O-linked Protein Glycosylation Research Articles

Sweet complexity: O-linked protein glycosylation in pathogenic Neisseria

Sweet complexity: O-linked protein glycosylation in pathogenic Neisseria

Clinical and biochemical footprints of congenital disorders of glycosylation: Proposed nosology

We have identified 200 congenital disorders of glycosylation (CDG) caused by 189 different gene defects and have proposed a classification system for CDG based on the mode of action. This classification includes 8 categories: 1. Disorders of monosaccharide synthesis and interconversion, 2. Disorders of nucleotide sugar synthesis and transport, 3. Disorders of N-linked protein glycosylation, 4. Disorders of O-linked protein glycosylation, 5. Disorders of lipid glycosylation, 6. Disorders of vesicular trafficking, 7. Disorders of multiple glycosylation pathways and 8. Disorders of glycoprotein/glycan degradation. Additionally, using information from IEMbase, we have described the clinical involvement of 19 organs and systems, as well as essential laboratory investigations for each type of CDG. Neurological, dysmorphic, skeletal, and ocular manifestations were the most prevalent, occurring in 81%, 56%, 53%, and 46% of CDG, respectively. This was followed by digestive, cardiovascular, dermatological, endocrine, and hematological symptoms (17–34%). Immunological, genitourinary, respiratory, psychiatric, and renal symptoms were less frequently reported (8–12%), with hair and dental abnormalities present in only 4–7% of CDG. The information provided in this study, including our proposed classification system for CDG, may be beneficial for healthcare providers caring for individuals with metabolic conditions associated with CDG.

Protein glycosylation in cardiovascular health and disease.

Protein glycosylation, which involves the attachment of carbohydrates to proteins, is one of the most abundant protein co-translational and post-translational modifications. Advances in technology have substantially increased our knowledge of the biosynthetic pathways involved in protein glycosylation, as well as how changes in glycosylation can affect cell function. In addition, our understanding of the role of protein glycosylation in disease processes is growing, particularly in the context of immune system function, infectious diseases, neurodegeneration and cancer. Several decades ago, cell surface glycoproteins were found to have an important role in regulating ion transport across the cardiac sarcolemma. However, with very few exceptions, our understanding of how changes in protein glycosylation influence cardiovascular (patho)physiology remains remarkably limited. Therefore, in this Review, we aim to provide an overview of N-linked and O-linked protein glycosylation, including intracellular O-linked N-acetylglucosamine protein modification. We discuss our current understanding of how all forms of protein glycosylation contribute to normal cardiovascular function and their roles in cardiovascular disease. Finally, we highlight potential gaps in our knowledge about the effects of protein glycosylation on the heart and vascular system, highlighting areas for future research.

Combining FAIMS based glycoproteomics and DIA proteomics reveals widespread proteome alterations in response to glycosylation occupancy changes in Neisseria gonorrhoeae.

Protein glycosylation is increasingly recognized as a common protein modification across bacterial species. Within the Neisseria genus O-linked protein glycosylation is conserved yet closely related Neisseria species express O-oligosaccharyltransferases (PglOs) with distinct targeting activities. Within this work, we explore the targeting capacity of different PglOs using Field Asymmetric Waveform Ion Mobility Spectrometry (FAIMS) fractionation and Data-Independent Acquisition (DIA) to allow the characterization of the impact of changes in glycosylation on the proteome of Neisseria gonorrhoeae. We demonstrate FAIMS expands the known glycoproteome of wild type N. gonorrhoeae MS11 and enables differences in glycosylation to be assessed across strains expressing different pglO allelic chimeras with unique substrate targeting activities. Combining glycoproteomic insights with DIA proteomics, we demonstrate that alterations within pglO alleles have widespread impacts on the proteome of N. gonorrhoeae. Examination of peptides known to be targeted by glycosylation using DIA analysis supports alterations in glycosylation occupancy occurs independently of changes in protein levels and that the occupancy of glycosylation is generally low on most glycoproteins. This work thus expands our understanding of the N. gonorrhoeae glycoproteome and the roles that pglO allelic variation may play in governing genus-level protein glycosylation.

Proteomic analysis of diabetic retinas.

