RESUMO
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.
Assuntos
Axônios , Drosophila , Manosiltransferases , Proteínas Tirosina Fosfatases , Animais , Axônios/metabolismo , Drosophila/enzimologia , Drosophila/metabolismo , Proteínas de Drosophila/genética , Distroglicanas/genética , Distroglicanas/metabolismo , Mamíferos/metabolismo , Manosiltransferases/metabolismo , Proteínas Tirosina Fosfatases/metabolismo , Proteínas Tirosina Fosfatases Semelhantes a Receptores/genéticaRESUMO
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.
Assuntos
Manose , Açúcares , Animais , Humanos , Glicosilação , Açúcares/metabolismo , Manose/metabolismo , Processamento de Proteína Pós-Traducional , Drosophila/metabolismo , Saccharomyces cerevisiae/metabolismo , Manosiltransferases/genética , Manosiltransferases/metabolismoRESUMO
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.
Assuntos
Axônios/fisiologia , Manosiltransferases/fisiologia , Postura/fisiologia , Células Receptoras Sensoriais/fisiologia , Animais , Animais Geneticamente Modificados , Proteínas de Drosophila/genética , Distroglicanas/genética , Embrião não Mamífero , Retroalimentação Fisiológica , Manosiltransferases/genética , Contração Muscular/fisiologia , Mutação , Sistema Nervoso Periférico/fisiologia , FenótipoRESUMO
CMP-sialic acid synthetase (CSAS) is a key enzyme of the sialylation pathway. CSAS produces the activated sugar donor, CMP-sialic acid, which serves as a substrate for sialyltransferases to modify glycan termini with sialic acid. Unlike other animal CSASs that normally localize in the nucleus, Drosophila melanogaster CSAS (DmCSAS) localizes in the cell secretory compartment, predominantly in the Golgi, which suggests that this enzyme has properties distinct from those of its vertebrate counterparts. To test this hypothesis, we purified recombinant DmCSAS and characterized its activity in vitro Our experiments revealed several unique features of this enzyme. DmCSAS displays specificity for N-acetylneuraminic acid as a substrate, shows preference for lower pH and can function with a broad range of metal cofactors. When tested at a pH corresponding to the Golgi compartment, the enzyme showed significant activity with several metal cations, including Zn(2+), Fe(2+), Co(2+) and Mn(2+), whereas the activity with Mg(2+) was found to be low. Protein sequence analysis and site-specific mutagenesis identified an aspartic acid residue that is necessary for enzymatic activity and predicted to be involved in co-ordinating a metal cofactor. DmCSAS enzymatic activity was found to be essential in vivo for rescuing the phenotype of DmCSAS mutants. Finally, our experiments revealed a steep dependence of the enzymatic activity on temperature. Taken together, our results indicate that DmCSAS underwent evolutionary adaptation to pH and ionic environment different from that of counterpart synthetases in vertebrates. Our data also suggest that environmental temperatures can regulate Drosophila sialylation, thus modulating neural transmission.
