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Tannerella forsythia, a member of the “red complex” bacteria implicated in severe periodontitis, employs various survival strategies and virulence factors to interact with the host. It thrives as a late colonizer in the oral biofilm, relying on its unique adaptation mechanisms for persistence. Essential to its survival are the type 9 protein secretion system and O-glycosylation of proteins, crucial for host interaction and immune evasion. Virulence factors of T. forsythia, including sialidase and proteases, facilitate its pathogenicity by degrading host glycoproteins and proteins, respectively. Moreover, cell surface glycoproteins like the S-layer and BspA modulate host responses and bacterial adherence, influencing colonization and tissue invasion. Outer membrane vesicles and lipopolysaccharides further induce inflammatory responses, contributing to periodontal tissue destruction. Interactions with specific host cell types, including epithelial cells, polymorphonuclear leukocytes macrophages, and mesenchymal stromal cells, highlight the multifaceted nature of T. forsythia's pathogenicity. Notably, it can invade epithelial cells and impair PMN function, promoting dysregulated inflammation and bacterial survival. Comparative studies with periodontitis-associated Porphyromonas gingivalis reveal differences in protease activity and immune modulation, suggesting distinct roles in disease progression. T. forsythia's potential to influence oral antimicrobial defense through protease-mediated degradation and interactions with other bacteria underscores its significance in periodontal disease pathogenesis. However, understanding T. forsythia's precise role in host-microbiome interactions and its classification as a keystone pathogen requires further investigation. Challenges in translating research data stem from the complexity of the oral microbiome and biofilm dynamics, necessitating comprehensive studies to elucidate its clinical relevance and therapeutic implications in periodontitis management.

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. [ABSTRACT FROM AUTHOR]

Tannerella serpentiformis is a health-associated Gram-negative oral anaerobe, while its closest phylogenetic relative is the periodontal pathogen Tannerella forsythia. The pathogen employs glycan mimicry through protein O-glycosylation, displaying a terminal nonulosonic acid aiding in evasion of host immune recognition. Like T. forsythia, T. serpentiformis cells are covered with a 2D-crystalline S-layer composed of two abundant S-layer glycoproteins-TssA and TssB. In this study, we elucidated the structure of the O-linked glycans of T. serpentiformis using 1D and 2D NMR spectroscopy analyzing S-layer glycopeptides and β-eliminated glycans. We found that T. serpentiformis produces two highly fucosylated, branched glycoforms carrying non-carbohydrate modifications, with the structure [2-OMe-Fuc-(α1,2)]-4-OMe-Glc-(β1,3)-[Fuc-(α1,4)]-2-NAc-GlcA-(β1,4)-[3-NH2, 2,4-OMe-Fuc-(α1,3)]-Fuc-(α1,4)-Xyl-(β1,4)-[3-OMe-Fuc-(α1,3)]-GlcA-(α1,2)-[Rha-(α1,4]-Gal, where the 3OMe-Fuc is variable; each glycoform contains a rare 2,4-methoxy, 3-amino-modified fucose. These glycoforms support the hypothesis that nonulosonic acid is a hallmark of pathogenic Tannerella species. A combined glycoproteomics and bioinformatics approach identified multiple sites within TssA (14 sites) and TssB (21 sites) to be O-glycosylated. LC-MS/MS confirmed the presence of the Bacteroidetes O-glycosylation motif (D)(S/T) (L/V/T/A/I) in Tannerella species, including the newly identified candidate "N" for the third position. Alphfold2 models of the S-layer glycoproteins were created revealing an almost uniform spatial distribution of the two glycoforms at the N-terminal two thirds of the proteins supported by glycoproteomics, with glycans facing outward. Glycoproteomics identified 921 unique glycopeptide sequences corresponding to 303 unique UniProt IDs. GO-term enrichment analysis versus the entire T. serpentiformis proteome classified these proteins as mainly membrane and cell periphery-associated glycoproteins, supporting a general protein O-glycosylation system in T. serpentiformis., (© The Author(s) 2024. Published by Oxford University Press.)

Tannerella forsythia , a member of the "red complex" bacteria implicated in severe periodontitis, employs various survival strategies and virulence factors to interact with the host. It thrives as a late colonizer in the oral biofilm, relying on its unique adaptation mechanisms for persistence. Essential to its survival are the type 9 protein secretion system and O -glycosylation of proteins, crucial for host interaction and immune evasion. Virulence factors of T. forsythia , including sialidase and proteases, facilitate its pathogenicity by degrading host glycoproteins and proteins, respectively. Moreover, cell surface glycoproteins like the S-layer and BspA modulate host responses and bacterial adherence, influencing colonization and tissue invasion. Outer membrane vesicles and lipopolysaccharides further induce inflammatory responses, contributing to periodontal tissue destruction. Interactions with specific host cell types, including epithelial cells, polymorphonuclear leukocytes macrophages, and mesenchymal stromal cells, highlight the multifaceted nature of T. forsythia's pathogenicity. Notably, it can invade epithelial cells and impair PMN function, promoting dysregulated inflammation and bacterial survival. Comparative studies with periodontitis-associated Porphyromonas gingivalis reveal differences in protease activity and immune modulation, suggesting distinct roles in disease progression. T. forsythia's potential to influence oral antimicrobial defense through protease-mediated degradation and interactions with other bacteria underscores its significance in periodontal disease pathogenesis. However, understanding T. forsythia's precise role in host-microbiome interactions and its classification as a keystone pathogen requires further investigation. Challenges in translating research data stem from the complexity of the oral microbiome and biofilm dynamics, necessitating comprehensive studies to elucidate its clinical relevance and therapeutic implications in periodontitis management., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest. The author(s) declared that they were an editorial board member of Frontiers, at the time of submission. This had no impact on the peer review process and the final decision., (© 2024 Schäffer and Andrukhov.)