As a metabolic disease, diabetes often leads to health complications such as heart failure, nephropathy, neurological disorders, and vision loss. Diabetic retinopathy (DR) affects as many as 100 million people worldwide. The mechanism of DR is complex and known to impact both neural and vascular components in the retina. While recent advances in the field have identified major cellular signaling contributing to DR pathogenesis, little has been reported on the protein post-translational modifications (PTM) - known to define protein localization, function, and activity - in the diabetic retina overall. Protein glycosylation is the enzymatic addition of carbohydrates to proteins, which can influence many protein attributes including folding, stability, function, and subcellular localization. O-linked glycosylation is the addition of sugars to an oxygen atom in amino acids with a free oxygen atom in their side chain (i.e., threonine, serine). To date, more than 100 congenital disorders of glycosylation have been described. However, no studies have identified the retinal O-linked glycoproteome in health or disease. With a critical need to expedite the discovery of PTMomics in diabetic retinas, we identified both global changes in protein levels and the retinal O-glycoproteome of control and diabetic mice. We used liquid chromatography/mass spectrometry-based proteomics and high throughput screening to identify proteins differentially expressed and proteins differentially O-glycosylated in the retinas of wildtype and diabetic mice. Changes in both global expression levels of proteins and proteins differentially glycosylated in the retinas of wild-type and diabetic mice have been identified. We provide evidence that diabetes shifts both global expression levels and O-glycosylation of metabolic and synaptic proteins in the retina. Here we report changes in the retinal proteome of diabetic mice. We highlight alterations in global proteins involved in metabolic processes, maintaining cellular structure, trafficking, and neuronal processes. We then showed changes in O-linked glycosylation of individual proteins in the diabetic retina.

CRISPRi-Mediated Silencing of Burkholderia O-Linked Glycosylation Systems Enables the Depletion of Glycosylation Yet Results in Modest Proteome Impacts.

The process of O-linked protein glycosylation is highly conserved across the Burkholderia genus and mediated by the oligosaccharyltransferase PglL. While our understanding of Burkholderia glycoproteomes has increased in recent years, little is known about how Burkholderia species respond to modulations in glycosylation. Utilizing CRISPR interference (CRISPRi), we explored the impact of silencing of O-linked glycosylation across four species of Burkholderia; Burkholderia cenocepacia K56-2, Burkholderia diffusa MSMB375, Burkholderia multivorans ATCC17616, and Burkholderia thailandensis E264. Proteomic and glycoproteomic analyses revealed that while CRISPRi enabled inducible silencing of PglL, this did not abolish glycosylation, nor recapitulate phenotypes such as proteome changes or alterations in motility that are associated with glycosylation null strains, despite inhibition of glycosylation by nearly 90%. Importantly, this work also demonstrated that CRISPRi induction with high levels of rhamnose leads to extensive impacts on the Burkholderia proteomes, which without appropriate controls mask the impacts specifically driven by CRISPRi guides. Combined, this work revealed that while CRISPRi allows the modulation of O-linked glycosylation with reductions up to 90% at a phenotypic and proteome levels, Burkholderia appears to demonstrate a robust tolerance to fluctuations in glycosylation capacity.

Genetic, Functional, and Immunogenic Analyses of the O-Linked Protein Glycosylation System in Neisseria meningitidis Serogroup A ST-7 Isolates.

Neisseria meningitidis exhibits a general O-linked protein glycosylation system in which pili and other extracytoplasmic proteins are glycosylated. To investigate glycan antigenicity in humans and the significance of high glycan diversity on immune escape mechanisms, we exploited serogroup A meningococcal strains and serum samples obtained from laboratory-confirmed Ethiopian patients with meningococcal disease. The 37 meningococcal isolates were sequenced, and their protein glycosylation (pgl) genotypes and protein glycosylation phenotypes were investigated in detail. An insertion sequence (IS1655) element in pglH reduced glycan variability in the majority of isolates, while phase variation strengthened glycan variability and microheterogeneity. Homologous recombination events within the pgl genes were identified in eight of the 37 isolates, and the phenotypic consequences ranged from none detected to altered glycoforms in two of the isolates in which the whole pgl locus was exchanged. Immunoblotting of sera against a complete panel of glycan-expressing mutant strains demonstrated that most of these patient sera had IgG antibodies against various neisserial protein glycan antigens. Furthermore, using a bactericidal assay comparing a wild-type meningococcal A strain and a glycosylation-null variant strain, we showed that these protein glycan antigens interfere with bactericidal killing by antibodies in patient sera. Altogether, we were largely able to link pgl genotype with glycosylation phenotype. Our study reveals that protein glycans seem to contribute to the ability of N. meningitidis to resist the bactericidal activity of human serum, possibly by masking protein epitopes important for bactericidal killing and thus protection against meningococcal disease. IMPORTANCE Bacterial meningitis is a serious global health problem, and one of the major causative organisms is Neisseria meningitidis. Extensive variability in protein glycan structure and antigenicity is due to phase variation of protein glycosylation genes and polymorphic gene content and function. The exact role(s) of glycosylation in Neisseria remains to be determined, but increasing evidence, supported by this study, suggests that glycan variability can be a strategy to escape the human immune system. The complexity of the O-linked protein glycosylation system requires further studies to fully comprehend how these bacteria utilize variation in pgl genes to produce such high glycoform diversity and to evade the human immune response.

