RESUMEN
Enfumafungin-type antibiotics, represented by enfumafungin and fuscoatroside, belong to a distinct group of triterpenoids derived from fungi. These compounds exhibit significant antifungal properties with ibrexafungerp, a semisynthetic derivative of enfumafungin, recently gaining FDA's approval as the first oral antifungal drug for treating invasive vulvar candidiasis. Enfumafungin-type antibiotics possess a cleaved E-ring with an oxidized carboxyl group and a reduced methyl group at the break site, suggesting unprecedented C-C bond cleavage chemistry involved in their biosynthesis. Here, we show that a 4-gene (fsoA, fsoD, fsoE, fsoF) biosynthetic gene cluster is sufficient to yield fuscoatroside by heterologous expression in Aspergillus oryzae. Notably, FsoA is an unheard-of terpene cyclase-glycosyltransferase fusion enzyme, affording a triterpene glycoside product that relies on enzymatic fusion. FsoE is a P450 enzyme that catalyzes successive oxidation reactions at C19 to facilitate a C-C bond cleavage, producing an oxidized carboxyl group and a reduced methyl group that have never been observed in known P450 enzymes. Our study thus sets the important foundation for the manufacture of enfumafungin-type antibiotics using biosynthetic approaches.
Asunto(s)
Antifúngicos , Antifúngicos/química , Antifúngicos/farmacología , Antifúngicos/metabolismo , Aspergillus oryzae/enzimología , Aspergillus oryzae/metabolismo , Familia de Multigenes , Triterpenos/química , Triterpenos/metabolismo , Sistema Enzimático del Citocromo P-450/metabolismoRESUMEN
ortho-Quinone methides (o-QMs) are a class of highly reactive intermediates that serve as important nonisolable building blocks (NBBs) in organic synthesis and small-molecule library construction. Because of their instability and nonisolability, most reported o-QMs are generated through in situ chemical synthesis, and only a few natural o-QMs have been reported due to the lack of directed discovery strategies. Herein, a new natural o-QM precursor (trichophenol A, 2) was identified from the fungal strain of Trichoderma sp. AT0167 through genome mining, which was generated by trilA (nonreducing polyketide synthase) and trilB (2-oxoglutarate dependent dioxygenase). Combinatorial biosynthesis via two other known NRPKS genes with trilA and trilB was performed, leading to the generation of five new trichophenol o-QM oligomers (trichophenols D-H, 5-9). The strategy combining genome mining with combinatorial biosynthesis not only targetedly uncovered a new natural o-QM precursor but also produced various new molecules through oligomerization of the new o-QM and its designated o-QM acceptors without chemical synthesis and isolation of intermediates, which was named NBB genome mining-combinatorial biosynthesis strategy for o-QM molecule library construction. This study provides a new strategy for the targeted discovery of natural o-QMs and small-molecule library construction with natural o-QMs.
RESUMEN
Bisabosqual-type meroterpenoids are fungi-derived polyketide-terpenoid hybrids bearing a 2,3,3a,3a1,9,9a-hexahydro-1H-benzofuro[4,3,2-cde]chromene skeleton (6/6/6/5 ring system) or its seco-C-ring structure, and exhibit diverse bioactivities. Their unique structural architecture and impressive biological activities have led to considerable interest in discovering new analogues. However, to date, only nine analogues have been identified. Herein, we reported the isolation and identification of six new bisabosqual-type meroterpenoids stachybisbins C-H (1-6), together with one known compound bisabosqual C (7), from Stachybotrys bisbyi PYH05-7. Intriguingly, we found that 7, which contains the intact tetracyclic skeleton, can be non-enzymatically converted into its seco derivative stachybisbin I (8), unveiling the biosynthetic relationship between bisabosquals and seco-bisabosquals. Moreover, based on CRISPR/Cas9-mediated gene disruption, we revealed that the three-gene cluster responsible for the formation of LL-Z1272ß is associated with the biosynthesis of bisabosqual-type meroterpenoids, and then proposed a plausible route to 1-8.
