Biology subjects

Azadi, P.

Publications and source records attributed to Azadi, P..

At least 19 recordsLinked to original sources

bioRxiv2026

N-glycoproteomic profiling of the hookworm Ancylostoma ceylanicum reveals heterogeneity and phosphorylcholine-modified glycans in larval and adult parasites.

Glycans of parasitic worms (helminths) have been previously described with roles in immunomodulation and the recognition of helminth proteins by the host immune system. Despite their significance in other systems, little is known about the glycosylation patterns of the hookworm Ancylostoma ceylanicum, an intestinal parasite of animals and humans, due to challenges in sample collection and structural complexity of many nematode glycans. Herein, we describe results of a released N-glycan analysis of adult hookworms, which revealed oligomannose, hybrid, and complex structures, including those with non-mammalian features. Next, we report the results of glycoproteomic analyses to identify hookworm glycoproteins and their associated glycan structures. We compared three enrichment methods and found that strong anion-exchange electrostatic repulsion liquid chromatography (SAX-ERLIC) led to the identification of the most N-glycopeptides. Analyses of larval and adult extracts revealed 82 larval and 157 adult N-glycoproteins, the majority of which were stage-specific. Of particular interest, phosphorylcholine (PC), a known immunomodulatory glycan modification, was detected in both stages, and several unique PC-modified structures were identified in each. Collectively, these results provide the first site-specific characterization of intact N-glycopeptides in A. ceylanicum and, with an adult-stage released glycan analysis, establish a method for glycoproteomic characterizations of other helminths.

microbiology↗

bioRxiv2026

Identification of glycosyltransferases that synthesize pectin heteroglycan block polymers and a structural motif required for their activity

Pectin is a family of plant cell wall complex polysaccharides and proteoglycans comprised of the glycan domains homogalacturonan (HG), rhamnogalacturonan I (RG-I), and the substituted HGs rhamnogalacturonan II (RG-II), xylogalacturonan and apiogalacturonan. The number, type, and arrangement of the pectic domains in different pectic polymers are unknown, as is the identity of the enzymes that link the domains together. Here we show that two Arabidopsis -1,4-galacturonosyltransferases, GAUT13 and GAUT14, in addition to previously reported HG acceptor-dependent and HG de novo synthesis activities, initiate and elongate HG on the non-reducing end of RG-I, identifying enzymes that synthesize pectic RG-I:HG heteroglycans. While other catalytic GAUTs did not initiate HG on RG-I acceptors, they were able to elongate HG once initiated on RG-I by GAUT13 and GAUT14. We show that the three different GAUT14 acceptors (UDP-GalA, HG, and RG-I) compete for the same binding pocket, and amino acids required for GAUT14 catalytic activity were identified using in vitro enzyme assays, computational modeling, and site-directed mutagenesis. The transfer of the GAUT14 hypervariable 1 (HV1) motif (RTFASKLQSRS) was sufficient and necessary to transfer RG-I:HG extension activity to GAUT10, which otherwise does not have this activity, representing a novel functional glycosyltransferase domain swapping strategy. The presence of all three catalytic activities in a GAUT13/14 homolog from the non-vascular seedless plant Physcomitrium established the early evolution of GAUT13/14 trifunctionality. The identification of GAUT13 and GAUT14 as RG-I:HG extension catalysts provides targets for probing in planta structure and function of RG-I:HG heteropolymers.

plant biology↗

bioRxiv2026

Genome-wide characterization of host factors involved in single-stranded RNA and DNA phage infection pathways

