact cancer cells before and after neratinib treatment. Glycoprotein gene signatures were also enriched in human brain metastases compared to primary tumors. We confirmed increased glycocalyx surrounding cocultures by immunofluorescence and showed that mucinase treatment increased sensitivity to neratinib by enabling a more efficient inhibition of EGFR/HER2 signaling in cancer cells. Overexpression of truncated MUC1 lacking the intracellular domain as a model of increased glycocalyx-induced resistance to neratinib both in cell culture and in experimental brain metastases in immunodeficient mice. Our results highlight the importance of glycoproteins as a resistance mechanism to HER2-targeting therapies in breast cancer brain metastases., Competing Interests: Competing interests statement:K.P. serves on the Scientific Advisory Boards of Novartis, Ideaya Biosciences, and Scorpion Therapeutics, holds equity options in Scorpion Therapeutics and Ideaya Biosciences. L.E.S. is a current employee of Astra-Zeneca. C.R.B. is a cofounder and member of the Scientific Advisory Boards of Palleon Pharmaceuticals, Lycia Therapeutics, InterVenn Bio, OliLux Bio, Enable Biosciences, Redwood Bioscience (a subsidiary of Catalent), GanNA Bio, Firefly Bio, Neuravid and Valora Therapeutics.

Targeted protein degradation is an emerging strategy for the elimination of classically undruggable proteins. Here, to expand the landscape of targetable substrates, we designed degraders that achieve substrate selectivity via recognition of a discrete peptide and glycan motif and achieve cell-type selectivity via antigen-driven cell-surface binding. We applied this approach to mucins, O-glycosylated proteins that drive cancer progression through biophysical and immunological mechanisms. Engineering of a bacterial mucin-selective protease yielded a variant for fusion to a cancer antigen-binding nanobody. The resulting conjugate selectively degraded mucins on cancer cells, promoted cell death in culture models of mucin-driven growth and survival, and reduced tumor growth in mouse models of breast cancer progression. This work establishes a blueprint for the development of biologics that degrade specific protein glycoforms on target cells., (© 2023. The Author(s).)

Galectin-3, well characterized as a glycan binding protein, has been identified as a putative RNA binding protein, possibly through participation in pre-mRNA maturation through interactions with splicosomes. Given recent developments with cell surface RNA biology, the putative dual-function nature of galectin-3 evokes a possible non-classical connection between glycobiology and RNA biology. However, with limited functional evidence of a direct RNA interaction, many molecular-level observations rely on affinity reagents and lack appropriate genetic controls. Thus, evidence of a direct interaction remains elusive. We demonstrate that antibodies raised to endogenous human galectin-3 can isolate RNA-protein crosslinks, but this activity remains insensitive to LGALS3 knock-out. Proteomic characterization of anti-galectin-3 IPs revealed enrichment of galectin-3, but high abundance of hnRNPA2B1, an abundant, well-characterized RNA-binding protein with weak homology to the N-terminal domain of galectin-3, in the isolate. Genetic ablation of HNRNPA2B1, but not LGALS3, eliminates the ability of the anti-galectin-3 antibodies to isolate RNA-protein crosslinks, implying either an indirect interaction or cross-reactivity. To address this, we introduced an epitope tag to the endogenous C-terminal locus of LGALS3. Isolation of the tagged galectin-3 failed to reveal any RNA-protein crosslinks. This result suggests that the galectin-3 does not directly interact with RNA and may be misidentified as an RNA-binding protein, at least in HeLa where the putative RNA associations were first identified. We encourage further investigation of this phenomenon employ gene deletions and, when possible, endogenous epitope tags to achieve the specificity required to evaluate potential interactions., (© The Author(s) 2023. Published by Oxford University Press.)

Siglec-7 (sialic acid-binding immunoglobulin-like lectin 7) is a glycan-binding immune receptor that is emerging as a significant target of interest for cancer immunotherapy. The physiological ligands that bind Siglec-7, however, remain incompletely defined. In this study, we characterized the expression of Siglec-7 ligands on peripheral immune cell subsets and assessed whether Siglec-7 functionally regulates interactions between immune cells. We found that disialyl core 1 O-glycans are the major immune ligands for Siglec-7 and that these ligands are particularly highly expressed on naïve T-cells. Densely glycosylated sialomucins are the primary carriers of these glycans, in particular a glycoform of the cell-surface marker CD43. Biosynthesis of Siglec-7-binding glycans is dynamically controlled on different immune cell subsets through a genetic circuit involving the glycosyltransferase GCNT1. Siglec-7 blockade was found to increase activation of both primary T-cells and antigen-presenting dendritic cells in vitro, indicating that Siglec-7 binds T-cell glycans to regulate intraimmune signaling. Finally, we present evidence that Siglec-7 directly activates signaling pathways in T-cells, suggesting a new biological function for this receptor. These studies conclusively demonstrate the existence of a novel Siglec-7-mediated signaling axis that physiologically regulates T-cell activity. Going forward, our findings have significant implications for the design and implementation of therapies targeting immunoregulatory Siglec receptors., Competing Interests: Conflict of interest C. R. B. is a cofounder and Scientific Advisory Board member of Palleon Pharmaceuticals, Enable Bioscience, Redwood Biosciences (a subsidiary of Catalent), InterVenn Biosciences, Lycia Therapeutics, Virsti Therapeutics, OliLux Biosciences, and GanNA Bio. S. W. and C. R. B. are coinventors on a patent application related to work reviewed in this article held by Stanford University (grant no.: PCT/US2020/041603)., (Copyright © 2023 The Authors. Published by Elsevier Inc. All rights reserved.)

Proteomics is the large scale study of protein structure and function from biological systems through protein identification and quantification. "Shotgun proteomics" or "bottom-up proteomics" is the prevailing strategy, in which proteins are hydrolyzed into peptides that are analyzed by mass spectrometry. Proteomics studies can be applied to diverse studies ranging from simple protein identification to studies of proteoforms, protein-protein interactions, protein structural alterations, absolute and relative protein quantification, post-translational modifications, and protein stability. To enable this range of different experiments, there are diverse strategies for proteome analysis. The nuances of how proteomic workflows differ may be challenging to understand for new practitioners. Here, we provide a comprehensive overview of different proteomics methods to aid the novice and experienced researcher. We cover from biochemistry basics and protein extraction to biological interpretation and orthogonal validation. We expect this work to serve as a basic resource for new practitioners in the field of shotgun or bottom-up proteomics.