Asunto(s)
Benzopiranos , Policétidos , Radiofármacos , TerpenosRESUMEN
Fernane-type triterpenoids are a small group of natural products mainly found in plants and fungi with a wide range of biological activities. Polytolypin is a representative fernane-type triterpenoid from fungi and possesses potent antifungal activity. So far, biosynthesis of fungal-derived fernane-type triterpenoids has not been characterized, which hinders the expansion of their structural diversity using biosynthetic approaches. Herein, we identified the biosynthetic gene cluster of polytolypin and elucidated its biosynthetic pathway through heterologous expression in Aspergillus oryzae NSAR1, which involves a new triterpene cyclase for the biosynthesis of the hydrocarbon skeleton motiol, followed by multiple oxidations via three P450 enzymes. Moreover, two new triterpene cyclases for the biosynthesis of two other fernane-type skeletons isomotiol and fernenol were identified from fungi, and were individually co-expressed with the three P450 enzymes involved in polytolypin biosynthesis. These studies led to the generation of 13 fernane-type triterpenoids including eight new compounds, and two of them showed stronger antifungal activity towards Candida albicans FIM709 than polytolypin.
Asunto(s)
Antifúngicos , Triterpenos , Antifúngicos/farmacología , Triterpenos/farmacología , Triterpenos/metabolismo , Sistema Enzimático del Citocromo P-450/metabolismo , Triterpenos Pentacíclicos , Vías Biosintéticas/genéticaRESUMEN
In fungi, there is a rare group of natural products harboring the 2,3,3a,9a-tetrahydro-4H-furo[2,3-b]chromene skeleton, represented by xyloketal B, which display a wide range of biological activities and have drawn significant attention. In this work, four new analogues simpliketals A-D (1-4), as well as two other new compounds simplilactones A and B (5 and 6), were isolated from Simplicillium sp. AHK071-01. Their structures were elucidated by extensive NMR spectroscopic methods, 13C NMR calculation, single-crystal X-ray diffraction, and ECD calculation. In addition, five known compounds (7-11) including alboatrin (7) were also obtained. Based on the structural similarity of the above compounds, we inferred that compounds 5, 6, and 8-11 might be biosynthetically related with 1-4 and 7, which allowed us to propose an alternative biosynthetic route to generate the furan-fused chromene skeleton of this class of compounds, instead of a previously presumed polyketide-terpenoid hybrid pathway. Finally, cytotoxicity assays showed that 1-4 exhibited weak inhibitory activity on PANC-1 cells and that 2 and 3 possessed moderate activity against SH-SY5Y cells.
Asunto(s)
Hypocreales , Neuroblastoma , Humanos , Benzopiranos/química , Estructura Molecular , FuranosRESUMEN
19-Hydroxybrevianamide M (1) and 6 R-methoxybrevianamide V (2), two new alkaloids, were isolated from an extract of the endophytic fungus Aspergillus sp. JNU18HC0517J, together with six known analogues (3- 8). Their structures were elucidated by extensive spectroscopic analyses, NMR calculations, and ECD calculations. 6 R-methoxybrevianamide V (2) was the first L-proline indole DKP alkaloid with substitution at C-6 on the proline ring. Furthermore, the cytotoxities and antimicrobial activities of these isolated compounds were also evaluated. Compound 8 exhibited moderate antibacterial activity against Staphylococcus aureus 209 P with a minimal inhibitory concentration (MIC) value of 16 µg/ml.[Figure: see text].
Asunto(s)
Alcaloides , Aspergillus , Estructura Molecular , Aspergillus/química , Alcaloides/química , Hongos , Alcaloides Indólicos/química , Antibacterianos/química , Pruebas de Sensibilidad MicrobianaRESUMEN
Twenty new malabaricane triterpenoids, astramalabaricosides A-T (1-20), were isolated from the roots of Astragalus membranaceus var. mongholicus (Astragali Radix). Their structures were determined by spectroscopic analysis, and the use of the circular dichroism exciton chirality method, quantum chemical calculations, and chemical methods. Malabaricane triterpenoids, an unusual group with the 6-6-5-tricyclic core, are distributed in plants (e.g., Simaroubaceae, Polypodiaceae, and Fabaceae), a marine sponge, and fungi, and their number obtained to date is limited. Compounds 1-20 were characterized as glycosides with a highly oxygenated side chain, and 13-20 were the first cyclic carbonate derivatives among the malabaricane triterpenoids. The stereocluster formed from the continuous hydroxylated chiral carbons in each highly oxygenated side chain and the 6-6-5-tricyclic core system were entirely segregated, and the independent identification of their stereoconfigurations required considerable effort. The migratory inhibitory and antiproliferative activities of 1-20 were evaluated by wound-healing and cell-viability assays, respectively. Most compounds showed significant migratory inhibitory activity, and a preliminary structure-activity relationship was developed. Malabaricane triterpenoids are being reported in the genus Astragalus for the first time.
