RESUMO
Development of novel anti-cancer therapeutics based on Golgi α-mannosidase II (GMII) inhibition is considerably impeded by an undesired co-inhibition of lysosomal α-mannosidase leading to severe side-effects. In this contribution, we describe a fully stereoselective synthesis of (5S)-5-[4-(halo)benzyl]swainsonines as highly potent and selective inhibitors of GMII. The synthesis starts from a previously reported aldehyde readily available from l-ribose, and the key features include an intramolecular reductive amination with substrate-controlled stereoselectivity and a late-stage derivatisation of the benzyl group via ipso-substitution. These novel swainsonine analogues were found to be nanomolar inhibitors of the Golgi-type α-mannosidase AMAN-2 (Ki = 23-75 nM) with excellent selectivity (selectivity index = 205-870) over the lysosomal-type Jack bean α-mannosidase. Finally, molecular docking and pKa calculations were performed to provide more insight into the structure of the inhibitor:enzyme complexes, and a pair interaction energy analysis (FMO-PIEDA) was carried out to rationalise the observed potency and selectivity of the inhibitors.
Assuntos
Inibidores Enzimáticos , Swainsonina , Humanos , Relação Dose-Resposta a Droga , Inibidores Enzimáticos/farmacologia , Inibidores Enzimáticos/síntese química , Inibidores Enzimáticos/química , Manosidases/antagonistas & inibidores , Manosidases/metabolismo , Modelos Moleculares , Simulação de Acoplamento Molecular , Estrutura Molecular , Relação Estrutura-Atividade , Swainsonina/farmacologia , Swainsonina/síntese química , Swainsonina/química , Compostos de Benzil/química , Compostos de Benzil/farmacologiaRESUMO
Heritable fungal endosymbiosis is underinvestigated in plant biology and documented in only three plant families (Convolvulaceae, Fabaceae, and Poaceae). An estimated 40% of morning glory species in the tribe Ipomoeeae (Convolvulaceae) have associations with one of two distinct heritable, endosymbiotic fungi (Periglandula and Chaetothyriales) that produce the bioactive metabolites ergot alkaloids, indole diterpene alkaloids, and swainsonine, which have been of interest for their toxic effects on animals and potential medical applications. Here, we report the occurrence of ergot alkaloids, indole diterpene alkaloids, and swainsonine in the Convolvulaceae; and the fungi that produce them based on synthesis of previous studies and new indole diterpene alkaloid data from 27 additional species in a phylogenetic, geographic, and life-history context. We find that individual morning glory species host no more than one metabolite-producing fungal endosymbiont (with one possible exception), possibly due to costs to the host and overlapping functions of the alkaloids. The symbiotic morning glory lineages occur in distinct phylogenetic clades, and host species have significantly larger seed size than nonsymbiotic species. The distinct and widely distributed endosymbiotic relationships in the morning glory family and their alkaloids provide an accessible study system for understanding heritable plant-fungal symbiosis evolution and their potential functions for host plants.
Assuntos
Alcaloides , Convolvulaceae , Alcaloides de Claviceps , Ipomoea , Animais , Convolvulaceae/metabolismo , Convolvulaceae/microbiologia , Swainsonina/metabolismo , Filogenia , Ipomoea/genética , Ipomoea/metabolismo , Ipomoea/microbiologia , Alcaloides de Claviceps/metabolismo , Alcaloides/metabolismo , Alcaloides DiterpenosRESUMO
Locoweeds are leguminous forbs known for their toxicity to livestock caused by the endophytic fungi Alternaria sect. Undifilum. Unlike the defensive mutualisms reported in many toxin-producing endophytes and their plant hosts, the benefits that A. sect. Undifilum can confer to it host plants remains unclear. Here, we conducted physiological and genetic analyses to show that A. (sect. Undifilum) oxytropis influences growth, especially root development, in its locoweed host Oxytropis ochrocephala and Arabidopsis. The presence of A. oxytropis significantly decreased primary root length while increasing the numbers of lateral roots and root hairs, and increasing plant leaf area and fresh weight. The fungus also increased the concentrations of plant endogenous auxin, and the expression of key genes for auxin biosynthesis, signaling, and transport. These effects on root development were abolished in mutants deficient in auxin signaling and polar transport. Alternaria oxytropis down-regulated expression of PIN1 but increased expression of PIN2, PIN7, and AUX1, which might reflect alterations in the spatial accumulation of auxin responsible for the changes in root architecture. Plant growth was insensitive to A. oxytropis when naphthylphthalamic acid was applied. Our findings indicate a function of A. oxytropis in promoting the growth and development of Arabidopsis via the regulation of auxin, which in turn suggests a possible role in benefiting its locoweed hosts via a process independent of its toxin production.