Neurons communicate with each other through electrochemical transmission at synapses. Microglia, the resident immune cells of the central nervous system, modulate this communication through a variety of contact-dependent and -independent means. Microglial secretion of active sialidase enzymes upon exposure to inflammatory stimuli is one unexplored mechanism of modulation. Recent work from our lab showed that treatment of neurons with bacterial sialidases disrupts neuronal network connectivity. Here, we find that activated microglia secrete neuraminidase-3 (Neu3) associated with fusogenic extracellular vesicles. Furthermore, we show that Neu3 mediates contact-independent disruption of neuronal network synchronicity through neuronal glycocalyx remodeling. We observe that NEU3 is transcriptionally upregulated upon exposure to inflammatory stimuli and that a genetic knockout of NEU3 abrogates the sialidase activity of inflammatory microglial secretions. Moreover, we demonstrate that Neu3 is associated with a subpopulation of extracellular vesicles, possibly exosomes, that are secreted by microglia upon inflammatory insult. Finally, we demonstrate that Neu3 is necessary and sufficient to both desialylate neurons and decrease neuronal network connectivity. These results implicate Neu3 in remodeling of the glycocalyx leading to aberrant network-level activity of neurons, with implications in neuroinflammatory diseases such as Parkinson's disease and Alzheimer's disease., Competing Interests: The authors declare the following competing financial interest(s): C.R.B. is a co-founder and Scientific Advisory Board member of Lycia Therapeutics, Palleon Pharmaceuticals, Enable Bioscience, Redwood Biosciences (a subsidiary of Catalent), InterVenn Bio, GanNa Bio, OliLux Bio, Neuravid Therapeutics, Valora Therapeutics, and Firefly Bio, and is a member of the Board of Directors of Alnylam Pharmaceuticals and OmniAb. R.U.K. is a co-inventor on a patent related to voltage-gated imaging dyes., (© 2023 The Authors. Published by American Chemical Society.)

Targeted protein degradation can provide advantages over inhibition approaches in the development of therapeutic strategies. Lysosome-targeting chimeras (LYTACs) harness receptors, such as the cation-independent mannose 6-phosphate receptor (CI-M6PR), to direct extracellular proteins to lysosomes. In this work, we used a genome-wide CRISPR knockout approach to identify modulators of LYTAC-mediated membrane protein degradation in human cells. We found that disrupting retromer genes improved target degradation by reducing LYTAC recycling to the plasma membrane. Neddylated cullin-3 facilitated LYTAC-complex lysosomal maturation and was a predictive marker for LYTAC efficacy. A substantial fraction of cell surface CI-M6PR remains occupied by endogenous M6P-modified glycoproteins. Thus, inhibition of M6P biosynthesis increased the internalization of LYTAC-target complexes. Our findings inform design strategies for next-generation LYTACs and elucidate aspects of cell surface receptor occupancy and trafficking.

Neurons communicate with each other through electrochemical transmission at synapses. Microglia, the resident immune cells of the central nervous system, can prune these synapses through a variety of contact-dependent and -independent means. Microglial secretion of active sialidase enzymes upon exposure to inflammatory stimuli is one unexplored mechanism of pruning. Recent work from our lab showed that treatment of neurons with bacterial sialidases disrupts neuronal network connectivity. Here, we find that activated microglia secrete Neuraminidase-3 (Neu3) associated with fusogenic extracellular vesicles. Furthermore, we show Neu3 mediates contact-independent pruning of neurons and subsequent disruption of neuronal networks through neuronal glycocalyx remodeling. We observe that NEU3 is transcriptionally upregulated upon exposure to inflammatory stimuli, and that a genetic knock-out of NEU3 abrogates the sialidase activity of inflammatory microglial secretions. Moreover, we demonstrate that Neu3 is associated with a subpopulation of extracellular vesicles, possibly exosomes, that are secreted by microglia upon inflammatory insult. Finally, we demonstrate that Neu3 is both necessary and sufficient to both desialylate neurons and decrease neuronal network connectivity. These results implicate Neu3 in remodeling of the glycocalyx leading to aberrant network-level activity of neurons, with implications in neuroinflammatory diseases such as Parkinson's disease and Alzheimer's disease., Competing Interests: Competing Interests. C.R.B. is a co-founder and Scientific Advisory Board member of Lycia Therapeutics, Palleon Pharmaceuticals, Enable Bioscience, Redwood Biosciences (a subsidiary of Catalent), InterVenn Bio, GanNa Bio, OliLux Bio, Neuravid Therapeutics, Valora Therapeutics, and Firefly Bio, and is a member of the Board of Directors of Alnylam Pharmaceuticals and OmniAb. R.L.K. is a co-inventor on a patent related voltage-gated imaging dyes.

Efforts to identify anti-cancer therapeutics and understand tumor-immune interactions are built with in vitro models that do not match the microenvironmental characteristics of human tissues. Using in vitro models which mimic the physical properties of healthy or cancerous tissues and a physiologically relevant culture medium, we demonstrate that the chemical and physical properties of the microenvironment regulate the composition and topology of the glycocalyx. Remarkably, we find that cancer and age-related changes in the physical properties of the microenvironment are sufficient to adjust immune surveillance via the topology of the glycocalyx, a previously unknown phenomenon observable only with a physiologically relevant culture medium., Competing Interests: Competing Interests J.R.C. is an inventor on an issued patent for Human Plasma-Like Medium (HPLM) assigned to the Whitehead Institute (Application number: PCT/US2017/061377. Patent number: 11453858. Issue date: 09/27/2022)

There is a significant interest in identifying blood-borne factors that mediate tissue crosstalk and function as molecular effectors of physical activity. Although past studies have focused on an individual molecule or cell type, the organism-wide secretome response to physical activity has not been evaluated. Here, we use a cell-type-specific proteomic approach to generate a 21-cell-type, 10-tissue map of exercise training-regulated secretomes in mice. Our dataset identifies >200 exercise training-regulated cell-type-secreted protein pairs, the majority of which have not been previously reported. Pdgfra-cre-labeled secretomes were the most responsive to exercise training. Finally, we show anti-obesity, anti-diabetic, and exercise performance-enhancing activities for proteoforms of intracellular carboxylesterases whose secretion from the liver is induced by exercise training., Competing Interests: Declaration of interests Stanford University has filed a provisional patent on extracellular CES2 proteins and methods of use., (Copyright © 2023 Elsevier Inc. All rights reserved.)