Single stranded RNA (ssRNA) and single stranded DNA (ssDNA) bacteriophages represent a key component of the global virome, yet the host genetic networks supporting their infection cycles remain poorly understood. Here, we present a comprehensive, genome-wide mapping of the genetic landscape regulating infection cycles for F pilus-dependent ssRNA and ssDNA phages in Escherichia coli. Genetic screens across ssRNA phages spanning all four genogroups of the Leviviricetes revealed a highly conserved network of host dependencies, with the notable exception of the F plasmid gene traD. While primary structural receptor components and dsbA mediated disulfide bond formation are universally required across all lineages to ensure F pilus integrity, traD exhibits a strict genogroup-specific requirement during entry, showing variable essentiality across different viral groups despite sharing an identical primary receptor. Our gene dosage screens revealed that an elevated copy number of the hslU protease or the RNA chaperone stpA restricts infection, identifying clear genetic barriers that can perturb the viral life cycle. Parallel assays with filamentous ssDNA phages produced host factor profiles consistent with published literature, while revealing additional variations in host dependency. These screens confirmed that ssDNA phages strictly rely on the host TolQRA complex for entry downstream of pilus engagement. The assays tracked prominent negative fitness signatures across homeostatic clusters, highlighting how the physiological burden of continuous, non-lytic virion extrusion strains the host envelope. Finally, this comparative approach traced the selectivity of our isolation host (E. coli HSF) to a horizontally acquired capsule architecture from Klebsiella. This surface shield excludes a large panel of double stranded DNA phages isolated on diverse E. coli strains, while allowing virions from ssDNA and ssRNA phages to engage the extended F pilus and bypass the barrier via native pilus retraction. Together, this work provides a systematic, class-wide map of single stranded phage-host interactions, bridging classical genetics with modern viral discovery while establishing a robust host platform to access uncultured viral diversity and a functional blueprint to design next generation diagnostics, protein antibiotics, and biocontrol tools to halt horizontal gene transfer.

microbiology↗

bioRxiv2026

Autoantibodies from recovered Guillain-Barre syndrome patients exhibit altered effector functions that prevent subsequent neuron degeneration

Guillain-Barre syndrome (GBS) is an autoimmune polyneuropathy that is the leading cause of nonpoliovirus-associated acute flaccid paralysis worldwide. In most cases, GBS occurs following an infection, most commonly Campylobacter jejuni, through the induction of antibodies recognizing bacterial ganglioside-mimicking lipooligosaccharides that cross-react with human neuronal gangliosides. This post-infectious autoimmune cascade causes neuropathy, from which patients can recover as their anti-ganglioside antibody titers diminish and immunostimulation decreases. In this study, we find 10% of clinically confirmed GBS patients maintain high titers of circulating anti-ganglioside antibodies more than one decade after recovery. These antibodies no longer cause neuropathy compared to acute sera from the same patients using a human pluripotent stem cell-derived sensory neuron model with human complement. We found both IgG subclass and glycoform differences between paired acute and recovered GBS patient sera, including anti-inflammatory modifications on isolated anti-GM1 ganglioside antibodies. Together, these data suggest that patients with GBS select for non-pathogenic variants of autoantibodies that are no longer capable of damaging their neurons, but may still protect against C. jejuni infection.

immunology↗

bioRxiv2026

Multi-factorial regulatory networks of placental antibody transfer by Fc receptors and maternal IgG Fc characteristics are modulated by clinical covariate profiles

Maternal immunoglobulin G (IgG) transferred across the placenta is crucial for newborn immunity. IgG transfer efficiency modulated by Fc characteristics including subclass and glycosylation gives rise to diverse transfer profiles across the population, yet the molecular mechanisms driving this variation are incompletely understood. To disentangle multimodal molecular relationships driving population heterogeneity in maternal-fetal antibody transfer, we characterized placental and serological antibody features in matched human tissues from two geographically distinct cohorts. Unsupervised clustering of maternal clinical covariates in both cohorts revealed a distinct patient profile with reduced plasma C-reactive protein and pregravid BMI and enhanced transfer efficiency of IgG subclasses. Quantification of IgG Fc glycan structures in matched maternal and cord plasma by liquid chromatography-mass spectrometry revealed subclass-specific IgG glycosylation patterns which impacted placental transfer efficiency and correlated with these key clinical features. We profiled expression and colocalization of key Fc {gamma} Receptors (Fc{gamma}Rs) by multiplex immunohistochemistry, revealing cell type-specific expression patterns. Variable Fc{gamma}R expression across gestation was consistent in both cohorts, implicating Fc{gamma}Rs as key drivers of temporal antibody transfer dynamics. While Fc{gamma}Rs were not strongly variable across the clinical profiles, partial correlation analysis of matched samples controlling for gestational age and demographic covariates revealed correlations between Fc{gamma}R expression frequencies and glycan- and subclass-specific transfer efficiency. These data systematically define multi-factorial regulatory networks of antibody transfer by placental Fc receptors and maternal IgG Fc characteristics, which are further modulated by clinical covariates. This study provides a basis for the rational design of prenatal vaccination strategies, administration schedules, and potential lifestyle interventions to improve maternal-fetal immunity.

