RESUMEN
Recent research has unveiled numerous important functions of protein glycosylation in development, homeostasis, and diseases. A type of glycosylation taking the center stage is protein O-mannosylation, a posttranslational modification conserved in a wide range of organisms, from yeast to humans. In animals, protein O-mannosylation plays a crucial role in the nervous system, whereas protein O-mannosylation defects cause severe neurological abnormalities and congenital muscular dystrophies. However, the molecular and cellular mechanisms underlying protein O-mannosylation functions and biosynthesis remain not well understood. This review outlines recent studies on protein O-mannosylation while focusing on the functions in the nervous system, summarizes the current knowledge about protein O-mannosylation biosynthesis, and discusses the pathologies associated with protein O-mannosylation defects. The evolutionary perspective revealed by studies in the Drosophila model system are also highlighted. Finally, the review touches upon important knowledge gaps in the field and discusses critical questions for future research on the molecular and cellular mechanisms associated with protein O-mannosylation functions.
Asunto(s)
Manosa , Azúcares , Animales , Humanos , Glicosilación , Azúcares/metabolismo , Manosa/metabolismo , Procesamiento Proteico-Postraduccional , Drosophila/metabolismo , Saccharomyces cerevisiae/metabolismo , Manosiltransferasas/genética , Manosiltransferasas/metabolismoRESUMEN
Mutations in protein O-mannosyltransferases (POMTs) result in severe brain defects and congenital muscular dystrophies characterized by abnormal glycosylation of α-dystroglycan (α-Dg). However, neurological phenotypes of POMT mutants are not well understood, and the functional substrates of POMTs other than α-Dg remain unknown. Using a Drosophila model, here we reveal that Dg alone cannot account for the phenotypes of POMT mutants, and identify Protein tyrosine phosphatase 69D (PTP69D) as a gene interacting with POMTs in producing the abdomen rotation phenotype. Using RNAi-mediated knockdown, mutant alleles, and a dominant-negative form of PTP69D, we reveal that PTP69D is required for the wiring of larval sensory axons. We also found that PTP69D and POMT genes interact in this process, and that their interactions lead to complex synergistic or antagonistic effects on axon wiring phenotypes, depending on the mode of genetic manipulation. Using glycoproteomic approaches, we further characterized the glycosylation of the PTP69D transgenic construct expressed in genetic strains with different levels of POMT activity. We found that the PTP69D construct carries many O-linked mannose modifications when expressed in Drosophila with wild-type or ectopically upregulated expression of POMTs. These modifications were absent in POMT mutants, suggesting that PTP69D is a substrate of POMT-mediated O-mannosylation. Taken together, our results indicate that PTP69D is a novel functional substrate of POMTs that is required for axon connectivity. This mechanism of POMT-mediated regulation of receptor-type protein tyrosine phosphatase functions could potentially be conserved in mammals and may shed new light on the etiology of neurological defects in muscular dystrophies.
Asunto(s)
Axones , Drosophila , Manosiltransferasas , Proteínas Tirosina Fosfatasas , Animales , Axones/metabolismo , Drosophila/enzimología , Drosophila/metabolismo , Proteínas de Drosophila/genética , Distroglicanos/genética , Distroglicanos/metabolismo , Mamíferos/metabolismo , Manosiltransferasas/metabolismo , Proteínas Tirosina Fosfatasas/metabolismo , Proteínas Tirosina Fosfatasas Similares a Receptores/genéticaRESUMEN
The human-pathogenic yeast Cryptococcus neoformans assembles two types of O-linked glycans on its proteins. In this study, we identified and functionally characterized the C. neoformans CAP6 gene, encoding an α1,3-mannosyltransferase responsible for the second mannose addition to minor O-glycans containing xylose in the Golgi apparatus. Two cell surface sensor proteins, Wml1 (WSC/Mid2-like) and Wml2, were found to be independent substrates of Cap6-mediated minor or Ktr3-mediated major O-mannosylation, respectively. The double deletion of KTR3 and CAP6 (ktr3Δ cap6Δ) completely blocked the mannose addition at the second position of O-glycans, resulting in the accumulation of proteins with O-glycans carrying only a single mannose. Tunicamycin (TM)-induced phosphorylation of the Mpk1 mitogen-activated protein kinase (MAPK) was greatly decreased in both ktr3Δ cap6Δ and wml1Δ wml2Δ strains. Transcriptome profiling of the ktr3Δ cap6Δ strain upon TM treatment revealed decreased expression of genes involved in the Mpk1-dependent cell wall integrity (CWI) pathway. Consistent with its defective growth under several stress conditions, the ktr3Δ cap6Δ strain was avirulent in a mouse model of cryptococcosis. Associated with this virulence defect, the ktr3Δ cap6Δ strain showed decreased adhesion to lung epithelial cells, decreased proliferation within macrophages, and reduced transcytosis of the blood-brain barrier (BBB). Notably, the ktr3Δ cap6Δ strain showed reduced induction of the host immune response and defective trafficking of ergosterol, an immunoreactive fungal molecule. In conclusion, O-glycan extension in the Golgi apparatus plays critical roles in various pathobiological processes, such as CWI signaling and stress resistance and interaction with host cells in C. neoformans. IMPORTANCE Cryptococcus neoformans assembles two types of O-linked glycans on its surface proteins, the more abundant major O-glycans that do not contain xylose residues and minor O-glycans containing xylose. Here, we demonstrate the role of the Cap6 α1,3-mannosyltransferase in the synthesis of minor O-glycans. Previously proposed to be involved in capsule biosynthesis, Cap6 works with the related Ktr3 α1,2-mannosyltransferase to synthesize O-glycans on their target proteins. We also identified two novel C. neoformans stress sensors that require Ktr3- and Cap6-mediated posttranslational modification for full function. Accordingly, the ktr3Δ cap6Δ double O-glycan mutant strain displays defects in stress signaling pathways, CWI, and ergosterol trafficking. Furthermore, the ktr3Δ cap6Δ strain is completely avirulent in a mouse infection model. Together, these results demonstrate critical roles for O-glycosylation in fungal pathogenesis. As there are no human homologs for Cap6 or Ktr3, these fungus-specific mannosyltransferases are novel targets for antifungal therapy.
Asunto(s)
Criptococosis , Cryptococcus neoformans , Animales , Ratones , Humanos , Cryptococcus neoformans/genética , Glicosilación , Manosiltransferasas/metabolismo , Xilosa/metabolismo , Manosa , Criptococosis/microbiología , Polisacáridos/metabolismo , Pared Celular/metabolismo , Aparato de Golgi/metabolismo , Proteínas Fúngicas/genéticaRESUMEN
Protein glycosylation is an essential covalent modification involved in protein secretion, stability, binding, folding, and activity. One or more sugars may be O-, N-, S-, or C-linked to specific amino acids by glycosyltransferases, which catalyze the transfer of these sugars from a phosphate-containing carrier molecule. Most glycosyltransferases are members of the GT-A, GT-B, or GT-C structural superfamilies. GT-C enzymes are integral membrane proteins that utilize a phospho-isoprenoid carrier for sugar transfer. To-date, two families of GT-Cs involved in protein glycosylation have been structurally characterized: the family represented by PglB, AglB, and Stt3, which catalyzes oligosaccharide transfer to Asn, and the family represented by Pmt1 and Pmt2, which catalyzes mannose transfer to Thr or Ser. This chapter reviews progress made over recent years on the structure and function of these two GT-C families.
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Glicosiltransferasas/química , Glicosiltransferasas/clasificación , Glicosilación , Glicosiltransferasas/metabolismo , Humanos , Proteínas de la Membrana/química , Proteínas de la Membrana/metabolismoRESUMEN
Protein O-mannosyltransferases (PMTs) represent a conserved family of multispanning endoplasmic reticulum membrane proteins involved in glycosylation of S/T-rich protein substrates and unfolded proteins. PMTs work as dimers and contain a luminal MIR domain with a ß-trefoil fold, which is susceptive for missense mutations causing α-dystroglycanopathies in humans. Here, we analyze PMT-MIR domains by an integrated structural biology approach using X-ray crystallography and NMR spectroscopy and evaluate their role in PMT function in vivo. We determine Pmt2- and Pmt3-MIR domain structures and identify two conserved mannose-binding sites, which are consistent with general ß-trefoil carbohydrate-binding sites (α, ß), and also a unique PMT2-subfamily exposed FKR motif. We show that conserved residues in site α influence enzyme processivity of the Pmt1-Pmt2 heterodimer in vivo. Integration of the data into the context of a Pmt1-Pmt2 structure and comparison with homologous ß-trefoil - carbohydrate complexes allows for a functional description of MIR domains in protein O-mannosylation.