Involvement of the Streptococcus mutans PgfE and GalE 4-epimerases in protein glycosylation, carbon metabolism, and cell division.

Streptococcus mutans is a key pathogen associated with dental caries and is often implicated in infective endocarditis. This organism forms robust biofilms on tooth surfaces and can use collagen-binding proteins (CBPs) to efficiently colonize collagenous substrates, including dentin and heart valves. One of the best characterized CBPs of S. mutans is Cnm, which contributes to adhesion and invasion of oral epithelial and heart endothelial cells. These virulence properties were subsequently linked to post-translational modification (PTM) of the Cnm threonine-rich repeat region by the Pgf glycosylation machinery, which consists of 4 enzymes: PgfS, PgfM1, PgfE, and PgfM2. Inactivation of the S. mutans pgf genes leads to decreased collagen binding, reduced invasion of human coronary artery endothelial cells, and attenuated virulence in the Galleria mellonella invertebrate model. The present study aimed to better understand Cnm glycosylation and characterize the predicted 4-epimerase, PgfE. Using a truncated Cnm variant containing only 2 threonine-rich repeats, mass spectrometric analysis revealed extensive glycosylation with HexNAc2. Compositional analysis, complemented with lectin blotting, identified the HexNAc2 moieties as GlcNAc and GalNAc. Comparison of PgfE with the other S. mutans 4-epimerase GalE through structural modeling, nuclear magnetic resonance, and capillary electrophoresis demonstrated that GalE is a UDP-Glc-4-epimerase, while PgfE is a GlcNAc-4-epimerase. While PgfE exclusively participates in protein O-glycosylation, we found that GalE affects galactose metabolism and cell division. This study further emphasizes the importance of O-linked protein glycosylation and carbohydrate metabolism in S. mutans and identifies the PTM modifications of the key CBP, Cnm.

Galnt17 loss-of-function leads to developmental delay and abnormal coordination, activity, and social interactions with cerebellar vermis pathology

GALNT17 encodes a N-acetylgalactosaminyltransferase (GalNAc-T) protein specifically involved in mucin-type O-linked glycosylation of target proteins, a process important for cell adhesion, cell signaling, neurotransmitter activity, neurite outgrowth, and neurite sensing. GALNT17, also known as WBSCR17, is located at the edge of the Williams-Beuren Syndrome (WBS) critical region and adjacent to the AUTS2 locus, genomic regions associated with neurodevelopmental phenotypes that are thought to be co-regulated. Although previous data have implicated Galnt17 in neurodevelopment, the in vivo functions of this gene have not been investigated. In this study, we have analyzed behavioral, brain pathology, and molecular phenotypes exhibited by Galnt17 knockout (Galnt17−/−) mice. We show that Galnt17−/− mutants exhibit developmental neuropathology within the cerebellar vermis, along with abnormal activity, coordination, and social interaction deficits. Transcriptomic and protein analysis revealed reductions in both mucin type O-glycosylation and heparan sulfate synthesis in the developing mutant cerebellum along with disruption of pathways central to neuron differentiation, axon pathfinding, and synaptic signaling, consistent with the mutant neuropathology. These brain and behavioral phenotypes and molecular data confirm a specific role for Galnt17 in brain development and suggest new clues to factors that could contribute to phenotypes in certain WBS and AUTS2 syndrome patients.

PGM3 regulates beta-catenin activity to promote colorectal cancer cell progression.