immunology↗

bioRxiv2026

Enterococcus faecalis autolysin, EpaU, binds the enterococcal polysaccharide antigen via its teichoic acid-like repeats and modulates c-di-AMP signaling

The cell wall of the Gram-positive bacterium Enterococcus faecalis is decorated with the enterococcal polysaccharide antigen (EPA), consisting of a core rhamnan backbone linked covalently with a strain-variable teichoic acid-like (TA) polymer. Current models propose that the TA decoration is a repeating polymer composed of two alternating subunits, designated TAI and TAII, which are attached to the rhamnan core via a mild-acid labile phosphodiester bond from the initiating TAI subunit. In this study, we characterize the EpaU autolysin encoded within the EPA biosynthetic gene cluster. We demonstrate that the cell wall-binding domain of EpaU associates with the intact TA domains of EPA synthesized with the aid of the glycosyltransferases EpaR and EpaX. We further show that EpaU is a potent autolysin that binds generally over the E. faecalis cell surface, suggesting that it functions as a remodeling peptidoglycan hydrolase. The absence of EpaU leads to increased ampicillin resistance and elevated intracellular levels of the second messenger c-di-AMP. These data suggest that E. faecalis possesses a mechanism that senses the integrity of the peptidoglycan meshwork and employs c-di-AMP to regulate cell turgor, potentially altering the antibiotic resistance. ImportanceEnterococcus faecalis is an important opportunistic pathogen that can cause severe nosocomial infections. Knowledge of how bacteria remodel the cell wall is key to understanding many important cellular processes, such as antibiotic resistance, cell division, biofilm formation, and stress resistance. In this study, we shed new light on the structural details of the main cell wall polysaccharide, Enterococcal Polysaccharide Antigen (EPA), and its interaction with EpaU, an autolysin that cleaves peptidoglycan during cell wall remodeling. We also report a link between EpaU and the regulation of turgor pressure via cyclic dinucleotide signaling. This work contributes to a more complete picture of E. faecalis cell wall and may provide insight into the development of antimicrobial agents based on autolysins.

microbiology↗

bioRxiv2026

Transposon insertion sequencing of Pseudomonas aeruginosa identifies multiple intersecting pathways essential for extreme colistin resistance

Colistin is used to treat antibiotic resistant gram-negative infections, including those caused by Pseudomonas aeruginosa (Pa). Using a diverse collection of clinical isolates, we identified BWH047, a colistin-resistant isolate with an extremely high minimum inhibitory concentration (MIC, 1280 {micro}g/mL). To characterize the genes conditionally essential for colistin resistance in BWH047, we employed transposon insertion sequencing and identified 20 gene candidates. In-frame deletion validated 75% of the candidates and identified genes in several novel pathways that contribute to colistin resistance, including algU and wapH. We also identified several candidate genes from previously reported colistin resistance pathways (e.g. arn, pmrAB). We further investigated the impact of a colistin resistance-associated inner membrane DedA-family undecaprenyl phosphate flippase, which we named DpcA (DedA of Pseudomonas necessary for colistin resistance A). Deletion of dpcA in BWH047 restored sensitivity to colistin (MIC = 0.5 {micro}g/mL) and resulted in several unique changes to the structure of lipopolysaccharide (LPS), including production of decreased amounts of the colistin resistance-conferring 4-amino-4-deoxy-L-arabinose (L-Ara4N) modification on lipid A. To date, this work represents the most complete analysis of colistin resistance in Pa and identifies novel intersecting pathways that contribute to extreme phenotypic resistance. Author summaryPseudomonas aeruginosa is a bacterium that causes a wide variety of infections. It is especially problematic given its propensity to become resistant to antibiotics. One antibiotic used to treat multidrug-resistant P. aeruginosa infections is colistin. In this study, we investigated colistin resistance mechanisms in a patient-derived, extremely phenotypically resistant P. aeruginosa isolate, BWH047, using transposon insertion sequencing and mass spectrometry. We identified 13 genes conditionally essential for colistin resistance and investigated the role of one of these genes, dpcA, on the composition of the bacterial outer membrane, the target of colistin. Additionally, our study identified novel colistin resistance genes residing in several intersecting pathways that could be targeted to prevent the development of antimicrobial resistance.