Mucin-type O-glycosylation (hereafter referred to as O-GalNAc glycosylation) is one of the most abundant glycosylation on proteins. It is initiated by the members of polypeptide N-acetyl-α-galactosaminyltransferases (ppGalNAc-Ts) family. The ppGalNAc-Ts could be used as tool enzymes to modify target proteins including therapeutic glycoprotein drugs with O-GalNAc glycosylation at specific glycosylated sites in vitro. Obtaining a large amount of ppGalNAc-T can greatly increase the yield of therapeutic O-glycoprotein and reduce the culture costs. In this study, we reported a simple Escherichia coli (E. coli) expression system capable of producing human ppGalNAc-Ts. By co-expressing human PDI, we could simply obtain active ppGalNAc-Ts with high efficiency. Using the E. coli expressed ppGalNAc-T2, we site-specifically synthesized O-glycosylated IL-2 at the native glycosylated site Thr23 residue. These results reveal the E. coli system we constructed is suitable to produce active ppGalNAc-Ts and thus has the potential value for basic research and production of therapeutic O-glycoproteins in vitro. • To provide a simple expression system of recombinant human ppGalNAc-T in E. coli. • High efficiently obtaining soluble and active ppGalNAc-Ts in E. coli. • Site-specific synthesis of O-glycosylated interleukin-2 in vitro. [ABSTRACT FROM AUTHOR]

The mechanisms by which methylated mammalian promoters are transcriptionally silenced even in the presence of all of the factors required for their expression have long been a major unresolved issue in the field of epigenetics. Repression requires the assembly of a methylation-dependent silencing complex that contains the TRIM28 protein (also known as KAP1 and TIF1β), a scaffolding protein without intrinsic repressive or DNA-binding properties. The identity of the key effector within this complex that represses transcription is unknown. We developed a methylation-sensitized interaction screen which revealed that TRIM28 was complexed with O-linked β-N-acetylglucosamine transferase (OGT) only in cells that had normal genomic methylation patterns. OGT is the only glycosyltransferase that modifies cytoplasmic and nuclear protein by transfer of N-acetylglucosamine (O-GlcNAc) to serine and threonine hydroxyls. Whole-genome analysis showed that O-glycosylated proteins and TRIM28 were specifically bound to promoters of active retrotransposons and to imprinting control regions, the two major regulatory sequences controlled by DNA methylation. Furthermore, genome-wide loss of DNA methylation caused a loss of O-GlcNAc from multiple transcriptional repressor proteins associated with TRIM28. A newly developed Cas9-based editing method for targeted removal of O-GlcNAc was directed against retrotransposon promoters. Local chromatin de-GlcNAcylation specifically reactivated the expression of the targeted retrotransposon family without loss of DNA methylation. These data revealed that O-linked glycosylation of chromatin factors is essential for the transcriptional repression of methylated retrotransposons. [ABSTRACT FROM AUTHOR]

Protein O-glycosylation is the attachment of carbohydrate structures to the oxygen atom in the hydroxyl group of Serine and Threonine residues. This post-translational modification is commonly found on the majority of proteins trafficking through the secretory pathway and is reported to influence protein characteristics such as folding, secretion, stability, solubility, oligomerization and intracellular localization. In addition, O-glycosylation is essential for cell-cell interactions, protein-protein interactions and many biological processes, such as stress response, immunization, phosphorylation, ubiquitination, cell division, metabolism and cell signaling. The availability of sequenced genomes and genetic tools to create mutants with clear phenotypes makes insects an interesting model system to study O-glycosylation. In this review, we provide an overview of the current knowledge of O-glycosylation, mainly obtained from the model organism Drosophila melanogaster, with a focus on the synthesis and biological roles of the common O-glycans in insects. [ABSTRACT FROM AUTHOR]

ABSTRACT The physiological role of protein O-glycosylation in prokaryotes is poorly understood due to our limited knowledge of the extent of their glycoproteomes. In Actinobacteria, defects in protein O-mannosyl transferase (Pmt)-mediated protein O-glycosylation have been shown to significantly retard growth (Mycobacterium tuberculosis and Corynebacterium glutamicum) or result in increased sensitivities to cell wall-targeting antibiotics (Streptomyces coelicolor), suggesting that protein O-glycosylation has an important role in cell physiology. Only a single glycoprotein (SCO4142, or PstS) has been identified to date in S. coelicolor. Combining biochemical and mass spectrometry-based approaches, we have isolated and characterized the membrane glycoproteome in S. coelicolor. A total of ninety-five high-confidence glycopeptides were identified which mapped to thirty-seven new S. coelicolor glycoproteins and a deeper understanding of glycosylation sites in PstS. Glycosylation sites were found to be modified with up to three hexose residues, consistent with what has been observed previously in other Actinobacteria. S. coelicolor glycoproteins have diverse roles and functions, including solute binding, polysaccharide hydrolases, ABC transporters, and cell wall biosynthesis, the latter being of potential relevance to the antibiotic-sensitive phenotype of pmt mutants. Null mutants in genes encoding a putative d-Ala-d-Ala carboxypeptidase (SCO4847) and an l,d-transpeptidase (SCO4934) were hypersensitive to cell wall-targeting antibiotics. Additionally, the sco4847 mutants displayed an increased susceptibility to lysozyme treatment. These findings strongly suggest that both glycoproteins are required for maintaining cell wall integrity and that glycosylation could be affecting enzyme function. IMPORTANCE In prokaryotes, the role of protein glycosylation is poorly understood due to our limited understanding of their glycoproteomes. In some Actinobacteria, defects in protein O-glycosylation have been shown to retard growth and result in hypersensitivity to cell wall-targeting antibiotics, suggesting that this modification is important for maintaining cell wall structure. Here, we have characterized the glycoproteome in Streptomyces coelicolor and shown that glycoproteins have diverse roles, including those related to solute binding, ABC transporters, and cell wall biosynthesis. We have generated mutants encoding two putative cell wall-active glycoproteins and shown them to be hypersensitive to cell wall-targeting antibiotics. These findings strongly suggest that both glycoproteins are required for maintaining cell wall integrity and that glycosylation affects enzyme function.