Assuntos
Proteínas de Arabidopsis , Arabidopsis , Endófitos/fisiologia , Alternaria , Arabidopsis/metabolismo , Swainsonina/análise , Swainsonina/metabolismo , Ácidos Indolacéticos/metabolismo , Raízes de Plantas/metabolismo , Proteínas de Arabidopsis/genética , Proteínas de Arabidopsis/metabolismoRESUMO
Astragalus adsurgens (A. adsurgens), which is considered a forage in China, grows widely in Eurasia and North America. However, Alternaria gansuense (A. gansuense) (synonym: Embellisia astragali) systematically infects A. adsurgens, producing swainsonine (SW), which poisons domesticated animals. In this study, we hypothesized that the A. gansuense SW-producing fungus is morphologically and molecularly related to the locoweed endophyte, Alternaria oxytropis (A. oxytropis), which systematically grows in host plants. Therefore, pure cultures of the fungi from diseased plants or endophytic interactions were collected from fields and assayed for SW via high-performance liquid chromatography linked to mass spectroscopy (HPLC-MS). The production of SW was also detected in A. adsurgens, A. oxytropis and diseased plants by assaying for the presence of the ß-ketoacyl synthase (KS) gene, which is required for SW synthesis. Diseased A. adsurgens and pure cultures of A. gansuense have SW and the healthy-looking A. adsurgens plants also contained SW, probably because they were infected with A. gansuense. Therefore, A. adsurgens-infected A. gansuense are not safe for livestock consumption.
Assuntos
Alternaria , Swainsonina , Animais , Swainsonina/análise , Alternaria/genética , Endófitos , ChinaRESUMO
OBJECTIVE: Swainsonine (SW) is the principal toxic ingredient of locoweeds, and is produced by multiple fungi. A key enzyme in the SW synthesis pathway is a hybrid swnk/nrps. To analyze the role of swnk in the SW biosynthesis pathway of Metarhizium anisopliae. RESULTS: The concentration of SW and the swnk expression in M. anisopliae fermentation from 1st to 7th day were determined using LC-MS and RT-qPCR, respectively. M. anisopliae had the highest SW content and swnk expression on the 5th day of fermentation; Mutant strain (MT) were obtained by PEG-mediated homologous recombination (HR) which knocked out swnk in the wild-type (WT) strain. Complemented-type (CT) strain were obtained by transforming a modified PUC19 complementation vector containing the geneticin (G418) resistance gene and swnK. SW was not detected in the MT strain and reverted to its original level in the CT strain; A Psilent-1 plasmid with Benomyl (ben)-resistant that was used interfered with swnk of WT strain. The level of SW was markedly diminished in the RNAi strain. RNAi of swnk affects the formation of the cell wall in M. anisopliae. CONCLUSION: These results indicate that swnk plays a crucial role in the SW biosynthesis of M. anisopliae.
Assuntos
Metarhizium , Swainsonina , Swainsonina/metabolismo , Metarhizium/genética , Metarhizium/metabolismo , Genes Fúngicos , FermentaçãoRESUMO
Plants produce various plant secondary compounds (PSCs) to deter the foraging of herbivorous mammals. However, little is known about whether PSCs can reshape gut microbiota and promote gut homeostasis of hosts. Using 16S rDNA sequencing to investigate the effects of PSCs on the gut microbiota of small herbivorous mammals, we studied plateau pikas (Ochotona curzoniae) fed diets containing swainsonine (SW) extracted from Oxytropis ochrocephala. Our results showed that both long- and short-term treatment of a single artificial diet in the laboratory significantly reduced alpha diversity and significantly affected beta diversity, core bacteria abundance, and bacterial functions in pikas. After SW was added to the artificial diet, the alpha diversity significantly increased in the long-term treatment, and core bacteria (e.g., Akkermansiaceae) with altered relative abundances in the two treatments showed no significant difference compared with pikas in the wild. The complexity of the co-occurrence network structure was reduced in the artificial diet, but it increased after SW was added in both treatments. Further, the abundances of bacteria related to altered alanine, aspartate, and glutamate metabolism in the artificial diet were restored in response to SW. SW further decreased the concentration of short-chain fatty acids (SCFAs) in both treatments. Our results suggest that PSCs play a key role in regulating gut microbiota community and intestinal homeostasis, thereby maintaining host health. KEY POINTS: ⢠Swainsonine improves the intestinal bacterial diversity of plateau pikas. ⢠Swainsonine promotes the recovery of core bacterial abundances in the gut of plateau pikas. ⢠Swainsonine promotes the restoration of intestinal bacterial functions of plateau pikas.