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Manosiltransferasas/química , Conformación Proteica , Animales , Glicosilación , Humanos , Dominios ProteicosRESUMEN
The transcription factor BrlA plays a central role in the production of asexual spores (conidia) in the fungus Aspergillus nidulans. BrlA levels are controlled by signal transducers known collectively as UDAs. Furthermore, it governs the expression of CDP regulators, which control most of the morphological transitions leading to the production of conidia. In response to the emergence of fungal cells in the air, the main stimulus triggering conidiation, UDA mutants such as the flbB deletant fail to induce brlA expression. Nevertheless, ΔflbB colonies conidiate profusely when they are cultured on a medium containing high H2PO4- concentrations, suggesting that the need for FlbB activity is bypassed. We used this phenotypic trait and an UV-mutagenesis procedure to isolate ΔflbB mutants unable to conidiate under these stress conditions. Transformation of mutant FLIP166 with a wild-type genomic library led to the identification of the putative transcription factor SocA as a multicopy suppressor of the FLIP (Fluffy, aconidial, In Phosphate) phenotype. Deregulation of socA altered both growth and developmental patterns. Sequencing of the FLIP166 genome enabled the identification and characterization of PmtCP282L as the recessive mutant form responsible for the FLIP phenotype. Overall, results validate this strategy for identifying genes/mutations related to the control of conidiation.
Asunto(s)
Aspergilosis/microbiología , Aspergillus nidulans/fisiología , Mutación , Fosfatos/metabolismo , Reproducción Asexuada , Estrés Fisiológico , Aspergillus nidulans/clasificación , Proteínas Fúngicas/química , Proteínas Fúngicas/genética , Regulación Fúngica de la Expresión Génica , Humanos , Modelos Moleculares , Fenotipo , Filogenia , Conformación ProteicaRESUMEN
In fungi, O-mannosylation is one type of conserved protein modifications that add the carbohydrate residues to specific residues of target proteins by protein O-mannosyltransferases. Previously, three members of O-mannosyltransferases were identified in Magnaporthe oryzae, with MoPmt2 playing important roles in fungal growth and pathogenicity. However, the biological roles of the rest Pmt proteins remain unclear. In this study, to understand if O-mannosyltransferases are crucial for fungal pathogenicity of M. oryzae, the Pmt-coding genes MoPmt1 and MoPmt4 were separately disrupted and their roles in pathogenesis were analyzed. Of the two genes, only MoPmt4 is specifically required for full virulence of M. oryzae. Deletion of MoPmt4 resulted in defects on radial growth, with more branching hyphae and septa as compared to Guy11. The MoPmt4 mutant was severely impaired not only in conidiation, but also in both penetration and biotrophic invasion in susceptible rice plants. This mutant also had defects in suppression of host-derived ROS-mediated plant defense responses that might be ascribed from the reduced activities of extracellular enzymes. Furthermore, like their fungi counterparts, MoPmt4 localized in the ER and had O-mannosyltransferase activity. Domain disruption analysis indicated that mannosyltransferase activity regulated by PMT domain of MoPmt4 is crucial for fungal development and pathogenicity of M. oryzae. Taken together, these data suggest that MoPmt4 is a protein O-mannosyltransferase essential for fungal development and full virulence of M. oryzae.