O-glycosylation of mammalian proteins is one of the important posttranslational modifications. We applied a support vector machine (SVM) to predict whether Ser or Thr is glycosylated, in order to elucidate the O-glycosylation mechanism. O-glycosylated sites were often found clustered along the sequence, whereas other sites were located sporadically. Therefore, we developed two types of SVMs for predicting clustered and isolated sites separately. We found that the amino acid composition was effective for predicting the clustered type, whereas the site-specific algorithm was effective for the isolated type. The highest prediction accuracy for the clustered type was 74%, while that for the isolated type was 79%. The existence frequency of amino acids around the O-glycosylation sites was different in the two types: namely, Pro, Val and Ala had high existence probabilities at each specific position relative to a glycosylation site, especially for the isolated type. Independent component analyses for the amino acid sequences around O-glycosylation sites showed the position-specific existences of the identified amino acids as independent components. The O-glycosylation sites were preferentially located within intrinsically disordered regions of extracellular proteins: particularly, more than 90% of the clustered O-GalNAc glycosylation sites were observed in intrinsically disordered regions. This feature could be the key for understanding the non-conservation property of O-glycosylation, and its role in functional diversity and structural stability.

We describe here the composition of the O-linked glycans on the Neurospora crassa cellobiohydrolase I (CBHI), which accounts for approximately 40% of the protein secreted by cells growing in the presence of cellulose. CBHI is O-glycosylated with six types of linear, and three types of branched, O-glycans containing approximately equal amounts of mannose and galactose. In addition to the classic fungal O-glycans with reducing end mannoses, we also identified reducing end galactoses which suggest the existence of a protein-O-galactosyltransferase in N. crassa. Because of the excellent genetic resources available for N. crassa, the knowledge of the CBHI O-glycans may enable the future evaluation of the role of O-glycosylation on cellulase function and the development of directed O-glycan/cellulase engineering. [ABSTRACT FROM AUTHOR]

Ralstonia solanacearum is one of the most lethal phytopathogens in the world. Due to its broad host range, it can causewilting disease inmany plant species of economic interest. In this work, we identified the O-oligosaccharyltransferase (O-OTase) responsible for protein O-glycosylation in R. solanacearum. An analysis of the glycoproteome revealed that 20 proteins, including type IV pilins are substrates of this general glycosylation system. Although multiple glycan forms were identified, the majority of the glycopeptides were modified with a pentasaccharide composed of HexNAc-(Pen)-dHex3, similar to the O antigen subunit present in the lipopolysaccharide ofmultiple R. solanacearumstrains. Disruption of the O-OTase led to the total loss of protein glycosylation, together with a defect in biofilm formation and reduced pathogenicity towards tomato plants. Comparative proteomic analysis revealed that the loss of glycosylation is not associated with widespread proteome changes. Only the levels of a single glycoprotein, the type IV pilin, were diminished in the absence of glycosylation. In parallel, disruption of glycosylation triggered an increase in the levels of a surface lectin homologous to Pseudomonas PA-IIL. These results reveal the important role of glycosylation in the pathogenesis of R. solanacearum. [ABSTRACT FROM AUTHOR]

Flagellin glycosylation impacts, in several documented cases, the functionality of bacterial flagella. The basis of flagellin glycosylation has been studied for various Gram-negative bacteria, but less is known about flagellin glycans of Gram-positive bacteria including Paenibacillus alvei, a secondary invader of honeybee colonies diseased with European foulbrood. Paenibacillus alvei CCM 2051T swarms vigorously on solidified culture medium, with swarming relying on functional flagella as evidenced by abolished biofilm formation of a non-motile P. alvei mutant defective in the flagellin protein Hag. Here, the glycobiology of the polar P. alvei flagella was investigated. Analysis on purified flagellin demonstrated that the 30-kDa Hag protein (PAV_2c01710) is modified with an O-linked trisaccharide comprised of one hexose and two N-acetyl-hexosamine residues, at three sites of glycosylation. Downstream of the hag gene on the bacterial chromosome, two open reading frames (PAV_2c01630, PAV_2c01640) encoding putative glycosyltransferases were shown to constitute a flagellin glycosylation island. Mutants defective in these genes exhibited altered migration in sodium dodecyl sulfate polyacrylamide gel electrophoresis as well as loss of extracellular flagella production and bacterial motility. This study reveals that flagellin glycosylation in P. alvei is pivotal to flagella formation and bacterial motility in vivo, and simultaneously identifies flagella glycosylation as a second protein O-glycosylation system in this bacterium, in addition to the well-investigated S-layer tyrosine O-glycosylation pathway. [ABSTRACT FROM AUTHOR]