microbiology↗

bioRxiv2026

Ctenophore mesoglea: building a mucus-like body

Ctenophora are one of the earliest, if not the earliest branching animal, making them crucial for insight into the early events of metazoan evolution. Ctenophores are largely composed of an extracellular mesoglea, but they lack homologs for the fibrous collagens that form a typical metazoan extracellular matrix (ECM). Therefore, the nature of this body-dominating material and its physiological role remains unknown. We found that ctenophore mesoglea is dominated by mucin-related proteins and novel sulfated polysaccharides. Mucus-like material that functions as an internal skeleton rather than as an external secretion indicates fundamental differences in epithelial function and ECM physiology between ctenophores and other Metazoa, suggesting an early period of biochemical and biomechanical diversity prior to the innovation of elastic connective tissue.

biochemistry↗

bioRxiv2025

Arabidopsis GALACTURONOSYLTRANSFERASE (GAUT) 1 synthesizes a homogalacturonan tightly bound to the cell wall and required for cell expansion

Arabidopsis GALACTURONOSYLTRANSFERASE1 (GAUT1) synthesizes homogalacturonan (HG), the most abundant pectin in growing plant cells. GAUT1 has the greatest in vitro enzyme activity of the six confirmed Arabidopsis HG biosynthetic GAUTs, but its biological activity remains elusive. Here we show that Arabidopsis GAUT1 homozygous mutants have a severe dwarfed seedling phenotype, survive several weeks as 2 to 3 mm seedlings, and have severely reduced shoot and root growth and hypocotyl epidermal, cortex and endodermal cell size. gaut1-1 pollen tubes are shorter than WT with increased bursting. Complementation of homozygous gaut1-1 with GAUT1 coding sequence driven by the GAUT1 promoter restored WT-like growth. The extreme dwarf phenotype of homozygous gaut1-1 seedlings precluded their use for detailed cell wall analysis, thus suspensions cultures were produced from callus generated from mutant and WT seedlings. Homozygous gaut1-1 suspension cells were smaller than WT with [~]30% reduced wall GalA content compared to WT. Sequential extraction of the walls with increasingly harsh solvents and sugar composition analysis revealed reduced GalA content in only the 4M KOH post-chlorite fraction, indicating that GAUT1-synthesized HG was held tightly in the wall by direct or indirect hydrogen bonding and/or oxidation-sensitive linkages. Treatment of wall fractions with endopolygalacturonase to hydrolyze HG and gel electrophoretic separation of hydrolysates exposed an HG-associated doublet band markedly downregulated in the homozygous gaut1-1 4M KOH post-chlorite fraction and to a lesser extent in 4M KOH and sodium chlorite fractions. NMR analysis identified the band as rhamnogalacturonan (RG)-II. Super resolution microscopy using anti-HG antibodies showed that, compared to WT, the homozygous gaut1-1 hypocotyl epidermal and callus cells had reduced content and length of HG nanofilaments, HG fibers associated with cell expansion in Arabidopsis. The results demonstrate that GAUT1-synthesized HG resides in a tightly-cell-wall-bound, RG-II-containing polymer required for HG nanofilament formation and seedling cell expansion.

plant biology↗

bioRxiv2025

Mass Spectrometric Determination of Site-Specific O-Acetylation in Rhamnogalacturonan-I Oligomers