Assuntos
Proteínas de Drosophila/química , Proteínas de Drosophila/metabolismo , N-Acilneuraminato Citidililtransferase/química , N-Acilneuraminato Citidililtransferase/metabolismo , Sequência de Aminoácidos , Animais , Ácido Aspártico/química , Ácido Aspártico/metabolismo , Células Cultivadas , Drosophila , Proteínas de Drosophila/classificação , Proteínas de Drosophila/genética , Humanos , Concentração de Íons de Hidrogênio , Cinética , Magnésio/metabolismo , Manganês/metabolismo , Mutação , N-Acilneuraminato Citidililtransferase/classificação , N-Acilneuraminato Citidililtransferase/genética , Filogenia , Relação Estrutura-Atividade , Especificidade por Substrato , TemperaturaRESUMO
While sialylation plays important functions in the nervous system, the complexity of glycosylation pathways and limitations of genetic approaches preclude the efficient analysis of these functions in mammalian organisms. Drosophila has recently emerged as a promising model for studying neural sialylation. Drosophila sialyltransferase, DSiaT, was shown to be involved in the regulation of neural transmission. However, the sialylation pathway was not investigated in Drosophila beyond the DSiaT-mediated step. Here we focused on the function of Drosophila cytidine monophosphate-sialic acid synthetase (CSAS), the enzyme providing a sugar donor for DSiaT. Our results revealed that the expression of CSAS is tightly regulated and restricted to the CNS throughout development and in adult flies. We generated CSAS mutants and analyzed their phenotypes using behavioral and physiological approaches. Our experiments demonstrated that mutant phenotypes of CSAS are similar to those of DSiaT, including decreased longevity, temperature-induced paralysis, locomotor abnormalities, and defects of neural transmission at neuromuscular junctions. Genetic interactions between CSAS, DSiaT, and voltage-gated channel genes paralytic and seizure were consistent with the hypothesis that CSAS and DSiaT function within the same pathway regulating neural excitability. Intriguingly, these interactions also suggested that CSAS and DSiaT have some additional, independent functions. Moreover, unlike its mammalian counterparts that work in the nucleus, Drosophila CSAS was found to be a glycoprotein-bearing N-glycans and predominantly localized in vivo to the Golgi compartment. Our work provides the first systematic analysis of in vivo functions of a eukaryotic CSAS gene and sheds light on evolutionary relationships among metazoan CSAS proteins.
Assuntos
Monofosfato de Citidina/metabolismo , Proteínas de Drosophila/genética , Drosophila/enzimologia , Ligases/genética , Ácido N-Acetilneuramínico/metabolismo , N-Acilneuraminato Citidililtransferase/genética , Fenômenos Fisiológicos do Sistema Nervoso/genética , Animais , Animais Geneticamente Modificados , Drosophila/genética , Proteínas de Drosophila/metabolismo , Evolução Molecular , Regulação da Expressão Gênica no Desenvolvimento/fisiologia , Ligases/metabolismo , Longevidade/genética , N-Acilneuraminato Citidililtransferase/metabolismo , Junção Neuromuscular/genética , Junção Neuromuscular/metabolismo , Paralisia/genética , Paralisia/metabolismo , Vesículas Secretórias/fisiologia , Sialiltransferases/genética , Sialiltransferases/metabolismo , Transmissão Sináptica/genética , Transmissão Sináptica/fisiologia , TemperaturaRESUMO
Recent studies have explored the function of N-linked glycosylation in the nervous system, demonstrating essential roles of carbohydrate structures in neural development. The function of N-glycans in neural physiology remains less understood; however, increasing evidence indicates that N-glycans can play specific modulatory roles controlling neural transmission and excitability of neural circuits. These roles are mediated via effects on synaptic proteins involved in neurotransmitter release, transporters that regulate nerotransmitter concentrations, neurotransmitter receptors, as well as via regulation of proteins that control excitability and response to milieu stimuli, such as voltage-gated ion channels and transient receptor potential channels, respectively. Sialylated N-glycan structures are among the most potent modulators of cell excitability, exerting prominent effects on voltage gated Na(+) and K(+) channels. This modulation appears to be underlain by complex molecular mechanisms involving electrostatic effects, as well as interaction modes based on more specific steric effects and interactions with lectins and other molecules. Data also indicate that particular features of N-glycans, such as their location on a protein and structural characteristics, can be specifically associated with the effect of glycosylation. These features and their functional implications can vary between different cell types, which highlight the importance of in vivo analyses of glycan functions. Experimental challenges are associated with the overwhelming complexity of the nervous system and glycosylation pathways in vertebrates, and thus model organisms like Drosophila should help elucidate evolutionarily conserved mechanisms underlying glycan functions. Recent studies supported this notion and shed light on functions of several glycosylation genes involved in the regulation of the nervous system.