Assuntos
Microbioma Gastrointestinal , Lagomorpha , Animais , Bactérias/genética , Fenômenos Fisiológicos Bacterianos , SwainsoninaRESUMO
Swainsonine (SW, 1), a unique indolizine with poly-hydroxyl groups, was re-isolated from the plant endophytic fungus Alternaria oxytropis. The structure (including planar structure and relative configuration) was systematically elucidated by NMR spectra (including 1 H, 13 C, 1 H-1 H COSY, HMQC, HMBC, and NOESY spectra in DMSO-d6 and in CD3 OD); 1 H NMR spectra of the modified Mosher's products were first used to determine the absolute configuration of SW. More importantly, the complex coupled features of H-7α, H-7ß, and H-6α in the 1 H NMR spectrum of (1) were analyzed in details, which will provide aids for the planar and relative configuration determination of analogs.
Assuntos
Micotoxinas/análise , Swainsonina/análise , Alternaria/química , Espectroscopia de Ressonância Magnética , Micotoxinas/química , Swainsonina/químicaRESUMO
N-Substituted derivatives of 1,4-dideoxy-1,4-imino-d-mannitol (DIM), the pyrrolidine core of swainsonine, have been synthesized efficiently and stereoselectively from d-mannose with 2,3:5,6-di-O-isopropylidene DIM (10) as a key intermediate. These N-substituted derivatives include N-alkylated, N-alkenylated, N-hydroxyalkylated and N-aralkylated DIMs with the carbon number of the alkyl chain ranging from one to nine. The obtained 33 N-substituted DIM derivatives were assayed against various glycosidases, which allowed a systematic evaluation of their glycosidase inhibition profiles. Though N-substitution of DIM decreased their α-mannosidase inhibitory activities, some of the derivatives showed significant inhibition of other glycosidases.
Assuntos
Inibidores Enzimáticos/síntese química , Inibidores Enzimáticos/farmacologia , Glicosídeo Hidrolases/antagonistas & inibidores , Manitol/análogos & derivados , Animais , Inibidores Enzimáticos/química , Glicosídeo Hidrolases/metabolismo , Humanos , Imino Furanoses/síntese química , Imino Furanoses/química , Imino Furanoses/farmacologia , Concentração Inibidora 50 , Manitol/síntese química , Manitol/química , Manitol/farmacologia , Ratos , Swainsonina/químicaRESUMO
Locoweeds are toxic leguminous plants in Astragalus and Oxytropis genera that contain fungal endophytes of Alternaria section Undifilum species. These fungi produce swainsonine, an alkaloid α-mannosidase inhibitor that causes a neurological syndrome, locoism in grazing animals. A SWN gene cluster has been identified in many swainsonine-producing fungi. The swnK gene, which is an essential component of the swainsonine biosynthetic pathway, encodes a polyketide synthase-nonribosomal peptide synthase (PKS-NRPS). To determine if swnK was conserved among Alternaria section Undifilum endophytes of locoweed, the sequence of the KS region of swnK was compared between various swainsonine-producing fungi. The internal transcribed spacer (ITS), and glyceraldehyde-3-phosphate dehydrogenase (GPD) regions from the same fungi were also assessed. Sequences were examined at the nucleotide and protein levels. Alternaria oxytropis, A. fulva, A. cinerea, and Alternaria sp. from Swainsona species produced distinct clades for all multigene data sets. swnK-KS sequence did not differ among fungi isolated from Astragalus mollissimus varieties or A. lentiginosus varieties. The swnK-KS amino acid sequence was essentially identical among all swainsonine-producing Alternaria sp. Two low swainsonine-producing fungi, Alternaria bornmuelleri and A. gansuense, clustered together, as did non-pathogen Alternaria endophytes. The swnK-KS sequence comparisons were effective in identifying swainsonine production capability and differentiating among swainsonine-producing fungal species.