The colony-stimulating factor 3 receptor (CSF3R) controls the growth of neutrophils, the most abundant type of white blood cell. In healthy neutrophils, signaling is dependent on CSF3R binding to its ligand, CSF3. A single amino acid mutation in CSF3R, T618I, instead allows for constitutive, ligand-independent cell growth and leads to a rare type of cancer called chronic neutrophilic leukemia. However, the disease mechanism is not well understood. Here, we investigated why this threonine to isoleucine substitution is the predominant mutation in chronic neutrophilic leukemia and how it leads to uncontrolled neutrophil growth. Using protein domain mapping, we demonstrated that the single CSF3R domain containing residue 618 is sufficient for ligand-independent activity. We then applied an unbiased mutational screening strategy focused on this domain and found that activating mutations are enriched at sites normally occupied by asparagine, threonine, and serine residues-the three amino acids which are commonly glycosylated. We confirmed glycosylation at multiple CSF3R residues by mass spectrometry, including the presence of GalNAc and Gal-GalNAc glycans at WT threonine 618. Using the same approach applied to other cell surface receptors, we identified an activating mutation, S489F, in the interleukin-31 receptor alpha chain. Combined, these results suggest a role for glycosylated hotspot residues in regulating receptor signaling, mutation of which can lead to ligand-independent, uncontrolled activity and human disease., Competing Interests: Conflict of interest C. R. B. is a cofounder and scientific advisory board member of Lycia Therapeutics, Palleon Pharmaceuticals, Enable Biosciences, Redwood Biosciences (a subsidiary of Catalent), OliLux Bio, Grace Science LLC, and InterVenn Biosciences. J. R. C. is a cofounder and equity holder of Combangio, Inc. (now Kala Pharmaceuticals), xCella Biosciences (now OmniAb), and Red Tree Venture Capital; has financial interests in Aravive, Inc.; is a member of the Board of Directors of OmniAb and Revel Pharmaceuticals; and is a Board Observer at Excellergy Therapeutics, Tachyon Therapeutics, and Acrigen Biosciences. J. E. M. receives funding from Gilead Sciences, Kura Oncology, Blueprint Medicines and is involved in a collaboration with Ionis Pharmaceuticals. S. A. M. is a consultant for InterVenn Biosciences. All other authors declare no conflicts of interest with the contents of this article., (Copyright © 2023 The Authors. Published by Elsevier Inc. All rights reserved.)

Food insecurity, poised to increase with burgeoning concerns related to climate change, may influence sleep, yet few studies examined the food security-sleep association among racially/ethnically diverse populations with multiple sleep dimensions. We determined overall and racial/ethnic-specific associations between food security and sleep health. Using National Health Interview Survey data, we categorised food security as very low, low, marginal and high. Sleep duration was categorised as very short, short, recommended and long. Sleep disturbances included trouble falling/staying asleep, insomnia symptoms, waking up feeling unrested and using sleep medication (all ≥3 d/times in the previous week). Adjusting for socio-demographic characteristics and other confounders, we used Poisson regression with robust variance to estimate prevalence ratios (PRs) and 95 % confidence intervals (95 % CIs) for sleep dimensions by food security. Among 177 435 participants, the mean age of 47⋅2 ± 0⋅1 years, 52⋅0 % were women, and 68⋅4 % were non-Hispanic (NH)-White. A higher percent of NH-Black (7⋅9 %) and Hispanic/Latinx (5⋅1 %) lived in very low food security households than NH-White (3⋅1 %) participants. Very low v. high food security was associated with a higher prevalence of very short (PR = 2⋅61 [95 % CI 2⋅44-2⋅80]) sleep duration as well as trouble falling asleep (PR = 2⋅21 [95 % CI 2⋅12-2⋅30]). Very low v. high food security was associated with a higher prevalence of very short sleep duration among Asian (PR = 3⋅64 [95 % CI 2⋅67-4⋅97]) and NH-White (PR = 2⋅73 [95 % CI 2⋅50-2⋅99]) participants compared with NH-Black (PR = 2⋅03 [95 % CI 1⋅80-2⋅31]) and Hispanic/Latinx (PR = 2⋅65 [95 % CI 2⋅30-3⋅07]) participants. Food insecurity was associated with poorer sleep in a racially/ethnically diverse US sample., (© Cambridge University Press 2023.)

The Siglecs (sialic acid-binding immunoglobulin-like lectins) are glycoimmune checkpoint receptors that suppress immune cell activation upon engagement of cognate sialoglycan ligands. The cellular drivers underlying Siglec ligand production on cancer cells are poorly understood. We find the MYC oncogene causally regulates Siglec ligand production to enable tumor immune evasion. A combination of glycomics and RNA-sequencing of mouse tumors revealed the MYC oncogene controls expression of the sialyltransferase St6galnac4 and induces a glycan known as disialyl-T. Using in vivo models and primary human leukemias, we find that disialyl-T functions as a "don't eat me" signal by engaging macrophage Siglec-E in mice or the human ortholog Siglec-7, thereby preventing cancer cell clearance. Combined high expression of MYC and ST6GALNAC4 identifies patients with high-risk cancers and reduced tumor myeloid infiltration. MYC therefore regulates glycosylation to enable tumor immune evasion. We conclude that disialyl-T is a glycoimmune checkpoint ligand. Thus, disialyl-T is a candidate for antibody-based checkpoint blockade, and the disialyl-T synthase ST6GALNAC4 is a potential enzyme target for small molecule-mediated immune therapy.

