Mucin-Type O-Glycosylation in the Drosophila Nervous System
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ABSTRACT:
SUBMITTER: Itoh K
PROVIDER: S-EPMC8558370 | biostudies-literature |
REPOSITORIES: biostudies-literature
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Project description:O-Glycosylation is one of the most important posttranslational modifications of proteins. It takes part in protein conformation, protein sorting, developmental processes and the modulation of enzymatic activities. In vertebrates, the basics of the biosynthetic pathway of O-glycans are already well understood. However, the regulation of the processes and the molecular aspects of defects, especially in correlation with cancer or developmental abnormalities, are still under investigation. The knowledge of the correlating invertebrate systems and evolutionary aspects of these highly conserved biosynthetic events may help improve the understanding of the regulatory factors of this pathway. Invertebrates display a broad spectrum of glycosylation varieties, providing an enormous potential for glycan modifications which may be used for the design of new pharmaceutically active substances. Here, overviews of the present knowledge of invertebrate mucin-type O-glycan structures and the currently identified enzymes responsible for the biosynthesis of these oligosaccharides are presented, and the few data dealing with functional aspects of O-glycans are summarised.
| S-EPMC6272458 | biostudies-literature
Project description:Mucin type O-glycosylation is a highly conserved form of post-translational modification initiated by the family of enzymes known as the polypeptide α-N-acetylgalactosaminyltransferases (ppGalNAcTs in mammals and PGANTs in Drosophila). To address the cellular functions of the many PGANT family members, RNA interference (RNAi) to each pgant gene was performed in two independent Drosophila cell culture lines. We demonstrate that RNAi to individual pgant genes results in specific reduction in gene expression without affecting the expression of other family members. Cells with reduced expression of individual pgant genes were then examined for changes in viability, morphology, adhesion, and secretion to assess the contribution of each family member to these cellular functions. Here we find that RNAi to pgant3, pgant6, or pgant7 resulted in reduced secretion, further supporting a role for O-glycosylation in proper secretion. Additionally, RNAi to pgant3 or pgant6 resulted in altered Golgi organization, suggesting a role for each in establishing or maintaining proper secretory apparatus structure. Other subcellular effects observed included multinucleated cells seen after RNAi to either pgant2 or pgant35A, suggesting a role for these genes in the completion of cytokinesis. These studies demonstrate the efficient and specific knockdown of pgant gene expression in two Drosophila cell culture systems, resulting in specific morphological and functional effects. Our work provides new information regarding the biological roles of O-glycosylation and illustrates a new platform for interrogating the cellular and subcellular effects of this form of post-translational modification.
| S-EPMC2966062 | biostudies-literature
Project description:Mucin-type O-glycosylation is an important post-translational modification that confers a variety of biological properties and functions to proteins. This post-translational modification has a particularly complex and differentially regulated biosynthesis rendering prediction and control of where O-glycans are attached to proteins, and which structures are formed, difficult. Because plants are devoid of GalNAc-type O-glycosylation, we have assessed requirements for establishing human GalNAc O-glycosylation de novo in plants with the aim of developing cell systems with custom-designed O-glycosylation capacity. Transient expression of a Pseudomonas aeruginosa Glc(NAc) C4-epimerase and a human polypeptide GalNAc-transferase in leaves of Nicotiana benthamiana resulted in GalNAc O-glycosylation of co-expressed human O-glycoprotein substrates. A chimeric YFP construct containing a 3.5 tandem repeat sequence of MUC1 was glycosylated with up to three and five GalNAc residues when co-expressed with GalNAc-T2 and a combination of GalNAc-T2 and GalNAc-T4, respectively, as determined by mass spectrometry. O-Glycosylation was furthermore demonstrated on a tandem repeat of MUC16 and interferon ?2b. In plants, prolines in certain classes of proteins are hydroxylated and further substituted with plant-specific O-glycosylation; unsubstituted hydroxyprolines were identified in our MUC1 construct. In summary, this study demonstrates that mammalian type O-glycosylation can be established in plants and that plants may serve as a host cell for production of recombinant O-glycoproteins with custom-designed O-glycosylation. The observed hydroxyproline modifications, however, call for additional future engineering efforts.