Assuntos
Policetídeos , Swainsonina , Alternaria/genética , Animais , Estados UnidosRESUMO
BACKGROUND: Swainsonine is a natural indolizidine alkaloid, its anti-tumor activity has been widely reported in varied cancers. This study aimed to investigate whether Swainsonine exerted anti-tumor impact on glioma cells, likewise uncovered the relative molecular mechanisms. METHODS: After administration with diverse concentrations of Swainsonine, cell growth, migration and invasion in U251 and LN444 cells were appraised by the common-used CCK-8, BrdU, flow cytometry and Transwell assays. MiR-92a mimic, inhibitor and the correlative NC were transfected into U251 and LN444 cells, and assessment of miR-92a expression was by utilizing qRT-PCR. Functions of miR-92a in above-mentioned cell biological processes were analyzed again in Swainsonine-treated cells. The momentous proteins of cell cycle, apoptosis and PI3K/AKT/mTOR pathway were ultimately examined by western blot. RESULTS: Swainsonine significantly hindered cell proliferation through decreasing cell viability, declining the percentage of BrdU cells, down-regulating CyclinD1 and up-regulating p16 expression. Enhancement of percentage of apoptotic cells was presented in Swainsonine-treated cells via activating cleaved-Caspase-3 and cleaved-Caspase-9. Additionally, Swainsonine impeded the abilities of migration and invasion by decreasing MMP-2, MMP-9, Vimentin and E-cadherin. Repression of miR-92a was observed in Swainsonine-treated cells, and miR-92a overexpression overturned the anti-tumor activity of Swainsonine in glioma cells. Finally, western blot assay displayed that Swainsonine hindered PI3K/AKT/mTOR pathway via regulating miR-92a. CONCLUSIONS: These discoveries corroborated that Swainsonine exerted anti-tumor impacts on glioma cells via repression of miR-92a, and inactivation of PI3K/AKT/mTOR signaling pathway.
Assuntos
Antineoplásicos Fitogênicos/farmacologia , Glioma/tratamento farmacológico , MicroRNAs/metabolismo , Transdução de Sinais/efeitos dos fármacos , Swainsonina/farmacologia , Antineoplásicos Fitogênicos/uso terapêutico , Linhagem Celular Tumoral , Movimento Celular/efeitos dos fármacos , Movimento Celular/genética , Proliferação de Células/efeitos dos fármacos , Proliferação de Células/genética , Ensaios de Seleção de Medicamentos Antitumorais , Regulação Neoplásica da Expressão Gênica/efeitos dos fármacos , Regulação Neoplásica da Expressão Gênica/genética , Glioma/patologia , Humanos , MicroRNAs/antagonistas & inibidores , MicroRNAs/genética , Invasividade Neoplásica/prevenção & controle , Fosfatidilinositol 3-Quinases/metabolismo , Proteínas Proto-Oncogênicas c-akt/metabolismo , Transdução de Sinais/genética , Swainsonina/uso terapêutico , Serina-Treonina Quinases TOR/metabolismoRESUMO
Many plant endophytes produce mycotoxins, but how host genetic variation influences endophyte colonization and mycotoxin production under natural conditions is poorly understood. This interaction has not been fully considered in many previous studies which used controlled experiments with agronomic or model plant species. Here, we investigated this interaction in a naturally occurring forb (a locoweed species) Oxytropis ochrocephala, its symbiotic endophyte Alternaria oxytropis, and the mycotoxin swainsonine. Host genetic variation was characterized by microsatellite markers. Endophyte infection rate and swainsonine levels were determined by PCR and HPLC, respectively. Genetic markers defined two distinct host populations and revealed that host genetics were significantly correlated with geographical location, elevation, and precipitation. As the host diverged, symbiotic interactions were reduced or failed to produce detectable swainsonine in one host population. Host genotype and precipitation had a significant impact in shaping swainsonine production at the population level. This study highlights the effect of host genotype in influencing this interaction in locoweeds.