The hexosamine biosynthetic pathway (HBP) is connected to abnormal N- and O-linked protein glycosylation in cancer, which performs critical roles in tumorigenesis. However, the regulation mechanisms of HBP and its role in colorectal cancer (CRC) progression remain unexplained. This study analyzed the expression level of phosphoglucomutase 3 (PGM3), a key enzyme in HBP, and identified its function in CRC cell lines. Analysis of publicly available CRC microarray data determined that PGM3 is upregulated in CRC tumor tissues. Furthermore, functional experiments emphasized the significant roles of PGM3 in facilitating CRC cell proliferation and migration. Mechanistically, we demonstrated that the activity of β-catenin in CRC was maintained by PGM3-mediated O-GlcNAcylation. PGM3 knockdown or inhibition of O-GlcNAc transferase decreased β-catenin activity and the expression levels of its downstream targets. Collectively, our findings indicate that PGM3 exhibits tumor-promoting roles by elevating O-GlcNAcylation level and maintaining β-catenin activity, and might serve as a prognostic biomarker and treatment target in CRC.

Sculpting the Bacterial O-Glycoproteome: Functional Analyses of Orthologous Oligosaccharyltransferases with Diverse Targeting Specificities.

ABSTRACTProtein glycosylation systems are widely recognized in bacteria, including members of the genus Neisseria. In most bacterial species, the molecular mechanisms and evolutionary contexts underpinning target protein selection and the glycan repertoire remain poorly understood. Broad-spectrum O-linked protein glycosylation occurs in all human-associated species groups within the genus Neisseria, but knowledge of their individual glycoprotein repertoires is limited. Interestingly, PilE, the pilin subunit of the type IV pilus (Tfp) colonization factor, is glycosylated in Neisseria gonorrhoeae and Neisseria meningitidis but not in the deeply branching species N. elongata subsp. glycolytica. To examine this in more detail, we assessed PilE glycosylation status across the genus and found that PilEs of commensal clade species are not modified by the gonococcal PglO oligosaccharyltransferase. Experiments using PglO oligosaccharyltransferases from across the genus expressed in N. gonorrhoeae showed that although all were capable of broad-spectrum protein glycosylation, those from a deep-branching group of commensals were unable to support resident PilE glycosylation. Further glycoproteomic analyses of these strains using immunoblotting and mass spectrometry revealed other proteins differentially targeted by otherwise remarkably similar oligosaccharyltransferases. Finally, we generated pglO allelic chimeras that begin to localize PglO protein domains associated with unique substrate targeting activities. These findings reveal previously unappreciated differences within the protein glycosylation systems of highly related bacterial species. We propose that the natural diversity manifest in the neisserial protein substrates and oligosaccharyltransferases has significant potential to inform the structure-function relationships operating in these and related bacterial protein glycosylation systems.

Linking inherent O-Linked Protein Glycosylation of YghJ to Increased Antigen Potential.

Enterotoxigenic Escherichia coli (ETEC) is a WHO priority pathogen and vaccine target which causes infections in low-income and middle-income countries, travelers visiting endemic regions. The global urgent demand for an effective preventive intervention has become more pressing as ETEC strains have become increasingly multiple antibiotic resistant. However, the vaccine development pipeline has been slow to address this urgent need. To date, vaccine development has focused mainly on canonical antigens such as colonization factors and expressed toxins but due to genomic plasticity of this enteric pathogen, it has proven difficult to develop effective vaccines. In this study, we investigated the highly conserved non-canonical vaccine candidate YghJ/SsLE. Using the mass spectrometry-based method BEMAP, we demonstrate that YghJ is hyperglycosylated in ETEC and identify 54 O-linked Set/Thr residues within the 1519 amino acid primary sequence. The glycosylation sites are evenly distributed throughout the sequence and do not appear to affect the folding of the overall protein structure. Although the glycosylation sites only constitute a minor subpopulation of the available epitopes, we observed a notable difference in the immunogenicity of the glycosylated YghJ and the non-glycosylated protein variant. We can demonstrate by ELISA that serum from patients enrolled in an ETEC H10407 controlled infection study are significantly more reactive with glycosylated YghJ compared to the non-glycosylated variant. This study provides an important link between O-linked glycosylation and the relative immunogenicity of bacterial proteins and further highlights the importance of this observation in considering ETEC proteins for inclusion in future broad coverage subunit vaccine candidates.