| S-EPMC3320939 | biostudies-literature
Project description:Changes in O-linked protein glycosylation are known to correlate with disease states but are difficult to monitor in a physiological setting because of a lack of experimental tools. Here, we report a technique for rapid profiling of O-linked glycoproteins in living animals by metabolic labeling with N-azidoacetylgalactosamine (GalNAz) followed by Staudinger ligation with phosphine probes. After injection of mice with a peracetylated form of GalNAz, azide-labeled glycoproteins were observed in a variety of tissues, including liver, kidney, and heart, in serum, and on isolated splenocytes. B cell glycoproteins were robustly labeled with GalNAz but T cell glycoproteins were not, suggesting fundamental differences in glycosylation machinery or metabolism. Furthermore, GalNAz-labeled B cells could be selectively targeted with a phosphine probe by Staudinger ligation within the living animal. Metabolic labeling with GalNAz followed by Staudinger ligation provides a means for proteomic analysis of this posttranslational modification and for identifying O-linked glycoprotein fingerprints associated with disease.
| S-EPMC1405625 | biostudies-literature
Project description:Acetylgalactosamine (GalNAc)-type O-glycosylation is an essential posttranslational modification (PTM) that plays fundamental roles in biology. Malfunction of this PTM is exemplified by the presence of truncated O-glycans in cancer. For instance, the glycoprotein MUC1 is overexpressed in many tumor tissues and tends to carry simple oligosaccharides that allow for the presentation of different tumor-associated antigens, such as the Tn or sTn antigens (GalNAc-α-1-O-Thr/Ser and Neu5Acα2-6GalNAcα1-O-Ser/Thr, respectively). In other cases, such as tumoral calcinosis associated with O-glycosylation of the fibroblast growth factor 23, O-glycans are absent or less abundant. Significant progress has been made in determining the three-dimensional structures of biomolecules that recognize GalNAc, such as antibodies, lectins, mucinases, GalNAc-transferases, and other glycosyltransferases. Analysis of the complexes between these entities and GalNAc-containing glycopeptides, in most cases derived from crystallographic or NMR analysis, provides an understanding of the key structural elements that control molecular recognition of these glycopeptides. Here, we describe and compare the binding sites of these proteins in detail, focusing on how the GalNAc moieties interact selectively with them. We also summarize the differences and similarities in GalNAc recognition. In general, the recognition of GalNAc-containing glycopeptides is determined by hydrogen bonds between hydroxyl groups and the N-acetyl group of GalNAc with proteins, as well as CH-π contacts in which the hydrophobic α-face of the sugar and the methyl group of NHAc can be involved. The latter interaction usually provides the basis for selectivity. It is worth noting that binding of these glycopeptides depends primarily on recognition of the sugar moiety, with some exceptions such as a few anti-MUC1 antibodies that primarily recognize the peptide backbone and use the sugar to facilitate shape complementarity or to establish a limited number of interactions with the protein. Focusing specifically on the GalNAc moiety, we can observe that there is some degeneracy of interactions within the same protein families, likely due to substrate flexibility. However, when all studied proteins are considered together, despite the commonalities within each protein family, no pattern can be discerned between the different families, apart from the presence of common residues such as Tyr, His, or Asp, which are responsible for hydrogen bonds. The lack of a pattern can be anticipated, given the diverse functions of mucinases, glycosyltransferases, antibodies, and lectins. Finally, it is important to point out that the conformational differences observed in solution in glycopeptides bearing GalNAc-α-1-O-Ser or GalNAc-α-1-O-Thr also can be found in the bound state. This unique characteristic is exploited, for instance, by the enzyme C1GalT1 to broadly glycosylate both acceptor substrates. The findings summarized in this review may contribute to the rational structure-guided development of therapeutic vaccines, novel diagnostic tools for early cancer detection, and new cancer treatments for cancer with tailored anti-Tn or anti-STn antibodies or new drugs to inhibit GalNAc-T isoenzymes.