Assuntos
Glicoproteínas de Membrana/metabolismo , Proteínas do Tecido Nervoso/metabolismo , Transmissão Sináptica/fisiologia , Animais , Proteínas de Drosophila/metabolismo , Drosophila melanogaster , Glicosilação , Humanos , Neurotransmissores/metabolismoRESUMO
Modification by sialylated glycans can affect protein functions, underlying mechanisms that control animal development and physiology. Sialylation relies on a dedicated pathway involving evolutionarily conserved enzymes, including CMP-sialic acid synthetase (CSAS) and sialyltransferase (SiaT) that mediate the activation of sialic acid and its transfer onto glycan termini, respectively. In Drosophila, CSAS and DSiaT genes function in the nervous system, affecting neural transmission and excitability. We found that these genes function in different cells: the function of CSAS is restricted to glia, while DSiaT functions in neurons. This partition of the sialylation pathway allows for regulation of neural functions via a glia-mediated control of neural sialylation. The sialylation genes were shown to be required for tolerance to heat and oxidative stress and for maintenance of the normal level of voltage-gated sodium channels. Our results uncovered a unique bipartite sialylation pathway that mediates glia-neuron coupling and regulates neural excitability and stress tolerance.
Assuntos
Drosophila , Fenômenos Fisiológicos do Sistema Nervoso , Animais , Drosophila/metabolismo , Transmissão Sináptica/fisiologia , Neurônios/metabolismo , Neuroglia/metabolismo , Polissacarídeos/metabolismoRESUMO
Protein O-mannosylation has a profound effect on the development and physiology of mammalian organisms. Mutations in genes affecting O-mannosyl glycan biosynthesis result in congenital muscular dystrophies. The main pathological mechanism triggered by O-mannosylation defects is a compromised interaction of cells with the extracellular matrix due to abnormal glycosylation of alpha-dystroglycan. Hypoglycosylation of alpha-dystroglycan impairs its ligand-binding activity and results in muscle degeneration and failure of neuronal migration. Recent experiments revealed the existence of compensatory mechanisms that could ameliorate defects of O-mannosylation. However, these mechanisms remain poorly understood. O-mannosylation and dystroglycan pathway genes show remarkable evolutionary conservation in a wide range of metazoans. Mutations and downregulation of these genes in zebrafish and Drosophila result in muscle defects and degeneration, also causing neurological phenotypes, which suggests that O-mannosylation has similar functions in mammals and lower animals. Thus, future studies in genetically tractable model organisms, such as zebrafish and Drosophila, should help to reveal molecular and genetic mechanisms of mammalian O-mannosylation and its role in the regulation of dystroglycan function.
Assuntos
Drosophila , Manose/metabolismo , Fenótipo , Polissacarídeos/química , Animais , Configuração de Carboidratos , Sequência de Carboidratos , Drosophila/genética , Drosophila/metabolismo , Distroglicanas/genética , Distroglicanas/metabolismo , Distrofina/metabolismo , Glicosilação , Humanos , Manose/química , Manosiltransferases/metabolismo , Dados de Sequência Molecular , Distrofias Musculares/genética , Distrofias Musculares/fisiopatologia , Polissacarídeos/metabolismoRESUMO
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.
Assuntos
Comportamento Animal/fisiologia , Sistema Nervoso Central/fisiologia , Drosophila , Sialiltransferases/fisiologia , Animais , Sistema Nervoso Central/anatomia & histologia , Sistema Nervoso Central/crescimento & desenvolvimento , Sistema Nervoso Central/metabolismo , Drosophila/enzimologia , Drosophila/fisiologia , Transtornos Neurológicos da Marcha/genética , Regulação da Expressão Gênica no Desenvolvimento , Genes de Insetos/fisiologia , Longevidade/genética , Mutação , Junção Neuromuscular/genética , Junção Neuromuscular/fisiologia , Neurônios/metabolismo , Neurônios/fisiologia , Sialiltransferases/genética , Canais de Sódio/genética , Canais de Sódio/fisiologia , Potenciais Sinápticos/genética , Potenciais Sinápticos/fisiologiaRESUMO
Numerous studies have revealed important functions for sialylation in both prokaryotes and higher animals. However, the genetic and biochemical potential for sialylation in Drosophila has only been confirmed recently. Recent studies suggest significant similarities between the sialylation pathways of vertebrates and insects and provide evidence for their common evolutionary origin. These new data support the hypothesis that sialylation in insects is a specialized and developmentally regulated process which likely plays a prominent role in the nervous system. Yet several key issues remain to be addressed in Drosophila, including the initiation of sialic acid de novo biosynthesis and understanding the structure and function of sialylated glycoconjugates. This review discusses our current knowledge of the Drosophila sialylation pathway, as compared to the pathway in bacteria and vertebrates. We arrive at the conclusion that Drosophila is emerging as a useful model organism that is poised to shed new light on the function of sialylation not only in protostomes, but also in a larger evolutionary context.