Flagellin glycosylation impacts, in several documented cases, the functionality of bacterial flagella. The basis of flagellin glycosylation has been studied for various Gram-negative bacteria, but less is known about flagellin glycans of Gram-positive bacteria including Paenibacillus alvei, a secondary invader of honeybee colonies diseased with European foulbrood. Paenibacillus alvei CCM 2051T swarms vigorously on solidified culture medium, with swarming relying on functional flagella as evidenced by abolished biofilm formation of a non-motile P. alvei mutant defective in the flagellin protein Hag. Here, the glycobiology of the polar P. alvei flagella was investigated. Analysis on purified flagellin demonstrated that the 30-kDa Hag protein (PAV_2c01710) is modified with an O-linked trisaccharide comprised of one hexose and two N-acetyl-hexosamine residues, at three sites of glycosylation. Downstream of the hag gene on the bacterial chromosome, two open reading frames (PAV_2c01630, PAV_2c01640) encoding putative glycosyltransferases were shown to constitute a flagellin glycosylation island. Mutants defective in these genes exhibited altered migration in sodium dodecyl sulfate polyacrylamide gel electrophoresis as well as loss of extracellular flagella production and bacterial motility. This study reveals that flagellin glycosylation in P. alvei is pivotal to flagella formation and bacterial motility in vivo, and simultaneously identifies flagella glycosylation as a second protein O-glycosylation system in this bacterium, in addition to the well-investigated S-layer tyrosine O-glycosylation pathway. [ABSTRACT FROM AUTHOR]

We report that fluorescently tagged arabinogalactan glycosyltransferases target not only the Golgi apparatus but also uncharacterized smaller compartments when transiently expressed in Nicotiana benthamiana. Approximately 80% of AtGALT31A [ Arabidopsis thaliana galactosyltransferase from family 31 (At1g32930)] was found in the small compartments, of which, 45 and 40% of AtGALT29A [ Arabidopsis thaliana galactosyltransferase from family 29 (At1g08280)] and AtGlcAT14A [ Arabidopsis thaliana glucuronosyltransferase from family 14 (At5g39990)] colocalized with AtGALT31A, respectively; in contrast, N-glycosylation enzymes rarely colocalized (3-18%), implicating a role of the small compartments in a part of arabinogalactan ( O-glycan) biosynthesis rather than N-glycan processing. The dual localization of AtGALT31A was also observed for fluorescently tagged AtGALT31A stably expressed in an Arabidopsis atgalt31a mutant background. Further, site-directed mutagenesis of a phosphorylation site of AtGALT29A ( Y144) increased the frequency of the protein being targeted to the AtGALT31A-localized small compartments, suggesting a role of Y144 in subcellular targeting. The AtGALT31A localized to the small compartments were colocalized with neither SYP61 (syntaxin of plants 61), a marker for trans-Golgi network (TGN), nor FM4-64-stained endosomes. However, 41% colocalized with EXO70E2 ( Arabidopsis thaliana exocyst protein Exo70 homolog 2), a marker for exocyst-positive organelles, and least affected by Brefeldin A and Wortmannin. Taken together, AtGALT31A localized to small compartments that are distinct from the Golgi apparatus, the SYP61-localized TGN, FM4-64-stained endosomes and Wortmannin-vacuolated prevacuolar compartments, but may be part of an unconventional protein secretory pathway represented by EXO70E2 in plants. [ABSTRACT FROM AUTHOR]

O-glycosylation of mammalian proteins is one of the important posttranslational modifications. We applied a support vector machine (SVM) to predict whether Ser or Thr is glycosylated, in order to elucidate the O-glycosylation mechanism. O-glycosylated sites were often found clustered along the sequence, whereas other sites were located sporadically. Therefore, we developed two types of SVMs for predicting clustered and isolated sites separately. We found that the amino acid composition was effective for predicting the clustered type, whereas the site-specific algorithm was effective for the isolated type. The highest prediction accuracy for the clustered type was 74%, while that for the isolated type was 79%. The existence frequency of amino acids around the O-glycosylation sites was different in the two types: namely, Pro, Val and Ala had high existence probabilities at each specific position relative to a glycosylation site, especially for the isolated type. Independent component analyses for the amino acid sequences around O-glycosylation sites showed the position-specific existences of the identified amino acids as independent components. The O-glycosylation sites were preferentially located within intrinsically disordered regions of extracellular proteins: particularly, more than 90% of the clustered O-GalNAc glycosylation sites were observed in intrinsically disordered regions. This feature could be the key for understanding the non-conservation property of O-glycosylation, and its role in functional diversity and structural stability. [ABSTRACT FROM AUTHOR]