| S-EPMC9996832 | biostudies-literature
Project description:Protein N-glycosylation can influence the nervous system in a variety of ways by affecting functions of glycoproteins involved in nervous system development and physiology. The importance of N-glycans for different aspects of neural development has been well documented. For example, some N-linked carbohydrate structures were found to play key roles in neural cell adhesion and axonal targeting during development. At the same time, the involvement of glycosylation in the regulation of neural physiology remains less understood. Recent studies have implicated N-glycosylation in the regulation of neural transmission, revealing novel roles of glycans in synaptic processes and the control of neural excitability. N-Glycans were found to markedly affect the function of several types of synaptic proteins involved in key steps of synaptic transmission, including neurotransmitter release, reception, and uptake. Glycosylation also regulates a number of channel proteins, such as TRP channels that control responses to environmental stimuli and voltage-gated ion channels, the principal determinants of neuronal excitability. Sialylated carbohydrate structures play a particularly prominent part in the modulation of voltage-gated ion channels. Sialic acids appear to affect channel functions via several mechanisms, including charge interactions, as well as other interactions that probably engage steric effects and interactions with other molecules. Experiments also indicated that some structural features of glycans can be particularly important for their function. Since glycan structures can vary significantly between different cell types and depend on the metabolic state of the cell, it is important to analyze glycan functions using in vivo approaches. While the complexity of the nervous system and intricacies of glycosylation pathways can create serious obstacles for in vivo experiments in vertebrates, recent studies have indicated that more simple and experimentally tractable model organisms like Drosophila should provide important advantages for elucidating evolutionarily conserved functions of N-glycosylation in the nervous system.
| S-EPMC4476505 | biostudies-literature
Project description:BACKGROUND:Increasing efforts have been made to assess the potential of Escherichia coli strains for the production of complex recombinant proteins. Since a considerable part of therapeutic proteins are glycoproteins, the lack of the post-translational attachment of sugar moieties in standard E. coli expression strains represents a major caveat, thus limiting the use of E. coli based cell factories. The establishment of an E. coli expression system capable of protein glycosylation could potentially facilitate the production of therapeutics with a putative concomitant reduction of production costs. RESULTS:The previously established E. coli strain expressing the soluble form of the functional human-derived glycosyltransferase polypeptide N-acetylgalactosaminyltransferase 2 (GalNAc-T2) was further modified by co-expressing the UDP-GlcNAc 4-epimerase WbgU derived from Plesiomonas shigelloides. This enables the conversion of uridine 5'-diphospho-N-acetylglucosamine (UDP-GlcNAc) to the sugar donor uridine 5'-diphospho-N-acetylgalactosamine (UDP-GalNAc) in the bacterial cytoplasm. Initially, the codon-optimised gene wbgU was inserted into a pET-derived vector and a Tobacco Etch Virus (TEV) protease cleavable polyhistidine-tag was translationally fused to the C- terminus of the amino acid sequence. The 4-epimerase was subsequently expressed and purified. Following the removal of the polyhistidine-tag, WbgU was analysed by circular dichroism spectroscopy to determine folding state and thermal transitions of the protein. The in vitro activity of WbgU was validated by employing a modified glycosyltransferase assay. The conversion of UDP-GlcNAc to UDP-GalNAc was shown by capillary electrophoresis analysis. Using a previously established chaperone pre-/co- expression platform, the in vivo activity of both glycosyltransferase GalNAc-T2 and 4-epimerase WbgU was assessed in E. coli, in combination with a mucin 10-derived target protein. Monitoring glycosylation by liquid chromatography electrospray ionization mass spectrometry (LC-ESI-MS), the results clearly indicated the in vivo glycosylation of the mucin-derived acceptor peptide. CONCLUSION:In the present work, the previously established E. coli- based expression system was further optimized and the potential for in vivo O-glycosylation was shown by demonstrating the transfer of sugar moieties to a mucin-derived acceptor protein. The results offer the possibility to assess the practical use of the described expression platform for in vivo glycosylations of important biopharmaceutical compounds in E. coli.
