RESUMEN
Protein O-linked mannose (O-Man) glycosylation is an evolutionary conserved posttranslational modification that fulfills important biological roles during embryonic development. Three nonredundant enzyme families, POMT1/POMT2, TMTC1-4, and TMEM260, selectively coordinate the initiation of protein O-Man glycosylation on distinct classes of transmembrane proteins, including α-dystroglycan, cadherins, and plexin receptors. However, a systematic investigation of their substrate specificities is lacking, in part due to the ubiquitous expression of O-Man glycosyltransferases in cells, which precludes analysis of pathway-specific O-Man glycosylation on a proteome-wide scale. Here, we apply a targeted workflow for membrane glycoproteomics across five human cell lines to extensively map O-Man substrates and genetically deconstruct O-Man initiation by individual and combinatorial knockout of O-Man glycosyltransferase genes. We established a human cell library for the analysis of substrate specificities of individual O-Man initiation pathways by quantitative glycoproteomics. Our results identify 180 O-Man glycoproteins, demonstrate new protein targets for the POMT1/POMT2 pathway, and show that TMTC1-4 and TMEM260 pathways widely target distinct Ig-like protein domains of plasma membrane proteins involved in cell-cell and cell-extracellular matrix interactions. The identification of O-Man on Ig-like folds adds further knowledge on the emerging concept of domain-specific O-Man glycosylation which opens for functional studies of O-Man-glycosylated adhesion molecules and receptors.
Asunto(s)
Manosa , Humanos , Glicosilación , Manosa/metabolismo , Especificidad por Sustrato , Glicoproteínas/metabolismo , Proteómica/métodos , Línea Celular , Glicosiltransferasas/metabolismo , Glicosiltransferasas/genética , Procesamiento Proteico-Postraduccional , Ingeniería Celular/métodosRESUMEN
Microalgae are a renewable and promising biomass for large-scale biofuel, food and nutrient production. However, their efficient exploitation depends on our knowledge of the cell wall composition and organization as it can limit access to high-value molecules. Here we provide an atomic-level model of the non-crystalline and water-insoluble glycoprotein-rich cell wall of Chlamydomonas reinhardtii. Using in situ solid-state and sensitivity-enhanced nuclear magnetic resonance, we reveal unprecedented details on the protein and carbohydrate composition and their nanoscale heterogeneity, as well as the presence of spatially segregated protein- and glycan-rich regions with different dynamics and hydration levels. We show that mannose-rich lower-molecular-weight proteins likely contribute to the cell wall cohesion by binding to high-molecular weight protein components, and that water provides plasticity to the cell-wall architecture. The structural insight exemplifies strategies used by nature to form cell walls devoid of cellulose or other glycan polymers.
Asunto(s)
Chlamydomonas reinhardtii , Chlamydomonas , Chlamydomonas reinhardtii/metabolismo , Glicoproteínas/metabolismo , Pared Celular/metabolismo , Celulosa/metabolismo , Agua/metabolismoRESUMEN
Mutations in the TMEM260 gene cause structural heart defects and renal anomalies syndrome, but the function of the encoded protein remains unknown. We previously reported wide occurrence of O-mannose glycans on extracellular immunoglobulin, plexin, transcription factor (IPT) domains found in the hepatocyte growth factor receptor (cMET), macrophage-stimulating protein receptor (RON), and plexin receptors, and further demonstrated that two known protein O-mannosylation systems orchestrated by the POMT1/2 and transmembrane and tetratricopeptide repeat-containing proteins 1-4 gene families were not required for glycosylation of these IPT domains. Here, we report that the TMEM260 gene encodes an ER-located protein O-mannosyltransferase that selectively glycosylates IPT domains. We demonstrate that disease-causing TMEM260 mutations impair O-mannosylation of IPT domains and that TMEM260 knockout in cells results in receptor maturation defects and abnormal growth of 3D cell models. Thus, our study identifies the third protein-specific O-mannosylation pathway in mammals and demonstrates that O-mannosylation of IPT domains serves critical functions during epithelial morphogenesis. Our findings add a new glycosylation pathway and gene to a growing group of congenital disorders of glycosylation.
Asunto(s)
Manosa , Manosiltransferasas , Animales , Glicosilación , Mamíferos/metabolismo , Manosa/metabolismo , Manosiltransferasas/genética , Manosiltransferasas/metabolismoRESUMEN
Small molecule inhibitors of glycosylation enzymes are valuable tools for dissecting glycan functions and potential drug candidates. Screening for inhibitors of glycosyltransferases are mainly performed by in vitro enzyme assays with difficulties moving candidates to cells and animals. Here, we circumvent this by employing a cell-based screening assay using glycoengineered cells expressing tailored reporter glycoproteins. We focused on GalNAc-type O-glycosylation and selected the GalNAc-T11 isoenzyme that selectively glycosylates endocytic low-density lipoprotein receptor (LDLR)-related proteins as targets. Our screen of a limited small molecule compound library did not identify selective inhibitors of GalNAc-T11, however, we identify two compounds that broadly inhibited Golgi-localized glycosylation processes. These compounds mediate the reversible fragmentation of the Golgi system without affecting secretion. We demonstrate how these inhibitors can be used to manipulate glycosylation in cells to induce expression of truncated O-glycans and augment binding of cancer-specific Tn-glycoprotein antibodies and to inhibit expression of heparan sulfate and binding and infection of SARS-CoV-2.
Asunto(s)
COVID-19 , SARS-CoV-2 , Animales , Glicosilación , SARS-CoV-2/metabolismo , Glicoproteínas/metabolismo , Polisacáridos/metabolismoRESUMEN
Mass spectrometry (MS) easily detects C-mannosylated peptides from purified proteins but not from complex biological samples. Enrichment of specific glycopeptides by lectin affinity prior to MS analysis has been widely applied to support glycopeptide identification but was until now not available for C-mannosylated peptides. Here, we used the α-mannose-specific Burkholderia cenocepacia lectin A (BC2L-A) and show that, in addition to its previously demonstrated high-mannose N-glycan binding capability, this lectin is able to retain C- and O-mannosylated peptides. Besides testing binding abilities to standard peptides, we applied BC2L-A affinity to enrich C-mannosylated peptides from complex samples of tryptic digests of HEK293 and MCF10A whole cell extracts, which led to the identification of novel C-mannosylation sites. In conclusion, BC2L-A enabled specific enrichment of C- and O-mannosylated peptides and might have superior properties over other mannose binding lectins for this purpose.
Asunto(s)
Burkholderia cenocepacia , Manosa , Burkholderia cenocepacia/química , Burkholderia cenocepacia/metabolismo , Glicopéptidos/metabolismo , Glicosilación , Células HEK293 , Humanos , Lectinas/química , Manosa/químicaRESUMEN
Mucins are a large family of heavily O-glycosylated proteins that cover all mucosal surfaces and constitute the major macromolecules in most body fluids. Mucins are primarily defined by their variable tandem repeat (TR) domains that are densely decorated with different O-glycan structures in distinct patterns, and these arguably convey much of the informational content of mucins. Here, we develop a cell-based platform for the display and production of human TR O-glycodomains (~200 amino acids) with tunable structures and patterns of O-glycans using membrane-bound and secreted reporters expressed in glycoengineered HEK293 cells. Availability of defined mucin TR O-glycodomains advances experimental studies into the versatile role of mucins at the interface with pathogenic microorganisms and the microbiome, and sparks new strategies for molecular dissection of specific roles of adhesins, glycoside hydrolases, glycopeptidases, viruses and other interactions with mucin TRs as highlighted by examples.