Abstract: The modification of serine and threonine residues of nuclear and cytoplasmic proteins by O-linked β-N-acetylglucosamine (O-GlcNAc) has emerged as a highly dynamic post-translational modification that plays a critical role in regulating numerous biological processes. Much of our understanding of the mechanisms underlying the role of O-GlcNAc on cellular function has been in the context of its adverse effects in mediating a range of chronic disease processes, including diabetes, cancer and neurodegenerative diseases. However, at the cellular level it has been shown that O-GlcNAc levels are increased in response to stress; augmentation of this response improved cell survival while attenuation decreased cell viability. Thus, it has become apparent that strategies that augment O-GlcNAc levels are pro-survival, whereas those that reduce O-GlcNAc levels decrease cell survival. There is a long history demonstrating the effectiveness of acute glucose–insulin–potassium (GIK) treatment and to a lesser extent glutamine in protecting against a range of stresses, including myocardial ischemia. A common feature of these approaches for metabolic cardioprotection is that they both have the potential to stimulate O-GlcNAc synthesis. Consequently, here we examine the links between metabolic cardioprotection with the ischemic cardioprotection associated with acute increases in O-GlcNAc levels. Some of the protective mechanisms associated with activation of O-GlcNAcylation appear to be transcriptionally mediated; however, there is also strong evidence to suggest that transcriptionally independent mechanisms also play a critical role. In this context we discuss the potential link between O-GlcNAcylation and cardiomyocyte calcium homeostasis including the role of non-voltage gated, capacitative calcium entry as a potential mechanism contributing to this protection. [Copyright &y& Elsevier]

The posttranslational modification of serine and threonine residues of nuclear and cytoplasmic proteins by the O-linked attachment of the monosaccharide β-N-acetylglucosamine (O-GIcNAc) is a highly dynamic and ubiquitous protein modification. Protein O-GlcNAcylation is rapidly emerging as a key regulator of critical biological processes including nuclear transport, translation and transcription, signal transduction, cytoskeletal reorganization, proteasomal degradation, and apoptosis. Increased levels of O-GlcNAc have been implicated as a pathogenic contributor to glucose toxicity and insulin resistance, which are both major hallmarks of diabetes mellitus and diabetes-related cardiovascular complications. Conversely, there is a growing body of data demon- strating that the acute activation of O-GlcNAc levels is an endogenous stress response designed to enhance cell survival. Reports on the effect of altered O-GlcNAc levels on the heart and cardiovascular system have been growing rapidly over the past few years and have implicated a role for O-GlcNAc in contributing to the adverse effects of diabetes on cardiovascular function as well as mediating the response to ischemic injury. Here, we summarize our present understanding of protein O-GlcNAcylation and its effect on the regulation of cardiovascular function. We examine the pathways regulating protein O-GlcNAcylation and discuss, in more detail, our understanding of the role of O-GIcNAc in both mediating the adverse effects of diabetes as well as its role in mediating cellular protective mechanisms in the cardiovascular system. In addition, we also explore the parallels between O-GlcNAc signaling and redox signaling, as an alternative paradigm for understanding the role of O-GlcNAcylation in regulating cell function. [ABSTRACT FROM AUTHOR]

We have previously shown that preischemic treatment with glucosamine improved cardiac functional recovery following ischemia-reperfusion, and this was mediated, at least in part, via enhanced flux through the hexosamine biosynthesis pathway and subsequently elevated O-linked N-acetylglucosamine (O-GlcNAc) protein levels. However, preischemic treatment is typically impractical in a clinical setting; therefore, the goal of this study was to investigate whether increasing protein O-GlcNAc levels only during reperfusion also improved recovery. Isolated perfused rat hearts were subjected to 20 min of global, no-flow ischemia followed by 60 min of reperfusion. Administration of glucosamine (10 mM) or an inhibitor of O-GlcNAcase, O-(2-acetamido-2-deoxy-D-glucopyranosylidene)amino-N-phenylcarbamate (PUGNAc; 200 µM), during the first 20 min of reperfusion significantly improved cardiac functional recovery and reduced troponin release during reperfusion compared with untreated control. Both interventions also significantly increased the levels of protein O-GlcNAc and ATP levels. We also found that both glucosamine and PUGNAc attenuated calpain-mediated proteolysis of α-fodrin as well as Ca2+/calmodulin-dependent protein kinase II during reperfusion. Thus two independent strategies for increasing protein O-GlcNAc levels in the heart during reperfusion significantly improved recovery, and this was correlated with attenuation of calcium-mediated proteolysis. These data provide further support for the concept that increasing cardiac O-GIcNAc levels may be a clinically relevant cardioprotective strategy and suggest that this protection could be due, at least in part, to inhibition of calcium-mediated stress responses. [ABSTRACT FROM AUTHOR]

Increased levels of O-linked attachment of N-acetylglucosamine (O-GIcNAc) on nucleocytoplasmic proteins are implicated in the development of diabetic cardiomyopathy and are regulated by O-GlcNAc transferase (OGT) expression and its substrate UDP-GlcNAc. Therefore, the goal of this study was to determine whether the development of diabetes in the Zucker diabetic fatty (ZDF) rat, a model of Type 2 diabetes, results in defects in cardiomyocyte mechanical function and, if so, whether this is associated with increased levels of O-GlcNAc and increased OGT expression. Six-week-old ZDF rats were hyperinsulinemic but normoglycemic, and there were no differences in cardiomyocyte mechanical function, UDP-GIcNAc, O-GIcNAc, or OGT compared with age-matched lean control rats. Cardiomyocytes isolated from 22-wk-old hyperglycemic ZDF rats exhibited significantly impaired relaxation, compared with both age-matched lean control and 6-wk-old ZDF groups. There was also a significant increase in O-GIcNAc levels in high-molecular-mass proteins in the 22-wk-old ZDF group compared with age-matched lean control and 6-wk-old ZDF groups; this was associated with increased UDP- GIcNAc levels but not increased OGT expression. Surprisingly, there was a significant decrease in overall O-GIcNAc levels between 6 and 22 wk of age in lean, ZDF, and Sprague-Dawley rats that was associated with decreased OGT expression. These results support the notion that an increase in O-GIcNAc on specific proteins may contribute to impaired cardiomyocyte function in diabetes. However, this study also indicates that in the heart the level of O-GlcNAc on proteins appears to be differentially regulated by age and diabetes. [ABSTRACT FROM AUTHOR]