Assuntos
Bioquímica , Drosophila/genética , Drosophila/metabolismo , Ácido N-Acetilneuramínico/metabolismo , Sequência de Aminoácidos , Animais , Proteínas de Drosophila/química , Proteínas de Drosophila/genética , Proteínas de Drosophila/metabolismo , Regulação da Expressão Gênica no Desenvolvimento , Dados de Sequência Molecular , Ácido N-Acetilneuramínico/biossínteseRESUMO
The family of mammalian O-mannosyltransferases includes two enzymes, POMT1 and POMT2, which are thought to be essential for muscle and neural development. Similar to mammalian organisms, Drosophila has two O-mannosyltransferase genes, rotated abdomen (rt) and DmPOMT2, encoding proteins with high homology to their mammalian counterparts. The previously reported mutant phenotype of the rt gene includes a clockwise rotation of the abdomen and defects in embryonic muscle development. No mutants have been described so far for the DmPOMT2 locus. In this study, we determined that the mutation in the twisted (tw) locus, tw(1), corresponds to a DmPOMT2 mutant. The twisted alleles represent a complementation group of recessive mutations that, similar to the rt mutants, exhibit a clockwise abdomen rotation phenotype. Several tw alleles were isolated in the past; however, none of them was molecularly characterized. We used an expression rescue approach to confirm that tw locus represents DmPOMT2 gene. We found that the tw1 allele represents an amino acid substitution within the conserved PMT domain of DmPOMT2 (TW) protein. Immunostaining experiments revealed that the protein products of both rt and tw genes colocalize within Drosophila cells where they reside in the ER subcellular compartment. In situ hybridization analysis showed that both genes have essentially overlapping patterns of expression throughout most of embryogenesis (stages 8-17), while only the rt transcript is present at early embryonic stages (5 and 6), suggesting its maternal origin. Finally, we analyzed the genetic interactions between rt and tw using several mutant alleles, RNAi, and ectopic expression approaches. Our data suggest that the two Drosophila O-mannosyltransferase genes, rt and tw, have nonredundant functions within the same developmental cascade and that their activities are required simultaneously for possibly the same biochemical process. Our results establish the possibility of using Drosophila as a model system for studying molecular and genetic mechanisms of protein O-mannosylation during development.