Objective: Childhood adversity has been associated with metabolic syndrome (MetS) and type 2 diabetes risk in adulthood. However, studies have yet to investigate traumatic childhood experiences (TCEs) beyond abuse and neglect (e.g., natural disaster) while considering potential racial/ethnic differences., Research Design and Methods: To investigate race/ethnicity as a potential modifier of the association between TCEs, MetS, and type 2 diabetes, we used prospectively collected data from 42,173 eligible non-Hispanic White (NHW; 88%), Black/African American (BAA; 7%), and Hispanic/Latina (4%) Sister Study participants (aged 35-74 years) enrolled from 2003 to 2009. A modified Brief Betrayal Trauma Survey captured TCEs. At least three prevalent metabolic abnormalities defined MetS, and self-report of a new diagnosis during the study period defined type 2 diabetes. We used adjusted Cox proportional hazards regression to estimate hazard ratios (HRs) and 95% CIs for type 2 diabetes over a mean ± SD follow-up of 11.1 ± 2.7 years, overall and by race/ethnicity. We also tested for modification and mediation by MetS., Results: Incident cases of type 2 diabetes were reported (n = 2,479 among NHW, 461 among BAA, and 281 among Latina participants). Reporting any TCEs (50% among NHW, 53% among BAA, and 51% among Latina participants) was associated with a 13% higher risk of type 2 diabetes (HR 1.13; 95% CI 1.04-1.22). Associations were strongest among Latina participants (HR 1.64 [95% CI 1.21-2.22] vs. 1.09 for BAA and NHW). MetS was not a modifier but mediated (indirect effect, HR 1.01 [95% CI 1.00-1.01]; P = 0.02) the overall association., Conclusions: TCE and type 2 diabetes associations varied by race/ethnicity and were partially explained by MetS., (© 2023 by the American Diabetes Association.)

Mucin-domain glycoproteins are highly O-glycosylated cell surface and secreted proteins that serve as both biochemical and biophysical modulators. Aberrant expression and glycosylation of mucins are known hallmarks in numerous malignancies, yet mucin-domain glycoproteins remain enigmatic in the broad landscape of cancer glycobiology. Here we review the multifaceted roles of mucins in cancer through the lens of the analytical and biochemical methods used to study them. We also describe a collection of emerging tools that are specifically equipped to characterize mucin-domain glycoproteins in complex biological backgrounds. These approaches are poised to further elucidate how mucin biology can be understood and subsequently targeted for the next generation of cancer therapeutics., (Copyright © 2023 Elsevier Inc. All rights reserved.)

O -Glycoproteases are an emerging class of enzymes that selectively digest glycoproteins at positions decorated with specific O -linked glycans. O -Glycoprotease substrates range from any O -glycoprotein (albeit with specific O -glycan modifications) to only glycoproteins harboring specific O -glycosylated sequence motifs, such as those found in mucin domains. Their utility for multiple glycoproteomic applications is driving the search to both discover new O -glycoproteases and to understand how structural features of characterized O -glycoproteases influence their substrate specificities. One challenge of defining O -glycoprotease specificity restraints is the need to characterize O -glycopeptides with site-specific analysis of O -glycosites. Here, we demonstrate how O-Pair Search, a recently developed O -glycopeptide-centric identification platform that enables rapid searches and confident O -glycosite localization, can be used to determine substrate specificities of various O -glycoproteases de novo from LC-MS/MS data of O -glycopeptides. Using secreted protease of C1 esterase inhibitor (StcE) from enterohemorrhagic Escherichia coli and O -endoprotease OgpA from Akkermansia mucinophila , we explore numerous settings that effect O -glycopeptide identification and show how non-specific and semi-tryptic searches of O -glycopeptide data can produce candidate cleavage motifs. These putative motifs can be further used to define new protease cleavage settings that lower search times and improve O -glycopeptide identifications. We use this platform to generate a consensus motif for the recently characterized immunomodulating metalloprotease (IMPa) from Pseudomonas aeruginosa and show that IMPa is a favorable O -glycoprotease for characterizing densely O -glycosylated mucin-domain glycoproteins.

Lysosomes are key degradative compartments of the cell. Transport to lysosomes relies on GlcNAc-1-phosphotransferase-mediated tagging of soluble enzymes with mannose 6-phosphate (M6P). GlcNAc-1-phosphotransferase deficiency leads to the severe lysosomal storage disorder mucolipidosis II (MLII). Several viruses require lysosomal cathepsins to cleave structural proteins and thus depend on functional GlcNAc-1-phosphotransferase. We used genome-scale CRISPR screens to identify lysosomal enzyme trafficking factor (LYSET, also named TMEM251) as essential for infection by cathepsin-dependent viruses including severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). LYSET deficiency resulted in global loss of M6P tagging and mislocalization of GlcNAc-1-phosphotransferase from the Golgi complex to lysosomes. Lyset knockout mice exhibited MLII-like phenotypes, and human pathogenic LYSET alleles failed to restore lysosomal sorting defects. Thus, LYSET is required for correct functioning of the M6P trafficking machinery and mutations in LYSET can explain the phenotype of the associated disorder.

Mucins are functionally implicated in a range of human pathologies, including cystic fibrosis, influenza, bacterial endocarditis, gut dysbiosis, and cancer. These observations have motivated the study of mucin biosynthesis as well as the development of strategies for inhibition of mucin glycosylation. Mammalian pathways for mucin catabolism, however, have remained underexplored. The canonical view, derived from analysis of N -glycoproteins in human lysosomal storage disorders, is that glycan degradation and proteolysis occur sequentially. Here, we challenge this view by providing genetic and biochemical evidence supporting mammalian proteolysis of heavily O -glycosylated mucin domains without prior deglycosylation. Using activity screening coupled with mass spectrometry, we ascribed mucin-degrading activity in murine liver to the lysosomal protease cathepsin D. Glycoproteomics of substrates digested with purified human liver lysosomal cathepsin D provided direct evidence for proteolysis within densely O -glycosylated domains. Finally, knockout of cathepsin D in a murine model of the human lysosomal storage disorder neuronal ceroid lipofuscinosis 10 resulted in accumulation of mucins in liver-resident macrophages. Our findings imply that mucin-degrading activity is a component of endogenous pathways for glycoprotein catabolism in mammalian tissues.