Abstract: There is growing recognition that the O-linked attachment of N-acetyl-glucosamine (O-GlcNAc) on serine and threonine residues of nuclear and cytoplasmic proteins is a highly dynamic post-translational modification that plays a key role in signal transduction pathways. Numerous proteins have been identified as targets of O-GlcNAc modifications including kinases, phosphatases, transcription factors, metabolic enzymes, chaperons, and cytoskeletal proteins. Modulation of O-GlcNAc levels has been shown to modify DNA binding, enzyme activity, protein–protein interactions, the half-life of proteins, and subcellular localization. The level of O-GlcNAc is regulated in part by the metabolism of glucose via the hexosamine biosynthesis pathway (HBP), and the metabolic abnormalities associated with insulin resistance and diabetes, such as hyperglycemia, hyperlipidemia, and hyperinsulinemia, are all associated with increased flux through the HBP and elevated O-GlcNAc levels. Increased HBP flux and O-GlcNAc levels have been implicated in the impaired relaxation of isolated cardiomyocytes, blunted response to angiotensin II and phenylephrine, hyperglycemia-induced cardiomyocyte apoptosis, and endothelial and vascular cell dysfunction. In contrast to these adverse effects, recent studies have also shown that O-GlcNAc levels increase in response to acute stress and that this is associated with increased cell survival. Thus, while the relationship between O-GlcNAc levels and cellular function is complex and not well-understood, it is clear that these pathways play a critical role in the regulation of cell function and survival in the cardiovascular system and may be implicated in the adverse effects of metabolic disease on the heart. [Copyright &y& Elsevier]

Increased levels of protein O-linked N-acetylglucosamine (O-GlcNAc) have been shown to increase cell survival following stress. Therefore, the goal of this study was to determine whether in isolated neonatal rat ventricular myocytes (NRVMs) an increase in protein O-GlcNAcylation resulted in improved survival and viability following ischemia-reperfusion (I/R). NRVMs were exposed to 4 h of ischemia and 16 h of reperfusion, and cell viability, necrosis, apoptosis, and O-GlcNAc levels were assessed. Treatment of cells with glucosamine, hyperglycemia, or O-(2-acetamido-2-deoxy-ᴅ-glucopyranosylidene)-amino-N-phenylcarbamate (PUGNAc), an inhibitor of O-GlcNAcase, significantly increased O-GlcNAc levels and improved cell viability, as well as reducing both necrosis and apoptosis compared with untreated cells following I/R. Alloxan, an inhibitor of O-GlcNAc transferase, markedly reduced O-GlcNAc levels and exacerbated I/R injury. The improved survival with hyperglycemia was attenuated by azaserine, which inhibits glucose metabolism via the hexosamine biosynthesis pathway. Reperfusion in the absence of glucose reduced O-GlcNAc levels on reperfusion compared with normal glucose conditions and decreased cell viability. O-GlcNAc levels significantly correlated with cell viability during reperfusion. The effects of glucosamine and PUGNAc on cellular viability were associated with reduced calcineurin activation as measured by translocation of nuclear factor of activated T cells, suggesting that increased O-GlcNAc levels may attenuate I/R induced increase in cytosolic Ca2+. These data support the concept that activation of metabolic pathways leading to an increase in O-GlcNAc levels is an endogenous stress-activated response and that augmentation of this response improves cell survival. Thus strategies designed to activate these pathways may represent novel interventions for inducing cardioprotection. [ABSTRACT FROM AUTHOR]

Abstract: Increased hexosamine biosynthesis pathway (HBP) flux and elevated levels of protein O-linked-N-acetylglucosamine (O-GlcNAc) decrease calcium influx into isolated cardiomyocytes. Increased O-GlcNAc levels also increase tolerance of cells to stress. Therefore, the goal of this study was to test the hypothesis that increasing HBP flux and protein O-GlcNAc levels in the intact heart will increase the tolerance to tissue injury resulting from the calcium paradox and ischemia. We used two strategies that have been shown to increase HBP flux in the intact heart, namely a brief period of streptozotocin-induced diabetes and acute pretreatment of the isolated perfused heart with glucosamine. Isolated perfused rat hearts were exposed to the calcium paradox or to ischemia and reperfusion. Both diabetes and glucosamine significantly improved recovery in the isolated perfused rat heart following the calcium paradox with left ventricular developed pressure (LVDP) returning to ~80% of baseline compared to 0% in controls (P <0.05), and lactate dehydrogenase release being reduced by approximately fivefold (P <0.05). In the diabetic group, azaserine, which inhibits the HBP, restored the sensitivity to the calcium paradox. Glucosamine treatment also improved functional recovery following ischemia/reperfusion (LVDP: 47±9% vs. 95±4%, P <0.05) and this was associated with a threefold increase in O-GlcNAc levels (P <0.05). Alloxan, an inhibitor of O-GlcNAc-transferase, blocked both the protection seen with glucosamine and the increase in O-GlcNAc. These data demonstrate that activation of the HBP with glucosamine may be a novel strategy for inducing cardioprotection, and that this appears to be mediated by an increase in protein O-GlcNAc levels. [Copyright &y& Elsevier]