O-Acetylation, a common modification in rhamnogalacturonan I (RG-I), is critical for various biological processes, including plant growth, stress responses, and pathogen defense. Precise determination of the degree and specific positions of acetylation is therefore essential. To date, nuclear magnetic resonance (NMR) and tandem mass spectrometry have been employed to identify acetyl positions in pectin oligosaccharides. Although NMR is effective, it requires pure, high-concentration samples. Tandem mass spectrometry (MS), which uses lower sample amounts, faces challenges due to acetyl migration between monosaccharide positions. The multiple steps in pectin sample analysis can further promote O-acetyl migration, especially near free hydroxyl groups. Moreover, during tandem MS, acetyl groups may detach, complicating accurate tracking. This study presents an approach to lock O-acetyl groups by introducing trideuteroacetyl and propionyl substituents onto free hydroxyls of RG-I or partially acetylated RG-I. By combining matrix-assisted laser desorption/ionization-time of flight (MALDI-TOF) MS and electrospray ionization (ESI) MS with MS/MS or tandem mass spectrometry (MSn), we devised a way to determine the monosaccharide sequence in the oligomer and precise positions of acetyl groups in partially acetylated RG-I. This method enables the study of the regiospecificity of recombinant pectin O-acetyltransferases and can be applied to other oligosaccharides to determine acyl positions. GRAPHICAL ABSTRACT O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=81 SRC="FIGDIR/small/694011v1_ufig1.gif" ALT="Figure 1"> View larger version (18K): org.highwire.dtl.DTLVardef@1384347org.highwire.dtl.DTLVardef@c8f21forg.highwire.dtl.DTLVardef@8fed52org.highwire.dtl.DTLVardef@125c4a1_HPS_FORMAT_FIGEXP M_FIG C_FIG

biophysics↗

bioRxiv2025

Persistent trade-offs balance competition and colonization across centuries

Microbial competition drives rapid adaptation, often forcing organisms to specialize in new ecological niches. Adaptations that improve competitive ability can reduce performance in other environments creating trade-offs. Whether such trade-offs persist in nature--or are eroded as lineages adapt through compensatory changes--remains largely unknown. Here we show that a trade-off between competitive ability and host colonization has been stably maintained in natural Pseudomonas populations for centuries. Wild plant-pathogenic Pseudomonas compete using tailocins--phage-derived molecular weapons that bind to specific cell-surface receptors. Genomic surveys and functional assays reveal that the most broadly lethal tailocins remain rare--while the tailocins production increases competitive killing, it also compromises plant colonization. We determine that the polymorphisms behind this trade-off are not transient -- historical genomes spanning two centuries show that the trade-off has been maintained for at least 10-10 generations. Our results demonstrate that, in natural populations, a trade-off between competition and pathogenicity is fundamental and not easily overcome. SignificanceWhen a microbe colonizes a host, it must both establish infection and outcompete other organisms. Short-term experiments show that gains in competitive ability can reduce colonization, creating trade-offs, but whether microbes resolve these conflicts over long evolutionary timescales is unknown. We show that a trade-off between competitive killing and host colonization has been stably maintained for centuries in natural Pseudomonas populations infecting Arabidopsis thaliana. Tailocins--phage-derived weapons--provide strong competitive advantages, yet their production reduces colonization success, explaining why the most broadly lethal variants remain rare. Genomic surveys and historical genomes spanning two centuries reveal that the polymorphisms underlying this trade-off have persisted across 10-10 generations. Understanding such long-lived constraints can inform antimicrobial strategies that exploit evolutionary trade-offs.

evolutionary biology↗

bioRxiv2025

Gram-positive bacterial cell wall components inhibit herpes simplex virus infection.

The role of the mucosal microbiome in viral infections remains unclear. Genital herpes, caused by herpes simplex virus 1 and 2 (HSV-1 and HSV-2), is among the most prevalent sexually transmitted infections worldwide. Despite evidence linking vaginal Lactobacillus to protection against sexually transmitted viruses, the specific microbial components and mechanisms that mediate this defense are not well understood. Here, we show that multiple cell wall components from diverse gram-positive bacteria, including lactobacilli, inhibit HSV-1 and HSV-2 infection in cells and in a mouse model of genital herpes infection. Peptidoglycan (PG) and lipoteichoic acid (LTA), both major components of the gram-positive bacterial cell wall, significantly reduced HSV infectivity in vitro and improved survival and disease outcomes in mice. We further showed that Lactobacillus crispatus surface layer proteins SlpA and SlpB bind HSV-1 and inhibit infection. Antiviral effects of cell wall components were dose-dependent, relied on intact PG structure, and, in the case of PG and LTA, were independent of TLR2-mediated host signaling. Collectively, our findings identify a species-independent antiviral function for gram-positive bacterial cell wall components against HSV and suggest that the composition of the mucosal microbiome may play an underappreciated role in suppressing mucosal herpes infection in humans.