Mucin domains are densely O-glycosylated modular protein domains found in various extracellular and transmembrane proteins. Mucin-domain glycoproteins play important roles in many human diseases, such as cancer and cystic fibrosis, but the scope of the mucinome remains poorly defined. Recently, we characterized a bacterial O-glycoprotease, StcE, and demonstrated that an inactive point mutant retains binding selectivity for mucin-domain glycoproteins. In this work, we leverage inactive StcE to selectively enrich and identify mucin-domain glycoproteins from complex samples like cell lysate and crude ovarian cancer patient ascites fluid. Our enrichment strategy is further aided by an algorithm to assign confidence to mucin-domain glycoprotein identifications. This mucinomics platform facilitates detection of hundreds of glycopeptides from mucin domains and highly overlapping populations of mucin-domain glycoproteins from ovarian cancer patients. Ultimately, we demonstrate our mucinomics approach can reveal key molecular signatures of cancer from in vitro and ex vivo sources., (© 2022. The Author(s).)

Abnormal glycosylation is a hallmark of cancer, and the hypersialylated tumor cell surface facilitates abnormal cell trafficking and drug resistance in several malignancies, including multiple myeloma (MM). Furthermore, hypersialylation has also been implicated in facilitating evasion of natural killer (NK) cell-mediated immunosurveillance but not in MM to date. In this study, we explore the role of hypersialylation in promoting escape from NK cells. We document strong expression of sialic acid-derived ligands for Siglec-7 (Siglec-7L) on primary MM cells and MM cell lines, highlighting the possibility of Siglec-7/Siglec-7L interactions in the tumor microenvironment. Interactomics experiments in MM cell lysates revealed PSGL-1 as the predominant Siglec-7L in MM. We show that desialylation, using both a sialidase and sialyltransferase inhibitor (SIA), strongly enhances NK cell-mediated cytotoxicity against MM cells. Furthermore, MM cell desialylation results in increased detection of CD38, a well-validated target in MM. Desialylation enhanced NK cell cytotoxicity against CD38+ MM cells after treatment with the anti-CD38 monoclonal antibody daratumumab. Additionally, we show that MM cells with low CD38 expression can be treated with all trans-retinoic acid (ATRA), SIA and daratumumab to elicit a potent NK cell cytotoxic response. Finally, we demonstrate that Siglec-7KO potentiates NK cell cytotoxicity against Siglec-7L+ MM cells. Taken together, our work shows that desialylation of MM cells is a promising novel approach to enhance NK cell efficacy against MM, which can be combined with frontline therapies to elicit a potent anti-MM response., (© 2022 by The American Society of Hematology. Licensed under Creative Commons Attribution-NonCommercial-NoDerivatives 4.0 International (CC BY-NC-ND 4.0), permitting only noncommercial, nonderivative use with attribution. All other rights reserved.)

Akkermansia muciniphila, a mucin-degrading microbe found in the human gut, is often associated with positive health outcomes. The abundance of A. muciniphila is modulated by the presence and accessibility of nutrients, which can be derived from diet or host glycoproteins. In particular, the ability to degrade host mucins, a class of proteins carrying densely O-glycosylated domains, provides a competitive advantage in the sustained colonization of niche mucosal environments. Although A. muciniphila is known to rely on mucins as a carbon and nitrogen source, the enzymatic machinery used by this microbe to process mucins in the gut is not yet fully characterized. Here, we focus on the mucin-selective metalloprotease, Amuc&#95;0627 (AM0627), which is known to cleave between adjacent residues carrying truncated core 1 O-glycans. We showed that this enzyme is capable of degrading purified mucin 2 (MUC2), the major protein component of mucus in the gut. An X-ray crystal structure of AM0627 (1.9 Å resolution) revealed O-glycan-binding residues that are conserved between structurally characterized enzymes from the same family. We further rationalized the substrate cleavage motif using molecular modeling to identify nonconserved glycan-interacting residues. We conclude that mutagenesis of these residues resulted in altered substrate preferences down to the glycan level, providing insight into the structural determinants of O-glycan recognition., Competing Interests: Conflict of interest C. R. B. is a cofounder and scientific advisory board member of Lycia Therapeutics, Palleon Pharmaceuticals, Enable Bioscience, Redwood Biosciences (a subsidiary of Catalent), OliLux Bio, Grace Science LLC, and InterVenn Biosciences., (Published by Elsevier Inc.)

The invasive leading edge represents a potential gateway for tumor metastasis. The role of fibroblasts from the tumor edge in promoting cancer invasion and metastasis has not been comprehensively elucidated. We hypothesize that cross-talk between tumor and stromal cells within the tumor microenvironment results in activation of key biological pathways depending on their position in the tumor (edge vs. core). Here we highlight phenotypic differences between tumor-adjacent-fibroblasts (TAF) from the invasive edge and tumor core fibroblasts from the tumor core, established from human lung adenocarcinomas. A multiomics approach that includes genomics, proteomics, and O-glycoproteomics was used to characterize cross-talk between TAFs and cancer cells. These analyses showed that O-glycosylation, an essential posttranslational modification resulting from sugar metabolism, alters key biological pathways including the cyclin-dependent kinase 4 (CDK4) and phosphorylated retinoblastoma protein axis in the stroma and indirectly modulates proinvasive features of cancer cells. In summary, the O-glycoproteome represents a new consideration for important biological processes involved in tumor-stroma cross-talk and a potential avenue to improve the anticancer efficacy of CDK4 inhibitors., Significance: A multiomics analysis of spatially distinct fibroblasts establishes the importance of the stromal O-glycoproteome in tumor-stroma interactions at the leading edge and provides potential strategies to improve cancer treatment. See related commentary by De Wever, p. 537., (©2021 American Association for Cancer Research.)

Ion-ion reactions are valuable tools in mass-spectrometry-based peptide and protein sequencing. To boost the generation of sequence-informative fragment ions from low charge-density precursors, supplemental activation methods, via vibrational and photoactivation, have become widely adopted. However, long-lived radical peptide cations undergo intramolecular hydrogen atom transfer from c-type ions to z • -type ions. Here we investigate the degree of hydrogen transfer for thousands of unique peptide cations where electron transfer dissociation (ETD) was performed and was followed by beam-type collisional activation (EThcD), resonant collisional activation (ETcaD), or concurrent infrared photoirradiation (AI-ETD). We report on the precursor charge density and the local amino acid environment surrounding bond cleavage to illustrate the effects of intramolecular hydrogen atom transfer for various precursor ions. Over 30% of fragments from EThcD spectra comprise distorted isotopic distributions, whereas over 20% of fragments from ETcaD have distorted distributions and less than 15% of fragments derived from ETD and AI-ETD reveal distorted isotopic distributions. Both ETcaD and EThcD give a relatively high degree of hydrogen migration, especially when D, G, N, S, and T residues were directly C-terminal to the cleavage site. Whereas all postactivation methods boost the number of c- and z • -type fragment ions detected, the collision-based approaches produce higher rates of hydrogen migration, yielding fewer spectral identifications when only c- and z • -type ions are considered. Understanding hydrogen rearrangement between c- and z • -type ions will facilitate better spectral interpretation.