Protein glycosylation in extracellular vesicles: Structural characterization and biological functions

Extracellular vesicles (EVs) are lipid bilayer-enclosed particles involved in intercellular communication, delivery of biomolecules from donor to recipient cells, cellular disposal and homeostasis, potential biomarkers and drug carriers. The content of EVs includes DNA, lipids, metabolites, proteins, and microRNA, which have been studied in various diseases, such as cancer, diabetes, pregnancy, neurodegenerative, and cardiovascular disorders. EVs are enriched in glycoconjugates and exhibit specific glycosignatures. Protein glycosylation is a co- and post-translational modification (PTM) that plays an important role in the expression and function of exosomal proteins. N- and O-linked protein glycosylation has been mapped in exosomal proteins. The purpose of this review is to highlight the importance of glycosylation in EVs proteins. Initially, we describe the main PTMs in EVs with a focus on glycosylation. Then, we explore glycan-binding proteins describing the main findings of studies that investigated the glycosylation of EVs in cancer, pregnancy, infectious diseases, diabetes, mental disorders, and animal fluids. We have highlighted studies that have developed innovative methods for studying the content of EVs. In addition, we present works related to lipid glycosylation. We explored the content of studies deposited in public databases, such as Exocarta and Vesiclepedia. Finally, we discuss analytical methods for structural characterization of glycoconjugates and present an overview of the critical points of the study of glycosylation EVs, as well as perspectives in this field.

LncRNA NKILA suppresses airway hyper reactivity via interfering the facilitation of MUC5AC and MUC5B mediated by GALNT2

Glycosylation of mucins mediated by N-acetylgalactosaminyltransferases (GALNTs) is closely related to respiratory diseases such as asthma and chronic obstructive pulmonary disease (COPD). In addition, long non-coding RNAs (LncRNAs) participate in physiological and pathological processes through various epigenetic mechanisms. In this study, we found that a novel LncRNA named NKILA combined with multiple mucins and GALNTs potentially by several bioinformatics methods, and we used quantitative real-time PCR (RT-qPCR) to detect the expressions of NKILA, MUC5AC, MUC5B, and GALNT2 mRNA in 50 cases of asthma samples and 19 cases of normal samples, whose results showed that the expression of NKILA was significantly decreased in asthmatic samples, negatively correlated with the severity of asthma and the expressions of MUC5AC and MUC5B, while GALNT2 was significantly increased in asthmatic tissues, and positively correlated with the severity of asthma and the expressions of MUC5AC and MUC5B. In vitro, we used transient transfection technology to overexpress or interfere with NKILA and GALNT2 and then detected the expressions of MUC5AC and MUC5B via RT-qPCR and Western blot, which demonstrated GALNT2 can promote the expressions of MUC5AC and MUC5B protein, while NKILA could inhibit this effect. Furthermore, co-immunoprecipitation results showed that GALNT2 could bind to MUC5AC and MUC5B protein. RNA immunoprecipitation and RNA pull-down experiments showed that NKILA could bind to GALNT2. These evidences suggested that there are correlations among the expression of NKILA, GALNT2, MUC5AC, and MUC5B proteins in asthmatic patients. Mechanically, we concluded that NKILA can suppress the O-linked glycosylation of MUC5AC and MUC5B proteins by binding to GALNT2 and inhibit the expression of MUC5AC and MUC5B proteins. Our researches provided a potential therapeutic target for AHR.

Allelic polymorphisms in a glycosyltransferase gene shape glycan repertoire in the O-linked protein glycosylation system of Neisseria.

Glycosylation of multiple proteins via O-linkage is well documented in bacterial species of Neisseria of import to human disease. Recent studies of protein glycosylation (pgl) gene distribution established that related protein glycosylation systems occur throughout the genus including nonpathogenic species. However, there are inconsistencies between pgl gene status and observed glycan structures. One of these relates to the widespread distribution of pglG, encoding a glycosyltransferase that in Neisseria elongata subsp. glycolytica is responsible for the addition of di-N-acetyl glucuronic acid at the third position of a tetrasaccharide. Despite pglG residing in strains of N. gonorrhoeae, N. meningitidis and N. lactamica, no glycan structures have been correlated with its presence in these backgrounds. Moreover, PglG function in N. elongata subsp. glycolytica minimally requires UDP-glucuronic acid (GlcNAcA), and yet N. gonorrhoeae, N. meningitidis and N. lactamica lack pglJ, the gene whose product is essential for UDP-GlcNAcA synthesis. We examined the functionality of pglG alleles from species spanning the Neisseria genus by genetic complementation in N. elongata subsp. glycolytica. The results indicate that select pglG alleles from N. meningitidis and N. lactamica are associated with incorporation of an N-acetyl-hexosamine at the third position and reveal the potential for an expanded glycan repertoire in those species. Similar experiments using pglG from N. gonorrhoeae failed to find any evidence of function suggesting that those alleles are missense pseudogenes. Taken together, the results are emblematic of how allelic polymorphisms can shape bacterial glycosyltransferase function and demonstrate that such alterations may be constrained to distinct phylogenetic lineages.