| S-EPMC6202839 | biostudies-literature
Project description:Mucin-type O-glycosylation is one of the most common posttranslational modifications of proteins. The abnormal expression of various polypeptide GalNAc-transferases (GalNAc-Ts) which initiate and define sites of O-glycosylation are linked to many cancers and other diseases. Current O-glycosyation prediction programs utilize O-glycoproteomics data obtained without regard to the transferase isoform (s) responsible for the glycosylation. With 20 different GalNAc-Ts in humans, having an ability to predict and interpret O-glycosylation sites in terms of specific GalNAc-T isoforms is invaluable. To fill this gap, ISOGlyP (Isoform-Specific O-Glycosylation Prediction) has been developed. Using position-specific enhancement values generated based on GalNAc-T isoform-specific amino acid preferences, ISOGlyP predicts the propensity that a site would be glycosylated by a specific transferase. ISOGlyP gave an overall prediction accuracy of 70% against in vivo data, which is comparable to that of the NetOGlyc4.0 predictor. Additionally, ISOGlyP can identify the known effects of long- and short-range prior glycosylation and can generate potential peptide sequences selectively glycosylated by specific isoforms. ISOGlyP is freely available for use at ISOGlyP.utep.edu. The code is also available on GitHub (https://github.com/jonmohl/ISOGlyP).
| S-EPMC8022967 | biostudies-literature
Project description:Chromatin organization and gene activity are responsive to developmental and environmental cues. Although many genes are transcribed throughout development and across cell types, much of gene regulation is highly cell-type specific. To readily track chromatin features at the resolution of cell types within complex tissues, we developed and validated chromatin affinity purification from specific cell types by chromatin immunoprecipitation (CAST-ChIP), a broadly applicable biochemical procedure. RNA polymerase II (Pol II) CAST-ChIP identifies ~1,500 neuronal and glia-specific genes in differentiated cells within the adult Drosophila brain. In contrast, the histone H2A.Z is distributed similarly across cell types and throughout development, marking cell-type-invariant Pol II-bound regions. Our study identifies H2A.Z as an active chromatin signature that is refractory to changes across cell fates. Thus, CAST-ChIP powerfully identifies cell-type-specific as well as cell-type-invariant chromatin states, enabling the systematic dissection of chromatin structure and gene regulation within complex tissues such as the brain.
| S-EPMC5877786 | biostudies-literature
Project description:In vertebrates, sialylated glycans participate in a wide range of biological processes and affect the development and function of the nervous system. While the complexity of glycosylation and the functional redundancy among sialyltransferases provide obstacles for revealing biological roles of sialylation in mammals, Drosophila possesses a sole vertebrate-type sialyltransferase, Drosophila sialyltransferase (DSiaT), with significant homology to its mammalian counterparts, suggesting that Drosophila could be a suitable model to investigate the function of sialylation. To explore this possibility and investigate the role of sialylation in Drosophila, we inactivated DSiaT in vivo by gene targeting and analyzed phenotypes of DSiaT mutants using a combination of behavioral, immunolabeling, electrophysiological, and pharmacological approaches. Our experiments demonstrated that DSiaT expression is restricted to a subset of CNS neurons throughout development. We found that DSiaT mutations result in significantly decreased life span, locomotor abnormalities, temperature-sensitive paralysis, and defects of neuromuscular junctions. Our results indicate that DSiaT regulates neuronal excitability and affects the function of a voltage-gated sodium channel. Finally, we showed that sialyltransferase activity is required for DSiaT function in vivo, which suggests that DSiaT mutant phenotypes result from a defect in sialylation of N-glycans. This work provided the first evidence that sialylation has an important biological function in protostomes, while also revealing a novel, nervous system-specific function of alpha2,6-sialylation. Thus, our data shed light on one of the most ancient functions of sialic acids in metazoan organisms and suggest a possibility that this function is evolutionarily conserved between flies and mammals.
| S-EPMC3354699 | biostudies-literature