Assuntos
Proteínas de Drosophila , Drosophila melanogaster , Manosiltransferases/genética , Manosiltransferases/metabolismo , Sequência de Aminoácidos , Substituição de Aminoácidos , Animais , Animais Geneticamente Modificados , Proteínas de Drosophila/genética , Proteínas de Drosophila/metabolismo , Drosophila melanogaster/embriologia , Drosophila melanogaster/enzimologia , Drosophila melanogaster/genética , Desenvolvimento Embrionário/fisiologia , Feminino , Regulação da Expressão Gênica no Desenvolvimento , Teste de Complementação Genética , Hibridização In Situ , Masculino , Dados de Sequência Molecular , Mutação , Fenótipo , RNA Interferente Pequeno/farmacologia , Homologia de Sequência de AminoácidosRESUMO
Aryl hydrocarbon receptor (AHR) is the key transcription factor that controls animal development and various adaptive processes. The AHR's target genes are involved in biodegradation of endogenous and exogenous toxins, regulation of immune response, organogenesis, and neurogenesis. Ligand binding is important for the activation of the AHR signaling pathway. Invertebrate AHR homologs are activated by endogenous ligands whereas vertebrate AHR can be activated by both endogenous and exogenous ligands (xenobiotics). Several studies using mammalian cultured cells have demonstrated that transcription of the AHR target genes can be activated by exogenous AHR ligands, but little is known about the effects of AHR in a living organism. Here, we examined the effects of human AHR and its ligands using transgenic Drosophila lines with an inducible human AhR gene. We found that exogenous AHR ligands can increase as well as decrease the transcription levels of the AHR target genes, including genes that control proliferation, motility, polarization, and programmed cell death. This suggests that AHR activation may affect the expression of gene networks that could be critical for cancer progression and metastasis. Importantly, we found that AHR target genes are also controlled by the enzymes that modify chromatin structure, in particular components of the epigenetic Polycomb Repressive complexes 1 and 2. Since exogenous AHR ligands (alternatively - xenobiotics) and small molecule inhibitors of epigenetic modifiers are often used as pharmaceutical anticancer drugs, our findings may have significant implications in designing new combinations of therapeutic treatments for oncological diseases.
RESUMO
Protein O-mannosylation is a special type of glycosylation that plays prominent roles in metazoans, affecting development and physiology of the nervous system and muscles. A major biological effect of O-mannosylation involves the regulation of α-dystroglycan, a membrane glycoprotein mediating cell-extracellular matrix interactions. Genetic defects of O-mannosylation result in the loss of ligand-binding activity of α-dystroglycan and cause congenital muscular dystrophies termed dystroglycanopathies. Recent progress in mass spectrometry and in vitro analyses has shed new light on the mechanism of α-dystroglycan glycosylation; however, this mechanism is underlain by complex genetic and molecular elements that remain poorly understood. Protein O-mannosylation is evolutionarily conserved in metazoans, yet the pathway is simplified and more amenable to genetic analyses in invertebrate organisms, indicating that genetically tractable in vivo models could facilitate research in this area. This unit describes recent methodological strategies for studying protein O-mannosylation using in vitro and in vivo approaches.
Assuntos
Western Blotting/métodos , Cromatografia de Afinidade/métodos , Distroglicanas/química , Glicosilação , Manose/química , Espectrometria de Massas/métodos , Humanos , Manose/análise , Distrofias MuscularesRESUMO
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.
RESUMO
Sialylated glycans play a prominent role in the Drosophila nervous system where they are involved in the regulation of neural transmission. However, the functional pathway of sialylation in invertebrates, including Drosophila, remains largely unknown. Here we used a combination of genetic and behavioral approaches to shed light on the Drosophila sialylation pathway. We examined genetic interactions between Drosophila sialyltransferase (DSiaT) and ß1,4-N-acetylgalactosaminyltransferase (ß4GalNAcT) genes. Our results indicated that ß4GalNAcTA and DSiaT cooperate within the same functional pathway that regulates neural transmission. We found that ß4GalNAcTA is epistatic to DSiaT. Our data suggest an intriguing possibility that ß4GalNAcTA may participate in the biosynthesis of sialylated glycans.
RESUMO
O-Fucose is an unusual form of glycosylation found on epidermal growth factor-like (EGF) repeats and thrombospondin type 1 repeats (TSRs) in many secreted and transmembrane proteins. Recently O-fucose on EGF repeats was shown to play important roles in Notch signaling. In contrast, physiological roles for O-fucose on TSRs are unknown. In the accompanying paper (Luo, Y., Nita-Lazar, A., and Haltiwanger, R. S. (2006) J. Biol. Chem. 281, 9385-9392), we demonstrated that an enzyme distinct from protein O-fucosyltransferase 1 adds O-fucose to TSRs. A known homologue of O-fucosyltransferase 1 is putative protein O-fucosyltransferase 2. The cDNA sequence encoding O-fucosyltransferase 2 was originally identified during a data base search for fucosyltransferases in Drosophila. Like O-fucosyltransferase 1, O-fucosyltransferase 2 is conserved from Caenorhabditis elegans to humans. Although O-fucosyltransferase 2 was assumed to be another protein O-fucosyltransferase, no biochemical characterization existed supporting this contention. Here we show that RNAi-mediated reduction of the O-fucosyltransferase 2 message significantly decreased TSR-specific O-fucosyltransferase activity in Drosophila S2 cells. We also found that O-fucosyltransferase 2 is predominantly localized in the endoplasmic reticulum compartment of these cells. Furthermore, we expressed recombinant Drosophila O-fucosyltransferase 2 and showed that it O-fucosylates TSRs but not EGF repeats in vitro. These results demonstrate that O-fucosyltransferase 2 is in fact a TSR-specific O-fucosyltransferase.