Engineering orthogonal human O-linked glycoprotein biosynthesis in bacteria.

A major objective of synthetic glycobiology is to re-engineer existing cellular glycosylation pathways from the top down or construct non-natural ones from the bottom up for new and useful purposes. Here, we have developed a set of orthogonal pathways for eukaryotic O-linked protein glycosylation in Escherichia coli that installed the cancer-associated mucin-type glycans Tn, T, sialyl-Tn and sialyl-T onto serine residues in acceptor motifs derived from different human O-glycoproteins. These same glycoengineered bacteria were used to supply crude cell extracts enriched with glycosylation machinery that permitted cell-free construction of O-glycoproteins in a one-pot reaction. In addition, O-glycosylation-competent bacteria were able to generate an antigenically authentic Tn-MUC1 glycoform that exhibited reactivity with antibody 5E5, which specifically recognizes cancer-associated glycoforms of MUC1. We anticipate that the orthogonal glycoprotein biosynthesis pathways developed here will provide facile access to structurally diverse O-glycoforms for a range of important scientific and therapeutic applications.

Genetic determinants of genus-level glycan diversity in a bacterial protein glycosylation system.

The human pathogens N. gonorrhoeae and N. meningitidis display robust intra- and interstrain glycan diversity associated with their O-linked protein glycosylation (pgl) systems. In an effort to better understand the evolution and function of protein glycosylation operating there, we aimed to determine if other human-restricted, Neisseria species similarly glycosylate proteins and if so, to assess the levels of glycoform diversity. Comparative genomics revealed the conservation of a subset of genes minimally required for O-linked protein glycosylation glycan and established those pgl genes as core genome constituents of the genus. In conjunction with mass spectrometric–based glycan phenotyping, we found that extant glycoform repertoires in N. gonorrhoeae, N. meningitidis and the closely related species N. polysaccharea and N. lactamica reflect the functional replacement of a progenitor glycan biosynthetic pathway. This replacement involved loss of pgl gene components of the primordial pathway coincident with the acquisition of two exogenous glycosyltransferase genes. Critical to this discovery was the identification of a ubiquitous but previously unrecognized glycosyltransferase gene (pglP) that has uniquely undergone parallel but independent pseudogenization in N. gonorrhoeae and N. meningitidis. We suggest that the pseudogenization events are driven by processes of compositional epistasis leading to gene decay. Additionally, we documented instances where inter-species recombination influences pgl gene status and creates discordant genetic interactions due ostensibly to the multi-locus nature of pgl gene networks. In summary, these findings provide a novel perspective on the evolution of protein glycosylation systems and identify phylogenetically informative, genetic differences associated with Neisseria species.

A Broad Spectrum Protein Glycosylation System Influences Type II Protein Secretion and Associated Phenotypes in Vibrio cholerae.

Protein secretion plays a crucial role for bacterial pathogens, exemplified by facultative human-pathogen Vibrio cholerae, which secretes various proteinaceous effectors at different stages of its lifecycle. Accordingly, the identification of factors impacting on protein secretion is important to understand the bacterial pathophysiology. PglLVc, a predicted oligosaccharyltransferase of V. cholerae, has been recently shown to exhibit O-glycosylation activity with relaxed glycan specificity in an engineered Escherichia coli system. By engineering V. cholerae strains to express a defined, undecaprenyl diphosphate-linked glycoform precursor, we confirmed functional O-linked protein glycosylation activity of PglLVc in V. cholerae. We demonstrate that PglLVc is required for the glycosylation of multiple V. cholerae proteins, including periplasmic chaperones such as DegP, that are required for efficient type II-dependent secretion. Moreover, defined deletion mutants and complementation strains provided first insights into the physiological role of O-linked protein glycosylation in V. cholerae. RbmD, a protein with structural similarities to PglLVc and other established oligosaccharyltransferases (OTases), was also included in this phenotypical characterization. Remarkably, presence or absence of PglLVc and RbmD impacts the secretion of proteins via the type II secretion system (T2SS). This is highlighted by altered cholera toxin (CT) secretion, chitin utilization and biofilm formation observed in ΔpglLVc and ΔrbmD single or double mutants. This work thus establishes a unique connection between broad spectrum O-linked protein glycosylation and the efficacy of type II-dependent protein secretion critical to the pathogen’s lifecycle.