Secreted polypeptides represent a fundamental axis of intercellular communication. Here, we present a protocol for the cell type-specific biotinylation, enrichment, and proteomic profiling of secreted plasma proteins directly in mice. This protocol uses conditional "turn-on" adeno-associated viruses expressing an endoplasmic reticulum-targeted biotin ligase to globally biotinylate proteins of the secretory pathway in a cell type-specific manner. Biotinylated secreted proteins can be directly purified from blood plasma and analyzed by SDS-PAGE gel or shotgun proteomics. For complete information on the generation and use of this protocol, please refer to Wei et al. (2021)., Competing Interests: The authors declare no competing interests., (© 2021 The Author(s).)

Lysosome dysfunction is a shared feature of rare lysosomal storage diseases and common age-related neurodegenerative diseases. Microglia, the brain-resident macrophages, are particularly vulnerable to lysosome dysfunction because of the phagocytic stress of clearing dying neurons, myelin, and debris. CD22 is a negative regulator of microglial homeostasis in the aging mouse brain, and soluble CD22 (sCD22) is increased in the cerebrospinal fluid of patients with Niemann-Pick type C disease (NPC). However, the role of CD22 in the human brain remains unknown. In contrast to previous findings in mice, here, we show that CD22 is expressed by oligodendrocytes in the human brain and binds to sialic acid–dependent ligands on microglia. Using unbiased genetic and proteomic screens, we identify insulin-like growth factor 2 receptor (IGF2R) as the binding partner of sCD22 on human myeloid cells. Targeted truncation of IGF2R revealed that sCD22 docks near critical mannose 6-phosphate–binding domains, where it disrupts lysosomal protein trafficking. Interfering with the sCD22-IGF2R interaction using CD22 blocking antibodies ameliorated lysosome dysfunction in human NPC1 mutant induced pluripotent stem cell–derived microglia-like cells without harming oligodendrocytes in vitro. These findings reinforce the differences between mouse and human microglia and provide a candidate microglia-directed immunotherapeutic to treat NPC.

Selective protein degradation platforms have afforded new development opportunities for therapeutics and tools for biological inquiry. The first lysosome-targeting chimeras (LYTACs) targeted extracellular and membrane proteins for degradation by bridging a target protein to the cation-independent mannose-6-phosphate receptor (CI-M6PR). Here, we developed LYTACs that engage the asialoglycoprotein receptor (ASGPR), a liver-specific lysosome-targeting receptor, to degrade extracellular proteins in a cell-type-specific manner. We conjugated binders to a triantenerrary N-acetylgalactosamine (tri-GalNAc) motif that engages ASGPR to drive the downregulation of proteins. Degradation of epidermal growth factor receptor (EGFR) by GalNAc-LYTAC attenuated EGFR signaling compared to inhibition with an antibody. Furthermore, we demonstrated that a LYTAC consisting of a 3.4-kDa peptide binder linked to a tri-GalNAc ligand degrades integrins and reduces cancer cell proliferation. Degradation with a single tri-GalNAc ligand prompted site-specific conjugation on antibody scaffolds, which improved the pharmacokinetic profile of GalNAc-LYTACs in vivo. GalNAc-LYTACs thus represent an avenue for cell-type-restricted protein degradation., (© 2021. The Author(s), under exclusive licence to Springer Nature America, Inc. part of Springer Nature.)

Severe cases of coronavirus disease 2019 (COVID-19), caused by infection with SARS-CoV-2, are characterized by a hyperinflammatory immune response that leads to numerous complications. Production of proinflammatory neutrophil extracellular traps (NETs) has been suggested to be a key factor in inducing a hyperinflammatory signaling cascade, allegedly causing both pulmonary tissue damage and peripheral inflammation. Accordingly, therapeutic blockage of neutrophil activation and NETosis, the cell death pathway accompanying NET formation, could limit respiratory damage and death from severe COVID-19. Here, we demonstrate that synthetic glycopolymers that activate signaling of the neutrophil checkpoint receptor Siglec-9 suppress NETosis induced by agonists of viral toll-like receptors (TLRs) and plasma from patients with severe COVID-19. Thus, Siglec-9 agonism is a promising therapeutic strategy to curb neutrophilic hyperinflammation in COVID-19., Competing Interests: The authors declare the following competing financial interest(s): C.S.D. and C.R.B. are coinventors on a patent application for cis-binding Siglec agonist glycopolymers as immune suppressants (USPTO63046140). C.R.B. is a co-founder and Scientific Advisory Board member of Lycia Therapeutics, Palleon Pharmaceuticals, Enable Bioscience, Redwood Biosciences (a subsidiary of Catalent), OliLux Inc., and InterVenn Bio, and a member of the Board of Directors of Eli Lily & Company. C.A.B. is a Scientific Advisory Board member of Catamaran Bio., (© 2021 The Authors. Published by American Chemical Society.)

Secreted polypeptides are a fundamental axis of intercellular and endocrine communication. However, a global understanding of the composition and dynamics of cellular secretomes in intact mammalian organisms has been lacking. Here, we introduce a proximity biotinylation strategy that enables labeling, detection and enrichment of secreted polypeptides in a cell type-selective manner in mice. We generate a proteomic atlas of hepatocyte, myocyte, pericyte and myeloid cell secretomes by direct purification of biotinylated secreted proteins from blood plasma. Our secretome dataset validates known cell type-protein pairs, reveals secreted polypeptides that distinguish between cell types and identifies new cellular sources for classical plasma proteins. Lastly, we uncover a dynamic and previously undescribed nutrient-dependent reprogramming of the hepatocyte secretome characterized by the increased unconventional secretion of the cytosolic enzyme betaine-homocysteine S-methyltransferase (BHMT). This secretome profiling strategy enables dynamic and cell type-specific dissection of the plasma proteome and the secreted polypeptides that mediate intercellular signaling.