Asunto(s)
Mucinas/metabolismo , Membrana Mucosa/metabolismo , Polisacáridos/genética , Polisacáridos/metabolismo , Ingeniería Genética , Glicosilación , Células HEK293 , Humanos , Microbiota , Mucina-1/genética , Mucina-1/metabolismoRESUMEN
The reversible posttranslational O-GlcNAc modification of serine or threonine residues of intracellular proteins is involved in many cellular events from signaling cascades to epigenetic and transcriptional regulation. O-GlcNAcylation is a conserved nutrient-dependent process involving two enzymes, with O-GlcNAc transferase (OGT) adding O-GlcNAc and with O-GlcNAcase (OGA) removing it in a manner that's protein- and context-dependent. O-GlcNAcylation is essential for epigenetic regulation of gene expression through its action on Polycomb and Trithorax and COMPASS complexes. However, the important role of O-GlcNAc in adult life and health span has been largely unexplored, mainly due the lack of available model systems. Cataloging the O-GlcNAc proteome has proven useful in understanding the biology of this modification in vivo. In this study, we leveraged a recently developed oga knockout fly mutant to identify the O-GlcNAcylated proteins in adult Drosophilamelanogaster. The adult O-GlcNAc proteome revealed many proteins related to cell and organismal growth, development, differentiation, and epigenetics. We identified many O-GlcNAcylated proteins that play a role in increased growth and decreased longevity, including HCF, SIN3A, LOLA, KISMET, ATX2, SHOT, and FOXO. Interestingly, oga mutant flies are larger and have a shorter life span compared to wild type flies, suggesting increased O-GlcNAc results in increased growth. Our results suggest that O-GlcNAc alters the function of many proteins related to transcription, epigenetic modification and signaling pathways that regulate growth rate and longevity. Therefore, our findings highlight the importance of O-GlcNAc in growth and life span in adult Drosophila.
Asunto(s)
Drosophila melanogaster/enzimología , Drosophila melanogaster/crecimiento & desarrollo , Glicoproteínas/metabolismo , Longevidad , Mutación/genética , Proteoma/metabolismo , beta-N-Acetilhexosaminidasas/genética , Animales , Tamaño Corporal , Proteínas de Drosophila/metabolismo , Drosophila melanogaster/anatomía & histología , Femenino , Ontología de Genes , N-Metiltransferasa de Histona-Lisina/metabolismo , Masculino , Fenotipo , Cromosomas Politénicos/metabolismo , Alas de Animales/enzimologíaRESUMEN
Aberrant mucin-type O-linked glycosylation is a common occurrence in cancer where the upregulation of sialyltransferases is often seen leading to the early termination of O-glycan chains. Mucin-type O-linked glycosylation is not limited to mucins and occurs on many cell surface glycoproteins including EGFR, where the number of sites can be limited. Upon EGF ligation, EGFR induces a signaling cascade and may also translocate to the nucleus where it directly regulates gene transcription, a process modulated by Galectin-3 and MUC1 in some cancers. Here, we show that upon EGF binding, breast cancer cells carrying different O-glycans respond by transcribing different gene expression signatures. MMP10, the principal gene upregulated when cells carrying sialylated core 1 glycans were stimulated with EGF, is also upregulated in ER-positive breast carcinoma reported to express high levels of ST3Gal1 and hence mainly core 1 sialylated O-glycans. In contrast, isogenic cells engineered to carry core 2 glycans upregulate CX3CL1 and FGFBP1 and these genes are upregulated in ER-negative breast carcinomas, also known to express longer core 2 O-glycans. Changes in O-glycosylation did not significantly alter signal transduction downstream of EGFR in core 1 or core 2 O-glycan expressing cells. However, striking changes were observed in the formation of an EGFR/galectin-3/MUC1/ß-catenin complex at the cell surface that is present in cells carrying short core 1-based O-glycans but absent in core 2 carrying cells.