microbiology↗

bioRxiv2025

A conserved glycan motif overcomes antigenic variation inducing broadly reactive antibodies against the zoonotic pathogen Streptococcus suis

Streptococcus suis is a largely neglected but emerging bacterial zoonotic pathogen of global concern for animal welfare, antibiotic resistance development and human health. No effective vaccines are currently available. Here, we identified and characterized the function and structure of two cell wall polysaccharide variants in pathogenic S. suis strains using genetic deletion and (heterologous) complementation, lectin staining, glycan composition analysis and specialized NMR spectroscopy. Both glycan variants were anionic polymers that differed in the presence of glucose in the side-chain as a result of allelic variation in a glycosyltransferase gene. Deletion of this variable glycosyltransferase revealed an identical glycan core and affected S. suis morphology and lysozyme resistance. Immunization of pigs with this core domain induced antibodies recognizing a wide range of antigenically-diverse pathogenic S. suis strains. This study provides new insights for developing next-generation glycoconjugate vaccines, whereby a single-glycan target could protect against the emerging zoonotic pathogen S. suis.

microbiology↗

bioRxiv2025

Hyper-Glycosylation as a Central Metabolic Driver of Alzheimers Disease

Alzheimers disease (AD) is a neurodegenerative disorder characterized by devastating degenerative decline. Metabolic disruptions are widely observed, yet their involvement in the molecular etiology of AD remains underexplored. Utilizing spatial metabolomics, lipidomics, and glycomics in both mouse models and human post-mortem samples, we identified a hyper-glycosylation phenotype as a hallmark of AD. To investigate the underlying mechanisms and whether the observed effect was a driver of the observed decline, we developed an advanced spatial isotopic tracing pulse-chase method to study the dynamics of N-linked glycans. Our analysis revealed enhanced glycan biosynthesis in AD mouse models. Based on these findings, we performed genetic and dietary interventions to modulate glycan biosynthesis. Genetic knockdown of glycan biosynthetic enzymes ameliorated the hyper-glycosylation and improved cognitive and behavioral outcomes in AD mice. In contrast, oral glucosamine supplementation drove hyper-glycosylation and exacerbated cognitive and behavioral deficits. To assess the clinical relevance of these findings, we conducted a retrospective analysis of a large population of patients with mild cognitive impairment (MCI), AD, and Alzheimers Disease Related Dementias (ADRD) stratified by glucosamine use, leveraging electronic health records. Consistently, glucosamine supplementation was associated with increased mortality in AD and ADRD patient cohorts, and significantly elevated progression from MCI to AD compared to age-matched controls. Collectively, our findings establish hyper-glycosylation as a pathological driver of AD and highlight glycan metabolism as an actional target in the fight against AD.

neuroscience↗

bioRxiv2025

Recognition of unique cell surface glycopolymers by diversified lectin domains specifies umbrella toxin targeting

The bacterial cell envelope provides structural integrity and is an essential conduit through which the organism interacts with its environment. However, the molecular structures of its constituents can also be exploited as receptors, permitting threats such as toxins and phage access to the cell. We previously demonstrated that Streptomyces coelicolor secretes an umbrella toxin particle that acts in a highly selective manner to inhibit the hyphal growth of competing Streptomyces strains. Here, we identify the receptor of umbrella toxins as teichuronic acid (TUA) oligosaccharides anchored to the cell surface through linkage to wall teichoic acids (WTA). We show that the carbohydrate portion of this previously undescribed hybrid TUA-WTA molecule is variable across species and that targeting specificity derives from its selective recognition by diversified lectin domains associated with umbrella particles. A cryo-EM structure of a lectin-TUA complex reveals the molecular basis for umbrella toxin cell targeting and, in conjunction with bioinformatic analyses, provides insights into a molecular arms race that we posit drives diversification of umbrella particles and the chemical composition of Streptomyces cell envelopes.