We have previously identified in the human EST sequence data base four overlapping clones that could be aligned with both a predicted protein sequence, deduced from the C. elegans genomic sequence, and partial amino acid sequences, obtained for a protein from canine pancreatic microsomes. We suggested that these proteins are homologs of yeast microsomal and DnaJ-like protein Scj1p and termed them ERj3p. Here we verified the predicted protein sequence of human ERj3p by sequence analysis of the corresponding cDNA. Multiple alignment of related sequences identified these proteins as true homologs of yeast Scj1p. Biochemical analysis of the canine protein characterized ERj3p as a soluble glycoprotein of the pancreatic endoplasmic reticulum. This pancreatic DnaJ-like protein was shown to interact with lumenal DnaK-like proteins, such as BiP. Furthermore, we found that ERj3p interacts with SDF2L1 protein that may be involved in protein O-glycosylation. We propose that ERj3p represents a cochaperone of DnaK-like chaperones of the mammalian endoplasmic reticulum and is involved in folding and maturation of newly synthesized proteins. [ABSTRACT FROM AUTHOR]

The structure of ecto-5′-nucleotidase from bull seminal plasma, containing a glycosyl-phosphatidylinositol anchor, was studied using mass spectrometry. MALDI-MS analysis of intact protein indicated a mass of 65 568.2 Da for the monomeric form, and it also showed a heterogeneous population of glycoforms with the glycosidic moiety accounting for ≈ 6000 Da. MALDI-MS analysis showed that Asn53, Asn311, Asn333 and Asn403 were four sites of N-glycosylation. GC-MS analysis provided information on the glycosidic structures linked to the four asparagines. Asn53, Asn311 and Asn333 were linked to high-mannose saccharide chains, whereas the glycan chains linked to Asn403 contained a heterogeneous mixture of oligosaccharides, the high-mannose type structure being the most abundant and hybrid or complex type glycans being minor components. By combining enzymatic and/or chemical hydrolysis with GC-MS analysis, detailed characterization of the glycosyl-phpsphatidylinositol anchor was obtained. MALDI spectral analysis indicated that the glycosyl-phosphatidylinositol core contained EtN(P)Man3GlcNH2-myo-inositol(P)-glycerol, principally modified by stearoyl and palmitoyl residues or by stearoyl and myristoyl residues to a minor extent. Moreover, 1-palmitoylglycerol and 1-stearoylglycerol outweighed 2-palmitoylglycerol and 2-stearoylglycerol. The combination of chemical and enzymatic digestions of the protein with the mass spectral analysis yielded a complete pattern of S–S bridges. The protein does not contain free thiols and its eight cysteines are linked by intramolecular disulfide bonds, the pairs being: Cys51–Cys57, Cys353–Cys358, Cys365–Cys387 and Cys476–Cys479. This work resolves details of the structure of ecto-5′-nucleotidase, with particular regard to the localization and composition of the glycidic moiety, number and localization of the disulfide bridges and characterization of the... [ABSTRACT FROM AUTHOR]

The physiological role of protein O-glycosylation in prokaryotes is poorly understood due to our limited knowledge of the extent of their glycoproteomes. In Actinobacteria , defects in protein O-mannosyl transferase (Pmt)-mediated protein O-glycosylation have been shown to significantly retard growth ( Mycobacterium tuberculosis and Corynebacterium glutamicum ) or result in increased sensitivities to cell wall-targeting antibiotics ( Streptomyces coelicolor ), suggesting that protein O-glycosylation has an important role in cell physiology. Only a single glycoprotein (SCO4142, or PstS) has been identified to date in S. coelicolor Combining biochemical and mass spectrometry-based approaches, we have isolated and characterized the membrane glycoproteome in S. coelicolor A total of ninety-five high-confidence glycopeptides were identified which mapped to thirty-seven new S. coelicolor glycoproteins and a deeper understanding of glycosylation sites in PstS. Glycosylation sites were found to be modified with up to three hexose residues, consistent with what has been observed previously in other Actinobacteria S. coelicolor glycoproteins have diverse roles and functions, including solute binding, polysaccharide hydrolases, ABC transporters, and cell wall biosynthesis, the latter being of potential relevance to the antibiotic-sensitive phenotype of pmt mutants. Null mutants in genes encoding a putative d-Ala-d-Ala carboxypeptidase (SCO4847) and an l,d-transpeptidase (SCO4934) were hypersensitive to cell wall-targeting antibiotics. Additionally, the sco4847 mutants displayed an increased susceptibility to lysozyme treatment. These findings strongly suggest that both glycoproteins are required for maintaining cell wall integrity and that glycosylation could be affecting enzyme function. IMPORTANCE , defects in protein O-glycosylation have been shown to retard growth and result in hypersensitivity to cell wall-targeting antibiotics, suggesting that this modification is important for maintaining cell wall structure. Here, we have characterized the glycoproteome in Actinobacteria , defects in protein O-glycosylation have been shown to retard growth and result in hypersensitivity to cell wall-targeting antibiotics, suggesting that this modification is important for maintaining cell wall structure. Here, we have characterized the glycoproteome in Streptomyces coelicolor and shown that glycoproteins have diverse roles, including those related to solute binding, ABC transporters, and cell wall biosynthesis. We have generated mutants encoding two putative cell wall-active glycoproteins and shown them to be hypersensitive to cell wall-targeting antibiotics. These findings strongly suggest that both glycoproteins are required for maintaining cell wall integrity and that glycosylation affects enzyme function., (Copyright © 2019 Keenan et al.)