Asunto(s)
Proteínas Fúngicas/genética , Pleiotropía Genética/genética , Magnaporthe/genética , Manosiltransferasas/genética , Proteínas Fúngicas/metabolismo , Eliminación de Gen , Regulación del Desarrollo de la Expresión Génica , Regulación Fúngica de la Expresión Génica , Hifa/genética , Hifa/crecimiento & desarrollo , Hifa/metabolismo , Magnaporthe/crecimiento & desarrollo , Magnaporthe/patogenicidad , Manosiltransferasas/metabolismo , Oryza/metabolismo , Oryza/microbiología , Especies Reactivas de Oxígeno/metabolismo , Esporas Fúngicas/genética , Esporas Fúngicas/crecimiento & desarrollo , Esporas Fúngicas/metabolismo , Virulencia/genéticaRESUMEN
Genetic defects in protein O-mannosyltransferase 1 (POMT1) and POMT2 underlie severe muscular dystrophies. POMT genes are evolutionarily conserved in metazoan organisms. In Drosophila, both male and female POMT mutants show a clockwise rotation of adult abdominal segments, suggesting a chirality of underlying pathogenic mechanisms. Here we described and analyzed a similar phenotype in POMT mutant embryos that shows left-handed body torsion. Our experiments demonstrated that coordinated muscle contraction waves are associated with asymmetric embryo rolling, unveiling a new chirality marker in Drosophila development. Using genetic and live-imaging approaches, we revealed that the torsion phenotype results from differential rolling and aberrant patterning of peristaltic waves of muscle contractions. Our results demonstrated that peripheral sensory neurons are required for normal contractions that prevent the accumulation of torsion. We found that POMT mutants show abnormal axonal connections of sensory neurons. POMT transgenic expression limited to sensory neurons significantly rescued the torsion phenotype, axonal connectivity defects, and abnormal contractions in POMT mutant embryos. Together, our data suggested that protein O-mannosylation is required for normal sensory feedback to control coordinated muscle contractions and body posture. This mechanism may shed light on analogous functions of POMT genes in mammals and help to elucidate the etiology of neurological defects in muscular dystrophies.SIGNIFICANCE STATEMENT Protein O-mannosyltransferases (POMTs) are evolutionarily conserved in metazoans. Mutations in POMTs cause severe muscular dystrophies associated with pronounced neurological defects. However, neurological functions of POMTs remain poorly understood. We demonstrated that POMT mutations in Drosophila result in abnormal muscle contractions and cause embryo torsion. Our experiments uncovered a chirality of embryo movements and a unique POMT-dependent mechanism that maintains symmetry of a developing system affected by chiral forces. Furthermore, POMTs were found to be required for proper axon connectivity of sensory neurons, suggesting that O-mannosylation regulates the sensory feedback controlling muscle contractions. This novel POMT function in the peripheral nervous system may shed light on analogous functions in mammals and help to elucidate pathomechanisms of neurological abnormalities in muscular dystrophies.
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Axones/fisiología , Manosiltransferasas/fisiología , Postura/fisiología , Células Receptoras Sensoriales/fisiología , Animales , Animales Modificados Genéticamente , Proteínas de Drosophila/genética , Distroglicanos/genética , Embrión no Mamífero , Retroalimentación Fisiológica , Manosiltransferasas/genética , Contracción Muscular/fisiología , Mutación , Sistema Nervioso Periférico/fisiología , FenotipoRESUMEN
Members of α-1,2-mannosyltransferase (Ktr) family are required for protein O-mannosylation for the elongation of Ser/Thr mannose residues in yeasts but functionally unknown in most filamentous fungi. Here we characterized the functions of the Ktr orthologues Ktr1, Ktr4 and Kre2/Mnt1 in Beauveria bassiana, a filamentous enotmopathogen, and found that they were positive, but differential, mediators of many biological traits. Inactivation of Ktr4 and Kre2 resulted in 92% reduction of conidial yield on a standard medium and growth defects on substrates with altered carbon or nitrogen sources and availability, accompanied with reduced conidial size and complexity. This contrasts to the dispensability of Ktr1 for fungal growth and conidiation. More cell wall damage occurred in Δktr4 and Δkre2 than in Δktr1, including altered contents of the cell wall components mannoproteins, α-glucans and chitin, more carbohydrate epitopes changed on conidial surfaces, much lower conidial hydrophobicity, and thinner cell walls. Consequently, Δktr4 and Δkre2 became more sensitive to oxidation and cell wall perturbation than Δktr1 during colony growth or conidial germination despite less difference in their sensitivities to two osmotic agents. Conidial thermotolerance, UV-B resistance and virulence were all lowered greatly in Δktr4 and Δkre2 but only the thermotolerance decreased in Δktr1. All the phenotypical changes were well restored to wild-type levels by the complementation of each target gene. Our results indicate that Ktr4 and Kre2 contribute more to the biocontrol potential of B. bassiana than Ktr1 although all of them are significant contributors.