Asunto(s)
Neoplasias de la Mama/metabolismo , Mucina-1/metabolismo , Neoplasias de la Mama/patología , Receptores ErbB/metabolismo , Femenino , Glicosilación , Humanos , Receptores de Estrógenos/metabolismoRESUMEN
Protein glycosylation plays essential roles in protein structure, stability, and activity such as cell adhesion. The cadherin superfamily of adhesion molecules carry O-linked mannose glycans at conserved sites and it was recently demonstrated that the transmembrane and tetratricopeptide repeat-containing proteins 1-4 (TMTC1-4) gene products contribute to the addition of these O-linked mannoses. Here, biochemical, cell biological, and organismal analysis was used to determine that TMTC3 supports the O-mannosylation of E-cadherin, cellular adhesion, and embryonic gastrulation. Using genetically engineered cells lacking all four TMTC genes, overexpression of TMTC3 rescued O-linked glycosylation of E-cadherin and cell adherence. The knockdown of the Tmtcs in Xenopus laevis embryos caused a delay in gastrulation that was rescued by the addition of human TMTC3. Mutations in TMTC3 have been linked to neuronal cell migration diseases including Cobblestone lissencephaly. Analysis of TMTC3 mutations associated with Cobblestone lissencephaly found that three of the variants exhibit reduced stability and missence mutations were unable to complement TMTC3 rescue of gastrulation in Xenopus embryo development. Our study demonstrates that TMTC3 regulates O-linked glycosylation and cadherin-mediated adherence, providing insight into its effect on cellular adherence and migration, as well the basis of TMTC3-associated Cobblestone lissencephaly.
Asunto(s)
Cadherinas/metabolismo , Proteínas Portadoras/metabolismo , Retículo Endoplásmico/metabolismo , Proteínas de la Membrana/metabolismo , Animales , Células COS , Proteínas Portadoras/genética , Adhesión Celular/fisiología , Moléculas de Adhesión Celular/metabolismo , Chlorocebus aethiops , Gastrulación/fisiología , Glicosilación , Células HEK293 , Humanos , Manosa/metabolismo , Proteínas de la Membrana/genética , Mutación , Neuronas/citología , Neuronas/metabolismo , Xenopus laevisRESUMEN
Most proteins trafficking the secretory pathway of metazoan cells will acquire GalNAc-type O-glycosylation. GalNAc-type O-glycosylation is differentially regulated in cells by the expression of a repertoire of up to twenty genes encoding polypeptide GalNAc-transferase isoforms (GalNAc-Ts) that initiate O-glycosylation. These GalNAc-Ts orchestrate the positions and patterns of O-glycans on proteins in coordinated, but poorly understood ways - guided partly by the kinetic properties and substrate specificities of their catalytic domains, as well as by modulatory effects of their unique GalNAc-binding lectin domains. Here, we provide the hereto most comprehensive characterization of nonredundant contributions of individual GalNAc-T isoforms to the O-glycoproteome of the human HEK293 cell using quantitative differential O-glycoproteomics on a panel of isogenic HEK293 cells with knockout of GalNAc-T genes (GALNT1, T2, T3, T7, T10, or T11). We confirm that a major part of the O-glycoproteome is covered by redundancy, whereas distinct O-glycosite subsets are covered by nonredundant GalNAc-T isoform-specific functions. We demonstrate that the GalNAc-T7 and T10 isoforms function in follow-up of high-density O-glycosylated regions, and that GalNAc-T11 has highly restricted functions and essentially only serves the low-density lipoprotein-related receptors in linker regions (C6XXXTC1) between the ligand-binding repeats.