Periodontitis is a polymicrobial, biofilm-caused, inflammatory disease affecting the tooth-supporting tissues. It is not only the leading cause of tooth loss worldwide, but can also impact systemic health. The development of effective treatment strategies is hampered by the complicated disease pathogenesis which is best described by a polymicrobial synergy and dysbiosis model. This model classifies the Gram-negative anaerobe Tannerella forsythia as a periodontal pathogen, making it a prime candidate for interference with the disease. Tannerella forsythia employs a protein O -glycosylation system that enables high-density display of nonulosonic acids via the bacterium's two-dimensional crystalline cell surface layer. Nonulosonic acids are sialic acid-like sugars which are well known for their pivotal biological roles. This review summarizes the current knowledge of T. forsythia' s unique cell envelope with a focus on composition, biosynthesis and functional implications of the cell surface O -glycan. We have obtained evidence that glycobiology affects the bacterium's immunogenicity and capability to establish itself in the polymicrobial oral biofilm. Analysis of the genomes of different T. forsythia isolates revealed that complex protein O -glycosylation involving nonulosonic acids is a hallmark of pathogenic T. forsythia strains and, thus, constitutes a valuable target for the design of novel anti-infective strategies to combat periodontitis., Competing Interests: The authors have no competing interests.

We describe here the composition of the O-linked glycans on the Neurospora crassa cellobiohydrolase I (CBHI), which accounts for approximately 40% of the protein secreted by cells growing in the presence of cellulose. CBHI is O-glycosylated with six types of linear, and three types of branched, O-glycans containing approximately equal amounts of mannose and galactose. In addition to the classic fungal O-glycans with reducing end mannoses, we also identified reducing end galactoses which suggest the existence of a protein-O-galactosyltransferase in N. crassa Because of the excellent genetic resources available for N. crassa, the knowledge of the CBHI O-glycans may enable the future evaluation of the role of O-glycosylation on cellulase function and the development of directed O-glycan/cellulase engineering., (© The Author 2016. Published by Oxford University Press. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.)

Flagellin glycosylation impacts, in several documented cases, the functionality of bacterial flagella. The basis of flagellin glycosylation has been studied for various Gram-negative bacteria, but less is known about flagellin glycans of Gram-positive bacteria including Paenibacillus alvei, a secondary invader of honeybee colonies diseased with European foulbrood. Paenibacillus alvei CCM 2051(T) swarms vigorously on solidified culture medium, with swarming relying on functional flagella as evidenced by abolished biofilm formation of a non-motile P. alvei mutant defective in the flagellin protein Hag. Here, the glycobiology of the polar P. alvei flagella was investigated. Analysis on purified flagellin demonstrated that the 30-kDa Hag protein (PAV&#95;2c01710) is modified with an O-linked trisaccharide comprised of one hexose and two N-acetyl-hexosamine residues, at three sites of glycosylation. Downstream of the hag gene on the bacterial chromosome, two open reading frames (PAV&#95;2c01630, PAV&#95;2c01640) encoding putative glycosyltransferases were shown to constitute a flagellin glycosylation island. Mutants defective in these genes exhibited altered migration in sodium dodecyl sulfate polyacrylamide gel electrophoresis as well as loss of extracellular flagella production and bacterial motility. This study reveals that flagellin glycosylation in P. alvei is pivotal to flagella formation and bacterial motility in vivo, and simultaneously identifies flagella glycosylation as a second protein O-glycosylation system in this bacterium, in addition to the well-investigated S-layer tyrosine O-glycosylation pathway., Competing Interests: statement None declared., (© The Author 2015. Published by Oxford University Press. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.)

Arabinogalactan proteins are abundant cell surface proteoglycans in plants and are implicated to act as developmental markers during plant growth. We previously reported that AtGALT31A, AtGALT29A, and AtGLCAT14A-C, which are involved in the biosynthesis of arabinogalactan proteins, localize not only to the Golgi cisternae but also to smaller compartments, which may be a part of the unconventional protein secretory pathway in plants. In Poulsen et al., (1) we have demonstrated increased targeting of AtGALT29A to small compartments when Y144 is substituted with another amino acid, and we implicated a role for Y144 in the subcellular targeting of AtGALT29A. In this paper, we are presenting another aspect of Y144 substitution in AtGALT29A; namely, Y144A construct demonstrated a 2.5-fold increase while Y144E construct demonstrated a 2-fold decrease in the galactosyltransferase activity of AtGALT29A. Therefore, the electrostatic status of Y144, which is regulated by an unknown kinase/phosphatase system, may regulate AtGALT29A enzyme activity. Moreover, we have identified additional proteins, apyrase 3 (APY3; At1g14240) and UDP-glucuronate epimerases 1 and 6 (GAE1, At4g30440; GAE6, At3g23820), from Arabidopsis thaliana that co-localize with AtGALT31A in the small compartments when expressed transiently in Nicotiana benthamiana. These proteins may play roles in nucleotide sugar metabolism in the small compartments together with arabinogalactan glycosyltransferases.