P6302GALNT4 enhances monocyte adherence and chemotaxis by regulating PSGL1 O-glycosylation in patients with acute coronary syndrome

Abstract Background GalNAc-T4 (GALNT4), a member of N-acetylgalactosaminyltransferases family, which transfers GalNAc to Serine and Threonine residues in the initial step of O-linked protein glycosylation. Genome wide association studies (GWAS) have predicted that GALNT4 gene play a causal role in the susceptibility to cardiovascular diseases, whereas the precise mechanisms of it in acute coronary disease (ACS) is unknown. Purpose In this study, we sought to determine the role of GALNT4 in regulating monocytes with ACS and the underlying mechanisms. Methods and results The expression of GALNT4 mRNA was significantly elevated in patience with STEMI (n=50) compare with stable coronary heart disease (SCHD) (n=50) and controls (n=50) (Clinicaltrial.gov: NCT 03335514). Moreover, GALNT4 siRNA knockdown suppressed monocytes adherence under static and flow condition with P-selectin stimulation, as well as chemotaxis triggered by monocyte chemotactic protein-1 (MCP-1). In contrast, adenovirus mediated GALNT4 overexpression enhanced monocytes adherence and chemotaxis. Furthermore, vicia villosa lectin (VVL) pull down and PSGL1 immunoprecipitation assays showed that GALNT4 overexpression increased VVL binding to PSGL-1 and P-selectin induce phosphorylation of Akt and mTOR. Conversely, knockdown of GALNT4 decreased VVL binding and attenuated the activity of Akt /mTOR. Additionally, mTOR inhibitor rapamycin blocked these effects of GALNT4 overexpression on monocytes. Conclusion Expression of GALNT4 was increased in patience with ACS, GALNT4 overexpression enhances the adherence and chemotaxis of monocytes via modifying O-glycosylation of PSGL-1 and activity Akt/mTOR pathway. These findings suggest that GALNT4 may be a potential therapeutic target for ACS. Acknowledgement/Funding The National Natural Science Foundation of China (No. 81100220 and No. 81770340)

A general protein O-glycosylation machinery conserved in Burkholderia species improves bacterial fitness and elicits glycan immunogenicity in humans

The Burkholderia genus encompasses many Gram-negative bacteria living in the rhizosphere. Some Burkholderia species can cause life-threatening human infections, highlighting the need for clinical interventions targeting specific lipopolysaccharide proteins. Burkholderia cenocepacia O-linked protein glycosylation has been reported, but the chemical structure of the O-glycan and the machinery required for its biosynthesis are unknown and could reveal potential therapeutic targets. Here, using bioinformatics approaches, gene-knockout mutants, purified recombinant proteins, LC-MS-based analyses of O-glycans, and NMR-based structural analyses, we identified a B. cenocepacia O-glycosylation (ogc) gene cluster necessary for synthesis, assembly, and membrane translocation of a lipid-linked O-glycan, as well as its structure, which consists of a β-Gal-(1,3)-α-GalNAc-(1,3)-β-GalNAc trisaccharide. We demonstrate that the ogc cluster is conserved in the Burkholderia genus, and we confirm the production of glycoproteins with similar glycans in the Burkholderia species: B. thailandensis, B. gladioli, and B. pseudomallei Furthermore, we show that absence of protein O-glycosylation severely affects bacterial fitness and accelerates bacterial clearance in a Galleria mellonella larva infection model. Finally, our experiments revealed that patients infected with B. cenocepacia, Burkholderia multivorans, B. pseudomallei, or Burkholderia mallei develop O-glycan-specific antibodies. Together, these results highlight the importance of general protein O-glycosylation in the biology of the Burkholderia genus and its potential as a target for inhibition or immunotherapy approaches to control Burkholderia infections.