Assuntos
Ascomicetos/crescimento & desenvolvimento , Astrágalo/microbiologia , Micotoxinas/biossíntese , Simbiose , Ascomicetos/metabolismo , Astrágalo/genética , Cromatografia Líquida de Alta Pressão , Variação Genética , Genótipo , Repetições de Microssatélites/genética , Micotoxinas/análise , Swainsonina/análise , Swainsonina/metabolismoRESUMO
Alternaria oxytropis is an endophytic fungus of locoweeds that synthesizes swainsonine toxin. In this work, we evaluated the effect of A. oxytropis on soybean seedlings and quantified swainsonine in different culture conditions. Soybean (Glycine max) seeds were co-cultured with A. oxytropis (at different concentrations of mycelial suspensions) in agar media and soil culture, and swainsonine was assayed using LC-MS/MS. The results showed evidence that A. oxytropis infected soybean seedlings produced detectable swainsonine in agar culture while the toxin was undetectable or below the detection limit (0.006% of swainsonine dry weight) in soil media even at higher concentrations of the fungus. These results suggest that swainsonine detection is highly dependent on culture conditions and that soybeans co-cultured with A. oxytropis in soil could potentially be used to limit toxin production.
Assuntos
Alternaria/patogenicidade , Glycine max/microbiologia , Swainsonina/análise , Cromatografia Líquida , Técnicas de Cocultura , Interações Hospedeiro-Patógeno , Limite de Detecção , Oxytropis , Plântula/crescimento & desenvolvimento , Plântula/microbiologia , Espectrometria de Massas em TandemRESUMO
Integrins are the major cell adhesion glycoproteins involved in cell-extracellular matrix (ECM) interaction and metastasis. Further, glycosylation on integrin is necessary for its proper folding and functionality. Herein, differential expression of integrins viz., αvß3 and αvß6 was examined in MDA-MB-231, MDA-MB-468 and MCF-10A cells, which signify three different stages of breast cancer development from highly metastatic to non-tumorigenic stage. The expression of αvß3 and αvß6 integrins at mRNA and protein levels was observed in all three cell lines and the results displayed a distinct pattern of expression. Highly metastatic cells showed enhanced expression of αvß3 than moderate metastatic and non-tumorigenic cells. The scenario was reversed in case of αvß6 integrin, which was strongly expressed in moderate metastatic and non-tumorigenic cells. N-glycosylation of αvß3 and αvß6 integrins is required for the attachment of cells to ECM proteins like fibronectin. The cell adhesion properties were found to be different in these cancer cells with respect to the type of integrins expressed. The results testify that αvß3 integrin in highly metastatic cells, αvß6 integrin in both moderate metastatic and non-tumorigenic cells play an important role in cell adhesion. The investigation typify that N-glycosylation on integrins is also necessary for cell-ECM interaction. Further, glycosylation inhibition by Swainsonine is found to be more detrimental to invasive property of moderate metastatic cells. Conclusively, types of integrins expressed as well as their N-glycosylation pattern alter during the course of breast cancer progression.
Assuntos
Antígenos de Neoplasias/metabolismo , Neoplasias da Mama/metabolismo , Neoplasias da Mama/patologia , Junções Célula-Matriz/metabolismo , Progressão da Doença , Integrina alfaVbeta3/metabolismo , Integrinas/metabolismo , Anticorpos Bloqueadores/farmacologia , Adesão Celular/efeitos dos fármacos , Linhagem Celular Tumoral , Junções Célula-Matriz/efeitos dos fármacos , Feminino , Fibronectinas/metabolismo , Glicosilação , Humanos , Invasividade Neoplásica , Swainsonina/química , Swainsonina/farmacologiaRESUMO
Swainsonine is an Astragalus membranaceus extract. It is indole, alkaloid, and soluble in water. Its effect on rat cardiomyocytes apoptosis, and the mechanisms underlying that effect, were investigated by inducing apoptosis in H9c2 cells. This was detected by MTT assay, Annexin V-FITC/propidium iodide double staining and western blotting. Flow cytometry and fluorescence microscopy were used to confirm swainsonine's effect on mitochondrial membrane potential and levels of reactive oxygen species, while an ATP-dependent bioluminescence assay kit served to find the ATP contents. Assessment was also carried out for peroxisome proliferator activated receptor γ co-activator 1α (PGC-1α) expression levels as well as those of such apoptosis-associated proteins as Cytochrome c, Caspase-3, B-cell lymphoma 2 (Bcl-2) and Bcl-2-associated X protein (Bax). Overall, indications were that swainsonine may have the potential to inhibit viability of cells, decrease expression of PGC-1α, induce mitochondrial dysfunction, upregulate Cytochrome c, Bax and Caspase-3, and downregulate Bcl-2. The suggestion would be that apoptosis may be induced through signalling pathways in H9c2 cells mediated by mitochondria.