Asunto(s)
Glicómica , Proteómica , Glicopéptidos/metabolismo , Glicosilación , Células HEK293 , Células Hep G2 , Humanos , Proteoma/metabolismoRESUMEN
Protein O-mannosylation (O-Man), originally discovered in yeast five decades ago, is an important post-translational modification (PTM) conserved from bacteria to humans, but not found in plants or nematodes. Until recently, the homologous family of ER-located protein O-mannosyl transferases (PMT1-7 in yeast; POMT1/POMT2 in humans), were the only known enzymes involved in directing O-Man biosynthesis in eukaryotes. However, recent studies demonstrate the existence of multiple distinct O-Man glycosylation pathways indicating that the genetic and biosynthetic regulation of O-Man in eukaryotes is more complex than previously envisioned. Introduction of sensitive glycoproteomics strategies provided an expansion of O-Man glycoproteomes in eukaryotes (yeast and mammalian cell lines) leading to the discovery of O-Man glycosylation on important mammalian cell adhesion (cadherin superfamily) and signaling (plexin family) macromolecules, and to the discovery of unique nucleocytoplasmic O-Man glycosylation in yeast. It is now evident that eukaryotes have multiple distinct O-Man glycosylation pathways including: i) the classical PMT1-7 and POMT1/POMT2 pathway conserved in all eukaryotes apart from plants; ii) a yet uncharacterized nucleocytoplasmic pathway only found in yeast; iii) an ER-located pathway directed by the TMTC1-4 genes found in metazoans and protists and primarily dedicated to the cadherin superfamily; and iv) a yet uncharacterized pathway found in metazoans primarily dedicated to plexins. O-Man glycosylation is thus emerging as a much more widespread and evolutionary diverse PTM with complex genetic and biosynthetic regulation. While deficiencies in the POMT1/POMT2 O-Man pathway underlie muscular dystrophies, the TMTC1-4 pathway appear to be involved in distinct congenital disorders with neurodevelopmental phenotypes. Here, we review and discuss the recent discoveries of the new non-classical O-Man glycosylation pathways, their substrates, functions and roles in disease.
Asunto(s)
Eucariontes/metabolismo , Manosa/metabolismo , Oxígeno/metabolismo , Glicoproteínas/química , Glicoproteínas/metabolismo , Glicosilación , Dominios ProteicosRESUMEN
Successful application of potent antibody-based T-cell engaging immunotherapeutic strategies is currently limited mainly to hematological cancers. One major reason is the lack of well-characterized antigens on solid tumors with sufficient cancer specific expression. Aberrantly O-glycosylated proteins contain promising cancer-specific O-glycopeptide epitopes suitable for immunotherapeutic applications, but currently only few examples of such antibody epitopes have been identified. We previously showed that chimeric antigen receptor T-cells directed towards aberrantly O-glycosylated MUC1 can control malignant growth in a mouse model. Here, we present a discovery platform for the generation of cancer-specific monoclonal antibodies targeting aberrant O-glycoproteins. The strategy is based on cancer cell lines engineered to homogeneously express the truncated Tn O-glycoform, the so-called SimpleCells. We used SimpleCells of different cancer origin to elicit monoclonal antibodies with selectivity for aberrant O-glycoproteins. For validation we selected and characterized one monoclonal antibody (6C5) directed to a Tn-glycopeptide in dysadherin (FXYD5), known to be upregulated in cancer and promote metastasis. While dysadherin is widely expressed also in normal cells, we demonstrated that the 6C5 epitope is specifically expressed in cancer.
Asunto(s)
Anticuerpos Monoclonales/análisis , Anticuerpos Monoclonales/biosíntesis , Glicoproteínas/metabolismo , Neoplasias/metabolismo , Animales , Anticuerpos Monoclonales/inmunología , Línea Celular Tumoral , Epítopos/inmunología , Epítopos/metabolismo , Glicoproteínas/inmunología , Humanos , Ratones , Neoplasias/inmunología , Neoplasias/patologíaAsunto(s)
Alérgenos/inmunología , Células Presentadoras de Antígenos/inmunología , Antígenos de Plantas/inmunología , Desensibilización Inmunológica , Linfocitos T/inmunología , Vacunas , Animales , Células CHO , Cricetulus , Escherichia coli/genética , Femenino , Ingeniería Genética , Glicosilación , Humanos , Ratones Endogámicos BALB C , Pichia/genéticaRESUMEN
O-glycosylation is one of the most abundant and diverse types of post-translational modifications of proteins. O-glycans modulate the structure, stability, and function of proteins and serve generalized as well as highly specific roles in most biological processes. This ShapShot presents types of O-glycans found in different organisms and their principle biosynthetic pathways. To view this SnapShot, open or download the PDF.