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Beauveria/enzimología , Manosiltransferasas/metabolismo , Animales , Beauveria/genética , Beauveria/crecimiento & desarrollo , Beauveria/patogenicidad , Pared Celular/química , Pared Celular/enzimología , Pared Celular/genética , Quitina/metabolismo , Glucanos/metabolismo , Hifa/enzimología , Hifa/crecimiento & desarrollo , Manosiltransferasas/genética , Morfogénesis , Mariposas Nocturnas/crecimiento & desarrollo , Mariposas Nocturnas/microbiología , Homología de Secuencia de Ácido Nucleico , Esporas Fúngicas/enzimología , Esporas Fúngicas/crecimiento & desarrollo , Estrés Fisiológico , VirulenciaRESUMEN
Mutations in POMT1 lead to a group of neuromuscular conditions ranging in severity from Walker-Warburg syndrome to limb girdle muscular dystrophy. We report two male siblings, ages 19 and 14, and an unrelated 6-year old female with early onset muscular dystrophy and intellectual disability with minimal structural brain anomalies and no ocular abnormalities. Compound heterozygous mutations in POMT1 were identified including a previously reported nonsense mutation (c.2167dupG; p.Asp723Glyfs*8) associated with Walker-Warburg syndrome and a novel missense mutation in a highly conserved region of the protein O-mannosyltransferase 1 protein (c.1958C>T; p.Pro653Leu). This novel variant reduces the phenotypic severity compared to patients with homozygous c.2167dupG mutations or compound heterozygous patients with a c.2167dupG mutation and a wide range of other mutant POMT1 alleles.
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Manosiltransferasas/genética , Distrofias Musculares/genética , Mutación Missense , Fenotipo , Adolescente , Encéfalo/patología , Células Cultivadas , Niño , Análisis Mutacional de ADN , Femenino , Fibroblastos/metabolismo , Humanos , Imagen por Resonancia Magnética , Masculino , Manosiltransferasas/metabolismo , Músculo Esquelético/patología , Distrofias Musculares/patología , Distrofias Musculares/fisiopatología , Índice de Severidad de la Enfermedad , Hermanos , Adulto JovenRESUMEN
Dystroglycanopathies form a subgroup of muscular dystrophies that arise from defects in enzymes that are implicated in the recently elucidated O-mannosylation pathway, thereby resulting in underglycosylation of α-dystroglycan. The emerging identification of additional brain proteins modified by O-mannosylation provides a broader context for interpreting the range of neurological consequences associated with dystroglycanopathies. This form of glycosylation is associated with protein mucin-like domains that present numerous serine and threonine residues as possible sites for modification. Furthermore, the O-Man glycans coexist in this region with O-GalNAc glycans (conventionally associated with such protein sequences), thus resulting in a complex glycoconjugate landscape. Sorting out the relationships between the various molecular defects in glycosylation and the modes of disease presentation, as well as the regulatory interplay among the O-Man glycans and the effects on other modes of glycosylation in the same domain, is challenging. Here we provide a perspective on chemical biology approaches employing synthetic and analytical methods to address these questions.
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Distroglicanos/metabolismo , Glicoconjugados/metabolismo , Manosa/metabolismo , Distrofias Musculares/metabolismo , Secuencia de Aminoácidos , Animales , Secuencia de Carbohidratos , Distroglicanos/química , Glicoconjugados/química , Glicopéptidos/química , Glicopéptidos/metabolismo , Humanos , Manosa/química , Datos de Secuencia Molecular , Distrofias Musculares/enzimología , Distrofias Musculares/terapiaRESUMEN
BACKGROUND: Protein O-mannosylation is a vital type of glycosylation that is conserved among fungi, animals, and humans. It is initiated in the endoplasmic reticulum (ER) where the synthesis of the mannosyl donor substrate and the mannosyltransfer to proteins take place. O-mannosylation defects interfere with cell wall integrity and ER homeostasis in yeast, and define a pathomechanism of severe neuromuscular diseases in humans. SCOPE OF REVIEW: On the molecular level, the O-mannosylation pathway and the function of O-mannosyl glycans have been characterized best in the eukaryotic model yeast Saccharomyces cerevisiae. In this review we summarize general features of protein O-mannosylation, including biosynthesis of the mannosyl donor, characteristics of acceptor substrates, and the protein O-mannosyltransferase machinery in the yeast ER. Further, we discuss the role of O-mannosyl glycans and address the question why protein O-mannosylation is essential for viability of yeast cells. GENERAL SIGNIFICANCE: Understanding of the molecular mechanisms of protein O-mannosylation in yeast could lead to the development of novel antifungal drugs. In addition, transfer of the knowledge from yeast to mammals could help to develop diagnostic and therapeutic approaches in the frame of neuromuscular diseases. This article is part of a Special Issue entitled: Functional and structural diversity of endoplasmic reticulum.