Assuntos
Antineoplásicos Fitogênicos/farmacologia , Regulação da Expressão Gênica/efeitos dos fármacos , Mitocôndrias/efeitos dos fármacos , Miócitos Cardíacos/efeitos dos fármacos , Swainsonina/farmacologia , Trifosfato de Adenosina/antagonistas & inibidores , Trifosfato de Adenosina/metabolismo , Animais , Apoptose/efeitos dos fármacos , Caspase 3/genética , Caspase 3/metabolismo , Linhagem Celular , Citocromos c/genética , Citocromos c/metabolismo , Potencial da Membrana Mitocondrial/efeitos dos fármacos , Mitocôndrias/metabolismo , Miócitos Cardíacos/citologia , Miócitos Cardíacos/metabolismo , Coativador 1-alfa do Receptor gama Ativado por Proliferador de Peroxissomo/antagonistas & inibidores , Coativador 1-alfa do Receptor gama Ativado por Proliferador de Peroxissomo/genética , Coativador 1-alfa do Receptor gama Ativado por Proliferador de Peroxissomo/metabolismo , Proteínas Proto-Oncogênicas c-bcl-2/antagonistas & inibidores , Proteínas Proto-Oncogênicas c-bcl-2/genética , Proteínas Proto-Oncogênicas c-bcl-2/metabolismo , Ratos , Espécies Reativas de Oxigênio/metabolismo , Transdução de Sinais , Proteína X Associada a bcl-2/agonistas , Proteína X Associada a bcl-2/genética , Proteína X Associada a bcl-2/metabolismoRESUMO
Sphingomyelin phosphodiesterase acid-like 3A (SMPDL3A) is a recently identified phosphodiesterase, which is a secreted N-linked glycoprotein. SMPDL3A is highly homologous to acid sphingomyelinase (aSMase), but unlike aSMase cannot cleave sphingomyelin. Rather, SMPDL3A hydrolyzes nucleotide tri- and diphosphates and their derivatives. While recent structural studies have shed light on these unexpected substrate preferences, many other aspects of SMPDL3A biology, which may give insight into its function in vivo, remain obscure. Here, we investigate the roles of N-glycosylation in the expression, secretion and activity of human SMPDL3A, using inhibitors of N-glycosylation and site-directed mutagenesis, with either THP-1 macrophages or CHO cells expressing human SMPDL3A. Tunicamycin (TM) treatment resulted in expression of non-glycosylated SMPDL3A that was not secreted, and was largely degraded by the proteasome. Proteasomal inhibition restored levels of SMPDL3A in TM-treated cells, although this non-glycosylated protein lacked phosphodiesterase activity. Enzymatic deglycosylation of purified recombinant SMPDL3A also resulted in significant loss of phosphodiesterase activity. Site-directed mutagenesis of individual N-glycosylation sites in SMPDL3A identified glycosylation of Asn69 and Asn222 as affecting maturation of its N-glycans and secretion. Glycosylation of Asn356 in SMPDL3A, an N-linked site conserved throughout the aSMase-like family, was critical for protection against proteasomal degradation and preservation of enzymatic activity. We provide the first experimental evidence for a predicted 22 residue N-terminal signal peptide in SMPDL3A, which is essential for facilitating glycosylation and is removed from the mature protein secreted from CHO cells. In conclusion, site-specific N-glycosylation is essential for the intracellular stability, secretion and activity of human SMPDL3A.