Asunto(s)
Evolución Molecular , Procesamiento Proteico-Postraduccional , Animales , Bacterias/genética , Bacterias/metabolismo , Drosophila/genética , Drosophila/metabolismo , Hongos/genética , Hongos/metabolismo , Glicosilación , Nematodos/genética , Nematodos/metabolismo , Plantas/metabolismo , Vertebrados/genética , Vertebrados/metabolismoRESUMEN
G protein-coupled receptors (GPCRs) are an important protein family of signalling receptors that govern a wide variety of physiological functions. The capacity to transmit extracellular signals and the extent of cellular response are largely determined by the amount of functional receptors at the cell surface that is subject to complex and fine-tuned regulation. Here, we demonstrate that the cell surface expression level of an inhibitory GPCR, the human δ-opioid receptor (hδOR) involved in pain and mood regulation, is modulated by site-specific N-acetylgalactosamine (GalNAc) -type O-glycosylation. Importantly, we identified one out of the 20 polypeptide GalNAc-transferase isoforms, GalNAc-T2, as the specific regulator of O-glycosylation of Ser6, Ser25 and Ser29 in the N-terminal ectodomain of the receptor. This was demonstrated by in vitro glycosylation assays using peptides corresponding to the hδOR N-terminus, Vicia villosa lectin affinity purification of receptors expressed in HEK293 SimpleCells capable of synthesizing only truncated O-glycans, GalNAc-T edited cell line model systems, and site-directed mutagenesis of the putative O-glycosylation sites. Interestingly, a single-nucleotide polymorphism, at residue 27 (F27C), was found to alter O-glycosylation of the receptor in efficiency as well as in glycosite usage. Furthermore, flow cytometry and cell surface biotinylation assays using O-glycan deficient CHO-ldlD cells revealed that the absence of O-glycans results in decreased receptor levels at the plasma membrane due to enhanced turnover. In addition, mutation of the identified O-glycosylation sites led to a decrease in the number of ligand-binding competent receptors and impaired agonist-mediated inhibition of cyclic AMP accumulation in HEK293 cells. Thus, site-specific O-glycosylation by a selected GalNAc-T isoform can increase the stability of a GPCR, in a process that modulates the constitutive turnover and steady-state levels of functional receptors at the cell surface.
Asunto(s)
Acetilgalactosamina/metabolismo , N-Acetilgalactosaminiltransferasas/metabolismo , Procesamiento Proteico-Postraduccional , Receptores Opioides delta/química , Proteínas Recombinantes de Fusión/química , Serina/metabolismo , Acetilgalactosamina/química , Secuencia de Aminoácidos , Animales , Células CHO , Línea Celular Tumoral , Membrana Celular/química , Membrana Celular/metabolismo , Cromatografía de Afinidad/métodos , Cricetulus , AMP Cíclico/metabolismo , Glicosilación , Células HEK293 , Células Hep G2 , Humanos , Mutagénesis Sitio-Dirigida , N-Acetilgalactosaminiltransferasas/genética , Neuronas/citología , Neuronas/metabolismo , Péptidos/síntesis química , Péptidos/metabolismo , Lectinas de Plantas/química , Polimorfismo de Nucleótido Simple , Estabilidad Proteica , Receptores Opioides delta/genética , Receptores Opioides delta/metabolismo , Proteínas Recombinantes de Fusión/genética , Proteínas Recombinantes de Fusión/metabolismo , Alineación de Secuencia , Polipéptido N-AcetilgalactosaminiltransferasaRESUMEN
The GlycoDomainViewer is a bioinformatic tool to aid in the mining of glycoproteomic datasets from different sources and facilitate incorporation of glycosylation into studies of protein structure and function. We present a version 2.0 of GlycoDomainViewer incorporating a number of advanced features, which enhances visibility and accessibility of the wealth of glycoproteomic data being generated. The GlycoDomainViewer enables visual exploration of glycoproteomic data, incorporating information from recent N- and O-glycoproteome studies on human and animal cell lines and some organs and body fluids. The initial data comprises sites of glycosylation for N-linked, O-GalNAc, O-Fucose, O-Xyl, O-Mannose (in both human and yeast) and cytosolic O-GlcNAc type. The data made available via this tool will be regularly updated to improve the coverage of known glycosylation sites and datasets, reflecting the advances currently being made in characterization of glycoproteomes. The tool is available at https://glycodomain.glycomics.ku.dk.