Glyco-immune checkpoint receptors , molecules that inhibit immune cell activity following binding to glycosylated cell-surface antigens, are emerging as attractive targets for cancer immunotherapy. Defining biologically relevant ligands that bind and activate such receptors, however, has historically been a significant challenge. Here, we present a CRISPRi genomic screening strategy that allowed unbiased identification of the key genes required for cell-surface presentation of glycan ligands on leukemia cells that bind the glyco-immune checkpoint receptors Siglec-7 and Siglec-9. This approach revealed a selective interaction between Siglec-7 and the mucin-type glycoprotein CD43. Further work identified a specific N-terminal glycopeptide region of CD43 containing clusters of disialylated O-glycan tetrasaccharides that form specific Siglec-7 binding motifs. Knockout or blockade of CD43 in leukemia cells relieves Siglec-7-mediated inhibition of immune killing activity. This work identifies a potential target for immune checkpoint blockade therapy and represents a generalizable approach to dissection of glycan-receptor interactions in living cells., Competing Interests: Competing interest statement: C.R.B. is a cofounder and Scientific Advisory Board member of Palleon Pharmaceuticals, Enable Biosciences, Redwood Bioscience (a subsidiary of Catalent), and InterVenn Biosciences, and a member of the Board of Directors of Eli Lilly and Company. S.W., B.A.H.S., and C.R.B. are co-inventors on a patent application related to this work held by Stanford University (PCT/US2020/041603).

Inflammatory pathologies caused by phagocytes lead to numerous debilitating conditions, including chronic pain and blindness due to age-related macular degeneration. Many members of the sialic acid-binding immunoglobulin-like lectin (Siglec) family are immunoinhibitory receptors whose agonism is an attractive approach for antiinflammatory therapy. Here, we show that synthetic lipid-conjugated glycopolypeptides can insert into cell membranes and engage Siglec receptors in cis , leading to inhibitory signaling. Specifically, we construct a cis -binding agonist of Siglec-9 and show that it modulates mitogen-activated protein kinase (MAPK) signaling in reporter cell lines, immortalized macrophage and microglial cell lines, and primary human macrophages. Thus, these cis -binding agonists of Siglecs present a method for therapeutic suppression of immune cell reactivity., Competing Interests: Competing interest statement: C.S.D. and C.R.B. are coinventors on a patent application related to this work (USPTO63046140). C.R.B. is a cofounder and scientific advisory board member of Lycia Therapeutics, Palleon Pharmaceuticals, Enable Bioscience, Redwood Biosciences (a subsidiary of Catalent), and InterVenn Biosciences, and a member of the board of directors of Eli Lilly & Company.

Glycosylation is a prevalent, yet heterogeneous modification with a broad range of implications in molecular biology. This heterogeneity precludes enrichment strategies that can be universally beneficial for all glycan classes. Thus, choice of enrichment strategy has profound implications on experimental outcomes. Here we review common enrichment strategies used in modern mass spectrometry-based glycoproteomic experiments, including lectins and other affinity chromatographies, hydrophilic interaction chromatography and its derivatives, porous graphitic carbon, reversible and irreversible chemical coupling strategies, and chemical biology tools that often leverage bioorthogonal handles. Interest in glycoproteomics continues to surge as mass spectrometry instrumentation and software improve, so this review aims to help equip researchers with the necessary information to choose appropriate enrichment strategies that best complement these efforts., Competing Interests: Conflict of interest The authors declare that they have no conflicts of interest with the contents of this article., (Copyright © 2020 The Authors. Published by Elsevier Inc. All rights reserved.)

Severe cases of coronavirus disease 2019 (COVID-19), caused by infection with SARS-Cov-2, are characterized by a hyperinflammatory immune response that leads to numerous complications. Production of proinflammatory neutrophil extracellular traps (NETs) has been suggested to be a key factor in inducing a hyperinflammatory signaling cascade, allegedly causing both pulmonary tissue damage and peripheral inflammation. Accordingly, therapeutic blockage of neutrophil activation and NETosis, the cell death pathway accompanying NET formation, could limit respiratory damage and death from severe COVID-19. Here, we demonstrate that synthetic glycopolymers that activate the neutrophil checkpoint receptor Siglec-9 suppress NETosis induced by agonists of viral toll-like receptors (TLRs) and plasma from patients with severe COVID-19. Thus, Siglec-9 agonism is a promising therapeutic strategy to curb neutrophilic hyperinflammation in COVID-19.
., Competing Interests: C.S.D. and C.R.B. are coinventors on a patent application for cis-binding Siglec agonist glycopolymers as immune suppressants (USPTO63046140). C.R.B. is a co-founder and Scientific Advisory Board member of Lycia Therapeutics, Palleon Pharmaceuticals, Enable Bioscience, Redwood Biosciences (a subsidiary of Catalent), and InterVenn Bio, and a member of the Board of Directors of Eli Lily & Company. C.A.B. is a Scientific Advisory Board member of Catamaran Bio.

Qualitative studies have identified haircare practices as important culturally specific barriers to physical activity (PA) among Black/African American (AA) women, but quantitative investigations are lacking. Using the Study of Environment, Lifestyle and Fibroids data among 1558 Black/AA women, we investigated associations between hair product usage/hair maintenance behaviors and PA during childhood and adulthood. Participants reported childhood and current chemical relaxer and leave-in conditioner use. Self-reported PA included childhood recreational sports participation, leisure-time PA engagement during adulthood, and, at each life stage, minutes of and intensity of PA. Adjusting for socioeconomic and health characteristics, we used Poisson regression with robust variance to estimate prevalence ratios (PRs) and 95% confidence intervals (CIs) for each PA measure for more vs. less frequent hair product use/hair maintenance. Thirty-four percent reported ≥twice/year chemical relaxer use and 22% reported ≥once/week leave-in conditioner use at age 10 years, and neither were associated with PA at age 10 years. In adulthood, ≥twice/year chemical relaxer users (30%) were less likely (PR = 0.90 [95% CI: 0.79-1.02]) and ≥once/week leave-in conditioner users (24%) were more likely (PR = 1.09 [95% CI: 0.99-1.20]) to report intense PA compared to counterparts reporting rarely/never use. Hair product use/maintenance may influence PA among Black/AA women and impact cardiometabolic health disparities.