Assuntos
Monócitos/enzimologia , Proteínas Recombinantes de Fusão/química , Esfingomielina Fosfodiesterase/química , Sequência de Aminoácidos , Animais , Células CHO , Linhagem Celular , Cricetulus , Glicosilação/efeitos dos fármacos , Humanos , Indolizinas/farmacologia , Leupeptinas/farmacologia , Monócitos/citologia , Monócitos/efeitos dos fármacos , Mutagênese Sítio-Dirigida , Mutação , Peptídeo-N4-(N-acetil-beta-glucosaminil) Asparagina Amidase/química , Inibidores de Proteases/farmacologia , Complexo de Endopeptidases do Proteassoma/efeitos dos fármacos , Complexo de Endopeptidases do Proteassoma/metabolismo , Sinais Direcionadores de Proteínas , Estabilidade Proteica/efeitos dos fármacos , Proteínas Recombinantes de Fusão/genética , Proteínas Recombinantes de Fusão/metabolismo , Alinhamento de Sequência , Homologia de Sequência de Aminoácidos , Esfingomielina Fosfodiesterase/genética , Esfingomielina Fosfodiesterase/metabolismo , Swainsonina/farmacologia , Tunicamicina/farmacologiaRESUMO
KEY POINTS: Ionotropic glutamate receptor (iGluR) subunits are N-glycosylated at 4-12 sites, and Golgi processing produces mature receptors that contain high-mannose, hybrid and complex oligosaccharides. N-glycosylation is crucial for receptor biogenesis, influences receptor trafficking and provides a binding site for carbohydrate binding proteins. Glycan moieties are large, polar and occasionally charged, and they are attached at sites along iGluRs that position them for involvement in the structural changes underlying gating. Altering glycan content on kainate receptors (KARs), a subfamily of iGluRs, changes functional properties of the receptor, such as desensitization, recovery from desensitization and deactivation. We report the first observation that the charged trisaccharide HNK-1 is conjugated to native KARs, and we find that it substantially alters recombinant KAR functional properties. Our results show that the molecular composition of N-glycans can influence KAR biophysical properties, revealing a potential mechanism for fine-tuning the function of these receptors. ABSTRACT: Ionotropic glutamate receptors (iGluRs) are tetrameric proteins with between four and 12 consensus sites for N-glycosylation on each subunit, which potentially allows for a high degree of structural diversity conferred by this post-translational modification. N-glycosylation is required for proper folding of iGluRs in mammalian cells, although the impact of oligosaccharides on the function of successfully folded receptors is less clear. Glycan moieties are large, polar, occasionally charged and mediate many protein-protein interactions throughout the nervous system. Additionally, they are attached at sites along iGluR subunits that position them for involvement in the structural changes underlying gating. In the present study, we show that altering glycan content on kainate receptors (KARs) changes the functional properties of the receptors in a manner dependent on the identity of both the modified sugars and the subunit composition of the receptor to which they are attached. We also report that native KARs carry the complex capping oligosaccharide human natural killer-1. Glycosylation patterns probably differ between cell types, across development or with pathologies, and thus our findings reveal a potential mechanism for context-specific fine-tuning of KAR function through diversity in glycan structure.
Assuntos
Polissacarídeos/química , Receptores de Ácido Caínico/química , Receptores de Ácido Caínico/fisiologia , Alcaloides/farmacologia , Animais , Feminino , Glicosilação , Células HEK293 , Humanos , Masculino , Camundongos Endogâmicos C57BL , Camundongos Knockout , Receptores de Ácido Caínico/genética , Swainsonina/farmacologia , alfa-Manosidase/antagonistas & inibidoresRESUMO
Rapid changes in the Earth's atmosphere and climate associated with human activity can have significant impacts on agriculture including livestock production. CO2 concentration has risen from the industrial revolution to the current time, and is expected to continue to rise. Climatic changes alter physiological processes, growth, and development in numerous plant species, potentially changing concentrations of plant secondary compounds. These physiological changes may influence plant population density, growth, fitness, and toxin concentrations and thus influence the risk of toxic plants to grazing livestock. Locoweeds, swainsonine-containing Astragalus species, are one group of plants that may be influenced by climate change. We evaluated how two different swainsonine-containing Astragalus species responded to elevated CO2 concentrations. Measurements of biomass, crude protein, water soluble carbohydrates and swainsonine concentrations were measured in two chemotypes (positive and negative for swainsonine) of each species after growth at CO2 levels near present day and at projected future concentrations. Biomass and water soluble carbohydrate concentrations responded positively while crude protein concentrations responded negatively to elevated CO2 in the two species. Swainsonine concentrations were not strongly affected by elevated CO2 in the two species. In the different chemotypes, biomass responded negatively and crude protein concentrations responded positively in the swainsonine-positive plants compared to the swainsonine-negative plants. Ultimately, changes in CO2 and endophyte status will likely alter multiple physiological responses in toxic plants such as locoweed, but it is difficult to predict how these changes will impact plant herbivore interactions.