Assuntos
Proteínas de Drosophila/genética , Proteínas de Drosophila/metabolismo , Fucosiltransferases/genética , Fucosiltransferases/metabolismo , Trombospondina 1/metabolismo , Sequência de Aminoácidos , Animais , Técnicas de Cultura de Células , Drosophila , Retículo Endoplasmático/enzimologia , Perfilação da Expressão Gênica , Glicosilação , Dados de Sequência Molecular , Interferência de RNA , Galactosídeo 2-alfa-L-FucosiltransferaseRESUMO
With the complete genome sequence of Drosophila melanogaster defined a systematic approach towards understanding the function of glycosylation has become possible. Structural assignment of the entire Drosophila glycome during specific developmental stages could provide information that would shed further light on the specific roles of different glycans during development and pinpoint the activity of certain glycosyltransferases and other glycan biosynthetic genes that otherwise might be missed through genetic analyses. In this paper the major glycoprotein N- and O-glycans of Drosophila embryos are described as part of our initial undertaking to characterize the glycome of Drosophila melanogaster. The N-glycans are dominated by high mannose and paucimannose structures. Minor amounts of mono-, bi- and tri-antennary complex glycans were observed with GlcNAc and Galbeta1-4GlcNAc non-reducing end termini. O-glycans were restricted to the mucin-type core 1 Galbeta1-3GalNAc sequence.
Assuntos
Drosophila melanogaster/química , Drosophila melanogaster/embriologia , Embrião não Mamífero/química , Glicoproteínas/química , Polissacarídeos/química , Animais , Sequência de Carboidratos , Dados de Sequência MolecularRESUMO
Sialylation is an important carbohydrate modification of glycoconjugates in the deuterostome lineage of animals. By contrast, the evidence for sialylation in protostomes has been scarce and somewhat controversial. In the present study, we characterize a Drosophila sialyltransferase gene, thus providing experimental evidence for the presence of sialylation in protostomes. This gene encodes a functional alpha2-6-sialyltransferase (SiaT) that is closely related to the vertebrate ST6Gal sialyltransferase family, indicating an ancient evolutionary origin for this family. Characterization of recombinant, purified Drosophila SiaT revealed a novel acceptor specificity as it exhibits highest activity toward GalNAcbeta1-4GlcNAc carbohydrate structures at the non-reducing termini of oligosaccharides and glycoprotein glycans. Oligosaccharides are preferred over glycoproteins as acceptors, and no activity toward glycolipid acceptors was detected. Recombinant Drosophila SiaT expressed in cultured insect cells possesses in vivo and in vitro autosialylation activity toward beta-linked GalNAc termini of its own N-linked glycans, thus representing the first example of a sialylated insect glycoconjugate. In situ hybridization revealed that Drosophila SiaT is expressed during embryonic development in a tissue- and stage-specific fashion, with elevated expression in a subset of cells within the central nervous system. The identification of a SiaT in Drosophila provides a new evolutionary perspective for considering the diverse functions of sialylation and, through the powerful genetic tools available in this system, a means of elucidating functions for sialylation in protostomes.