The recently described O-glycoprotease OpeRATOR presents exciting opportunities for O-glycoproteomics. This bacterial enzyme purified from Akkermansia muciniphila cleaves N-terminally to serine and threonine residues that are modified with (preferably asialylated) O-glycans. This provides orthogonal cleavage relative to canonical proteases (e.g., trypsin) for improved O-glycopeptide characterization with tandem mass spectrometry (MS/MS). O-glycopeptides with a modified N-terminal residue, such as those generated by OpeRATOR, present several potential benefits, perhaps the most notable being de facto O-glycosite localization without the need of glycan-retaining fragments in MS/MS spectra. Indeed, O-glycopeptides modified exclusively at the N-terminus would enable O-glycoproteomic methods to rely solely on collision-based fragmentation rather than electron-driven dissociation because glycan-retaining peptide fragments would not be required for localization. The caveat is that modified peptides would need to reliably contain only a single O-glycosite. Here, we use methods that combine collision- and electron-based fragmentation to characterize the number of O-glycosites that are present in O-glycopeptides derived from the OpeRATOR digestion of four known O-glycoproteins. Our data show that over 50% of O-glycopeptides in our sample generated from combined digestion using OpeRATOR and trypsin contain multiple O-glycosites, indicating that collision-based fragmentation alone is not sufficient. Electron-based dissociation methods are necessary to capture the O- glycopeptide diversity present in OpeRATOR digestions.

We report O-Pair Search, an approach to identify O-glycopeptides and localize O-glycosites. Using paired collision- and electron-based dissociation spectra, O-Pair Search identifies O-glycopeptides via an ion-indexed open modification search and localizes O-glycosites using graph theory and probability-based localization. O-Pair Search reduces search times more than 2,000-fold compared to current O-glycopeptide processing software, while defining O-glycosite localization confidence levels and generating more O-glycopeptide identifications. Beyond the mucin-type O-glycopeptides discussed here, O-Pair Search also accepts user-defined glycan databases, making it compatible with many types of O-glycosylation. O-Pair Search is freely available within the open-source MetaMorpheus platform at https://github.com/smith-chem-wisc/MetaMorpheus .

Photoactivation and photodissociation have long proven to be useful tools in tandem mass spectrometry, but implementation often involves cumbersome and potentially dangerous configurations. Here, we redress this problem by using a fiber-optic cable to couple an infrared (IR) laser to a mass spectrometer for robust, efficient, and safe photoactivation experiments. Transmitting 10.6 μm IR photons through a hollow-core fiber, we show that such fiber-assisted activated ion-electron transfer dissociation (AI-ETD) and IR multiphoton dissociation (IRMPD) experiments can be carried out as effectively as traditional mirror-based implementations. We report on the transmission efficiency of the hollow-core fiber for conducting photoactivation experiments and perform various intact protein and peptide analyses to illustrate the benefits of fiber-assisted AI-ETD, namely, a simplified system for irradiating the two-dimensional linear ion trap volume concurrent with ETD reactions to limit uninformative nondissociative events and thereby amplify sequence coverage. We also describe a calibration scheme for the routine analysis of IR laser alignment and power through the fiber and into the dual cell quadrupolar linear ion trap. In all, these advances allow for a more robust, straightforward, and safe instrumentation platform, permitting implementation of AI-ETD and IRMPD on commercial mass spectrometers and broadening the accessibility of these techniques.

Site-specific characterization of glycosylation requires intact glycopeptide analysis, and recent efforts have focused on how to best interrogate glycopeptides using tandem mass spectrometry (MS/MS). Beam-type collisional activation, i.e., higher-energy collisional dissociation (HCD), has been a valuable approach, but stepped collision energy HCD (sceHCD) and electron transfer dissociation with HCD supplemental activation (EThcD) have emerged as potentially more suitable alternatives. Both sceHCD and EThcD have been used with success in large-scale glycoproteomic experiments, but they each incur some degree of compromise. Most progress has occurred in the area of N -glycoproteomics. There is growing interest in extending this progress to O -glycoproteomics, which necessitates comparisons of method performance for the two classes of glycopeptides. Here, we systematically explore the advantages and disadvantages of conventional HCD, sceHCD, ETD, and EThcD for intact glycopeptide analysis and determine their suitability for both N - and O -glycoproteomic applications. For N -glycopeptides, HCD and sceHCD generate similar numbers of identifications, although sceHCD generally provides higher quality spectra. Both significantly outperform EThcD methods in terms of identifications, indicating that ETD-based methods are not required for routine N -glycoproteomics even if they can generate higher quality spectra. Conversely, ETD-based methods, especially EThcD, are indispensable for site-specific analyses of O -glycopeptides. Our data show that O -glycopeptides cannot be robustly characterized with HCD-centric methods that are sufficient for N -glycopeptides, and glycoproteomic methods aiming to characterize O -glycopeptides must be constructed accordingly.

Monoclonal antibodies (mAbs) are important therapeutic glycoproteins, but their large size and structural complexity make them difficult to rapidly characterize. Top-down mass spectrometry (MS) has the potential to overcome challenges of other common approaches by minimizing sample preparation and preserving endogenous modifications. However, comprehensive mAb characterization requires generation of many, well-resolved fragments and remains challenging. While ETD retains modifications and cleaves disulfide bonds-making it attractive for mAb characterization-it can be less effective for precursors having high m / z values. Activated ion electron transfer dissociation (AI-ETD) uses concurrent infrared photoactivation to promote product ion generation and has proven effective in increasing sequence coverage of intact proteins. Here, we present the first application of AI-ETD to mAb sequencing. For the standard NIST mAb, we observe a high degree of complementarity between fragments generated using standard ETD with a short reaction time and AI-ETD with a long reaction time. Most importantly, AI-ETD reveals disulfide-bound regions that have been intractable, thus far, for sequencing with top-down MS. We conclude AI-ETD has the potential to rapidly and comprehensively analyze intact mAbs.