Asunto(s)
Glicoproteínas/análisis , Glicoproteínas/química , Proteoma/análisis , Proteoma/química , Proteómica/métodos , Animales , Línea Celular , Glicoproteínas/metabolismo , Glicosilación , Humanos , Proteoma/metabolismoRESUMEN
The cadherin (cdh) superfamily of adhesion molecules carry O-linked mannose (O-Man) glycans at highly conserved sites localized to specific ß-strands of their extracellular cdh (EC) domains. These O-Man glycans do not appear to be elongated like O-Man glycans found on α-dystroglycan (α-DG), and we recently demonstrated that initiation of cdh/protocadherin (pcdh) O-Man glycosylation is not dependent on the evolutionary conserved POMT1/POMT2 enzymes that initiate O-Man glycosylation on α-DG. Here, we used a CRISPR/Cas9 genetic dissection strategy combined with sensitive and quantitative O-Man glycoproteomics to identify a homologous family of four putative protein O-mannosyltransferases encoded by the TMTC1-4 genes, which were found to be imperative for cdh and pcdh O-Man glycosylation. KO of all four TMTC genes in HEK293 cells resulted in specific loss of cdh and pcdh O-Man glycosylation, whereas combined KO of TMTC1 and TMTC3 resulted in selective loss of O-Man glycans on specific ß-strands of EC domains, suggesting that each isoenzyme serves a different function. In addition, O-Man glycosylation of IPT/TIG domains of plexins and hepatocyte growth factor receptor was not affected in TMTC KO cells, suggesting the existence of yet another O-Man glycosylation machinery. Our study demonstrates that regulation of O-mannosylation in higher eukaryotes is more complex than envisioned, and the discovery of the functions of TMTCs provide insight into cobblestone lissencephaly caused by deficiency in TMTC3.
Asunto(s)
Cadherinas/metabolismo , Proteínas Portadoras/genética , Glicosiltransferasas/genética , Manosa/metabolismo , Proteínas de la Membrana/genética , Proteínas Portadoras/metabolismo , Glicosilación , Glicosiltransferasas/metabolismo , Células HEK293 , Humanos , Proteínas de la Membrana/metabolismo , Familia de MultigenesRESUMEN
Many Gram-negative bacterial pathogens use a syringe-like apparatus called a type III secretion system to inject virulence factors into host cells. Some of these effectors are enzymes that modify host proteins to subvert their normal functions. NleB is a glycosyltransferase that modifies host proteins with N-acetyl-d-glucosamine to inhibit antibacterial and inflammatory host responses. NleB is conserved among the attaching/effacing pathogens enterohemorrhagic Escherichia coli (EHEC), enteropathogenic E. coli (EPEC), and Citrobacter rodentium Moreover, Salmonella enterica strains encode up to three NleB orthologs named SseK1, SseK2, and SseK3. However, there are conflicting reports regarding the activities and host protein targets among the NleB/SseK orthologs. Therefore, here we performed in vitro glycosylation assays and cell culture experiments to compare the activities and substrate specificities of these effectors. SseK1, SseK3, EHEC NleB1, EPEC NleB1, and Crodentium NleB blocked TNF-mediated NF-κB pathway activation, whereas SseK2 and NleB2 did not. C. rodentium NleB, EHEC NleB1, and SseK1 glycosylated host GAPDH. C. rodentium NleB, EHEC NleB1, EPEC NleB1, and SseK2 glycosylated the FADD (Fas-associated death domain protein). SseK3 and NleB2 were not active against either substrate. We also found that EHEC NleB1 glycosylated two GAPDH arginine residues, Arg197 and Arg200, and that these two residues were essential for GAPDH-mediated activation of TNF receptor-associated factor 2 ubiquitination. These results provide evidence that members of this highly conserved family of bacterial virulence effectors target different host protein substrates and exhibit distinct cellular modes of action to suppress host responses.