Assuntos
Astrágalo/efeitos dos fármacos , Astrágalo/metabolismo , Dióxido de Carbono/farmacologia , Swainsonina/metabolismo , Astrágalo/crescimento & desenvolvimento , Biomassa , Metabolismo dos Carboidratos/efeitos dos fármacos , Mudança Climática , Relação Dose-Resposta a Droga , Proteínas de Plantas/metabolismo , SolubilidadeRESUMO
Swainsonine is found in several plant species worldwide, and causes severe toxicosis in livestock grazing these plants, leading to a chronic condition characterized by weight loss, altered behavior, depression, decreased libido, infertility, and death. Swainsonine has been detected in 13 North American Astragalus species of which eight belong to taxa in four taxonomic sections, the Densifolii, Diphysi, Inflati, and Trichopodi. These sections belong to two larger groups representing several morphologically related species, the Pacific Piptolobi and the small-flowered Piptolobi. The objective of this study was to screen the other 31 species for swainsonine in sections Densifolii, Diphysi, Inflati, and Trichopodi previously not known to contain swainsonine. Furthermore, to broaden the scope further, 21 species within the 8 sections of the Pacific Piptolobi and the small flowered Piptolobi were screened for swainsonine. Swainsonine was detected for the first time in 36 Astragalus taxa representing 29 species using liquid and gas chromatography coupled with mass spectrometry. Several taxonomic sections were highly enriched in species that contain swainsonine while others were not. A systematic examination for swainsonine in these species will provide important information on the toxic risk of these species and may be a valuable reference for diagnosticians and land managers.
Assuntos
Astrágalo/química , Swainsonina/análise , Classificação , Cromatografia Gasosa-Espectrometria de Massas , América do Norte , Ilhas do Pacífico , Swainsonina/toxicidadeRESUMO
The fungus Slafractonia leguminicola, the causal agent of blackpatch disease of legumes produces two mycotoxins slaframine and swainsonine, causing slobbers' symptoms and locoism of grazing animals, respectively. The genetics of this important fungus is poorly understood. This work aimed to develop a genetic transformation system and evaluate the efficacy of RNA interference (RNAi) in S. leguminicola. In this study, S. leguminicola was transformed using a PEG-mediated method with a fungal construct that carries a hygromycin resistance cassette. To assess the use of RNAi, a silencing construct pSilentPKS1-AS was constructed which includes inverted repeat transgenes of the polyketide synthase gene (pks1) that is involved in melanin biosynthesis. Transformation of S. leguminicola with the IRT pks1 vector decreased pks1 transcripts levels 82-92% in knockdown mutants when compared with the wild type and was accompanied with a reduction in melanin and swainsonine production. These results demonstrate that RNAi can be a useful tool for studying gene function in S. leguminicola.
Assuntos
Ascomicetos/enzimologia , Regulação para Baixo , Policetídeo Sintases/genética , Ascomicetos/genética , Regulação Enzimológica da Expressão Gênica , Regulação Fúngica da Expressão Gênica , Técnicas de Silenciamento de Genes , Melaninas/biossíntese , Interferência de RNA , Swainsonina/metabolismoRESUMO
Epimerization of C5 of an N-hydroxypyrrolidine ring by regioselective oxidation to a nitrone followed by diastereoselective reduction provides a new approach to the synthesis of swainsonine and related compounds. The only protection in the synthesis of the potent mannosidase inhibitor DIM (1,4-dideoxy-1,4-imino-d-mannitol) was the acetonation of d-mannose.