microbiology↗

bioRxiv2025

Glycoengineering of the hepatitis C virus E2 glycoprotein leads to improved biochemical properties and enhanced immunogenicity

An effective vaccine against hepatitis C virus (HCV) must elicit the production of broadly neutralizing antibodies (bnAbs) reproducibly against the E1E2 glycoprotein complex. Little is known about how glycan content affects this process. Ideally, glycans would maximize epitope exposure without compromising antigen stability or exposing new epitopes. However, typical recombinant vaccines contain considerable heterogeneity in glycan content, which can affect the antibody response and neutralization potency. Here we employed glycoengineered Chinese hamster ovary (geCHO) cell lines that impart nearly homogeneous glycosylation as a means to test how specific glycan features influence antigenicity and immunogenicity for the secreted HCV E2 ectodomain (sE2). Specific geCHO antigens exhibited a modest but reproducible increase in affinity for some mAbs relative to CHO- and HEK293-produced sE2. Surprisingly, one geCHO sE2 antigen failed to bind the CD81 receptor, indicating the potential for significant glycan effects on biochemical properties. We immunized mice with the four antigens and found the total antibody response to be the same for all groups. However, sera from one geCHO group exhibited a 7-fold improvement in neutralization against the homologous HCV pseudovirus and had the most mice whose sera exhibited neutralization activity against genotypes 1b, 2a, 2b, and 3. Further analysis identified beneficial and deleterious glycan features, and the glycan that correlated the most with decreased potency was relatively small. However, size was not the sole determinant of glycan-driven effects on the antibody response. In summary, glycan content impacts biochemical properties of antigens to varying degrees and such effects can influence immune response quality and uniformity.

immunology↗

bioRxiv2024

Identification of the Regulatory Elements and Protein Substrates of Lysine Acetoacetylation

Short chain fatty acylations establish connections between cell metabolism and regulatory pathways. Lysine acetoacetylation (Kacac) was recently identified as a new histone mark. However, regulatory elements, substrate proteins, and epigenetic functions of Kacac are not yet fully understood, hindering further in-depth understanding of acetoacetate modulated (patho)physiological processes. Here, we created a chemo-immunological approach for reliable detection of Kacac, and demonstrated that acetoacetate serves as the primary precursor for histone Kacac. We report the enzymatic addition of the Kacac mark by the acyltransferases GCN5, p300, and PCAF, and its removal by deacetylase HDAC3. Furthermore, we establish acetoacetyl-CoA synthetase (AACS) as a key regulator of cellular Kacac levels. A comprehensive proteomic analysis has identified 139 Kacac sites on 85 human proteins. Bioinformatics analysis of Kacac substrates and RNA-seq data reveal the broad impacts of Kacac on multifaceted cellular processes. These findings unveil pivotal regulatory mechanisms for the acetoacetate-mediated Kacac pathway, opening a new avenue for further investigation into ketone body functions in various pathophysiological states.

biochemistry↗

bioRxiv2024

Deep structural analysis of rhamnogalacturonan-I (RG-I) in Arabidopsis reveals new insights into pectin diversity

Pectic polysaccharides are an integral part of primary plant cell walls, where they are perfrom crucial structural and biological functions. Pectin is generally divided into four distinct structural categories, including homogalacturonan, xylogalacturonan, rhamnogalacturonan I (RG-I) and rhamnogalacturonan II. Among the four main pectin domains, the structural intricacies of rhamnogalacturonan-I (RG-I) remain the least understood, especially in widely employed plant models. We employed multiple complementary analytical techniques to present a detailed structural analysis of RG-I in the model system Arabidopsis thaliana. Using highly purified RG-I from different tissues, we conducted comparative linkage and NMR analyses, complemented by mass spectrometry of enzymatically digested RG-I oligosaccharides. Our findings present the most comprehensive structural overview of Arabidopsis RG-I to date, revealing novel structural features. Notably, we identified O-acetylation of rhamnose backbone residues as a predominant feature, a modification previously unreported in this species. The combined results present a comprehensive structural overview of Arabidopsis thaliana RG-I that will serve as a roadmap for studying pectin biosynthesis and function.

biochemistry↗