Assuntos
Drosophila melanogaster/enzimologia , Sialiltransferases/metabolismo , Sequência de Aminoácidos , Animais , Cátions Bivalentes , Sistema Nervoso Central/embriologia , Sistema Nervoso Central/enzimologia , Drosophila melanogaster/embriologia , Drosophila melanogaster/genética , Evolução Molecular , Regulação da Expressão Gênica no Desenvolvimento , Genes de Insetos , Concentração de Íons de Hidrogênio , Cinética , Dados de Sequência Molecular , Filogenia , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Homologia de Sequência de Aminoácidos , Sialiltransferases/química , Sialiltransferases/genética , Especificidade por Substrato , Temperatura , beta-D-Galactosídeo alfa 2-6-SialiltransferaseRESUMO
Two glycosyltransferases that transfer sugars to EGF domains, OFUT1 and Fringe, regulate Notch signaling. However, sites of O-fucosylation on Notch that influence Notch activation have not been previously identified. Moreover, the influences of OFUT1 and Fringe on Notch activation can be positive or negative, depending on their levels of expression and on whether Delta or Serrate is signaling to Notch. Here, we describe the consequences of eliminating individual, highly conserved sites of O-fucose attachment to Notch. Our results indicate that glycosylation of an EGF domain proposed to be essential for ligand binding, EGF12, is crucial to the inhibition of Serrate-to-Notch signaling by Fringe. Expression of an EGF12 mutant of Notch (N-EGF12f) allows Notch activation by Serrate even in the presence of Fringe. By contrast, elimination of three other highly conserved sites of O-fucosylation does not have detectable effects. Binding assays with a soluble Notch extracellular domain fusion protein and ligand-expressing cells indicate that the NEGF12f mutation can influence Notch activation by preventing Fringe from blocking Notch-Serrate binding. The N-EGF12f mutant can substitute for endogenous Notch during embryonic neurogenesis, but not at the dorsoventral boundary of the wing. Thus, inhibition of Notch-Serrate binding by O-fucosylation of EGF12 might be needed in certain contexts to allow efficient Notch signaling.
Assuntos
Drosophila/embriologia , Drosophila/genética , Fucose/metabolismo , Regulação da Expressão Gênica no Desenvolvimento , Proteínas de Membrana/química , Proteínas de Membrana/genética , Sequência de Aminoácidos , Animais , Sítios de Ligação , Padronização Corporal/genética , Cruzamentos Genéticos , Proteínas de Drosophila/antagonistas & inibidores , Proteínas de Drosophila/química , Proteínas de Drosophila/genética , Embrião não Mamífero/fisiologia , Ligantes , Proteínas de Membrana/antagonistas & inibidores , Conformação Proteica , Receptores NotchRESUMO
O-Fucose has been identified on epidermal growth factor-like (EGF) repeats of Notch, and elongation of O-fucose has been implicated in the modulation of Notch signaling by Fringe. O-Fucose modifications are also predicted to occur on Notch ligands based on the presence of the C(2)XXGG(S/T)C(3) consensus site (where S/T is the modified amino acid) in a number of the EGF repeats of these proteins. Here we establish that both mammalian and Drosophila Notch ligands are modified with O-fucose glycans, demonstrating that the consensus site was useful for making predictions. The presence of O-fucose on Notch ligands raised the question of whether Fringe, an O-fucose specific beta 1,3-N-acetylglucosaminyltransferase, was capable of modifying O-fucose on the ligands. Indeed, O-fucose on mammalian Delta 1 and Jagged1 can be elongated with Manic Fringe in vivo, and Drosophila Delta and Serrate are substrates for Drosophila Fringe in vitro. These results raise the interesting possibility that alteration of O-fucose glycans on Notch ligands could play a role in the mechanism of Fringe action on Notch signaling. As an initial step to begin addressing the role of the O-fucose glycans on Notch ligands in Notch signaling, a number of mutations in predicted O-fucose glycosylation sites on Drosophila Serrate have been generated. Interestingly, analysis of these mutants has revealed that O-fucose modifications occur on some EGF repeats not predicted by the C(2)XXGGS/TC(3) consensus site. A revised, broad consensus site, C(2)X(3-5)S/TC(3) (where X(3-5) are any 3-5 amino acid residues), is proposed.