Asunto(s)
Proteínas Bacterianas/metabolismo , Citrobacter rodentium/enzimología , Escherichia coli Enterohemorrágica/enzimología , Escherichia coli Enteropatógena/enzimología , Proteínas de Escherichia coli/metabolismo , Salmonella enterica/enzimología , Factores de Virulencia/metabolismo , Animales , Proteínas Bacterianas/genética , Citrobacter rodentium/genética , Citrobacter rodentium/patogenicidad , Escherichia coli Enterohemorrágica/genética , Escherichia coli Enterohemorrágica/patogenicidad , Escherichia coli Enteropatógena/genética , Escherichia coli Enteropatógena/patogenicidad , Proteínas de Escherichia coli/genética , Proteína de Dominio de Muerte Asociada a Fas/genética , Proteína de Dominio de Muerte Asociada a Fas/metabolismo , Gliceraldehído-3-Fosfato Deshidrogenasas/genética , Gliceraldehído-3-Fosfato Deshidrogenasas/metabolismo , Glicosilación , Ratones , Células RAW 264.7 , Receptores Tipo II del Factor de Necrosis Tumoral/genética , Receptores Tipo II del Factor de Necrosis Tumoral/metabolismo , Salmonella enterica/genética , Salmonella enterica/patogenicidad , Factor 2 Asociado a Receptor de TNF/genética , Factor 2 Asociado a Receptor de TNF/metabolismo , Ubiquitinación , Factores de Virulencia/genéticaRESUMEN
Protein O-mannosylation is found in yeast and metazoans, and a family of conserved orthologous protein O-mannosyltransferases is believed to initiate this important post-translational modification. We recently discovered that the cadherin superfamily carries O-linked mannose (O-Man) glycans at highly conserved residues in specific extracellular cadherin domains, and it was suggested that the function of E-cadherin was dependent on the O-Man glycans. Deficiencies in enzymes catalyzing O-Man biosynthesis, including the two human protein O-mannosyltransferases, POMT1 and POMT2, underlie a subgroup of congenital muscular dystrophies designated α-dystroglycanopathies, because deficient O-Man glycosylation of α-dystroglycan disrupts laminin interaction with α-dystroglycan and the extracellular matrix. To explore the functions of O-Man glycans on cadherins and protocadherins, we used a combinatorial gene-editing strategy in multiple cell lines to evaluate the role of the two POMTs initiating O-Man glycosylation and the major enzyme elongating O-Man glycans, the protein O-mannose ß-1,2-N-acetylglucosaminyltransferase, POMGnT1. Surprisingly, O-mannosylation of cadherins and protocadherins does not require POMT1 and/or POMT2 in contrast to α-dystroglycan, and moreover, the O-Man glycans on cadherins are not elongated. Thus, the classical and evolutionarily conserved POMT O-mannosylation pathway is essentially dedicated to α-dystroglycan and a few other proteins, whereas a novel O-mannosylation process in mammalian cells is predicted to serve the large cadherin superfamily and other proteins.
Asunto(s)
Cadherinas/metabolismo , Moléculas de Adhesión Celular/metabolismo , Distroglicanos/metabolismo , Manosiltransferasas/metabolismo , Proteínas del Tejido Nervioso/metabolismo , Animales , Células CHO , Cadherinas/genética , Moléculas de Adhesión Celular/genética , Cricetinae , Cricetulus , Glicosilación , Células HEK293 , Humanos , Manosiltransferasas/genética , Proteínas del Tejido Nervioso/genéticaRESUMEN
Mass spectrometry-based "-omics" technologies are important tools for global and detailed mapping of post-translational modifications. Protein glycosylation is an abundant and important post translational modification widespread throughout all domains of life. Characterization of glycoproteins, including identification of glycan structure and components, their attachment sites and protein carriers, remains challenging. However, recent advances in glycoproteomics, a subbranch that studies and categorizes protein glycosylations, have greatly expanded the known protein glycosylation space and research in this area is rapidly accelerating. Here, we review recent developments in glycoproteomic technologies with a special focus on microbial protein glycosylation.