Asunto(s)
Planta del Astrágalo , Triterpenos , Astragalus propinquus/química , Triterpenos/farmacología , Triterpenos/análisis , Raíces de Plantas/químicaRESUMEN
Isoniazid (INH) is a first-line anti-tuberculosis drug which can cause idiosyncratic liver injury, while the underlying mechanisms need to be further elucidated. In this study, we explored the time series gene expression profiling of a hepatocyte cell line under isoniazid treatment. Through cluster analysis and enrichment analysis of differentially expressed genes, we revealed a total of 6 gene clusters and a series of pathways related to hepatotoxicity, and 13 key candidate genes were identified according to the protein-protein interaction (PPI) network analysis and maSigPro analysis. These findings lay a foundation for understanding the mechanisms of isoniazid -induced liver toxicity and provide new target genes for the monitoring and treatment of INH-induced hepatotoxicity in the future.
Asunto(s)
Enfermedad Hepática Inducida por Sustancias y Drogas , Isoniazida , Antituberculosos/toxicidad , Enfermedad Hepática Inducida por Sustancias y Drogas/genética , Expresión Génica , Humanos , Isoniazida/metabolismo , Isoniazida/toxicidad , Hígado/metabolismo , Factores de TiempoRESUMEN
Fusicoccane-type terpenoids are a subgroup of diterpenoids featured with a unique 5-8-5 ring system. They are widely distributed in nature and possess a variety of biological activities. Up to date, only five fusicoccane-type diterpene synthases have been identified. Here, we identify a two-gene biosynthetic gene cluster containing a new fusicoccane-type diterpene synthase gene tadA and an associated cytochrome P450 gene tadB from Talaromyces wortmannii ATCC 26942. Heterologous expression reveals that TadA catalyzes the formation of a new fusicoccane-type diterpene talaro-7,13-diene. D2O isotope labeling combined with site-directed mutagenesis indicates that TadA might employ a different C2,6 cyclization strategy from the known fusicoccane-type diterpene synthases, in which a neutral intermediate is firstly formed and then protonated by an environmental proton. In addition, we demonstrate that the associated cytochrome P450 enzyme TadB is able to catalyze multiple oxidation of talaro-7,13-diene to yield talaro-6,13-dien-5,8-dione.
RESUMEN
To systematically study the susceptible genetic markers for liver injury induced by anti-tuberculosis drugs in the Chinese population, 109 genes related to drug metabolism, transport and immunity were captured by Haloplex capture technique from DNA samples of 41 patients with liver injury induced by anti-tuberculosis drugs and 39 healthy controls, and sequenced completely. Association study was conducted using Plink software. To verify the significant candidate SNPs, the χ 2 study was expanded to the control group from the 1000-person Genome Project of the East Asian population. SIFT and Polyphen2 software were used to predict the functional significance of the associated SNPs. Our results identified the UGT1A4 rs2011404 (χ 2 = 4.6809, P = 0.0305) as a susceptible genetic marker for liver injury induced by anti-tuberculosis drugs, and rs2011404 mutation might contribute to UGT1A4 protein dysfunction. This study has provided a potentially useful reference for establishing the precision medicine in rational uses of anti-tuberculosis drugs in the clinic.
Asunto(s)
Antituberculosos/efectos adversos , Enfermedad Hepática Inducida por Sustancias y Drogas/genética , Marcadores Genéticos , Predisposición Genética a la Enfermedad , Pueblo Asiatico , Estudios de Casos y Controles , China , Glucuronosiltransferasa/genética , Humanos , Polimorfismo de Nucleótido Simple , Tuberculosis/tratamiento farmacológicoRESUMEN
A novel valerenane sesquiterpenoid sinulaspirolactam A (1), together with five known compounds, was isolated from the soft coral Sinularia sp. Their structures were determined by spectroscopic analyses. The absolute configuration of 1 was established by ECD calculation. Compound 1 was the first example of valerenane sesquiterpenoid bearing an aza-spiro[4.5] ring moiety, the plausible biogenetic pathway of which was proposed. Cytotoxic activities of these compounds were also evaluated.
Asunto(s)
Antozoos/química , Antineoplásicos/química , Antineoplásicos/farmacología , Sesquiterpenos/química , Sesquiterpenos/farmacología , Animales , Línea Celular Tumoral , Supervivencia Celular/efectos de los fármacos , Modelos Moleculares , Estructura MolecularRESUMEN
Malectin is a newly discovered endoplasmic reticulum (ER)-resident lectin, which specifically recognizes Glc2Man9GlcNAc2 on newly synthesized glycoproteins. We have previously demonstrated that malectin forms a complex with ribophorin I for selective retention of misfolded glycoproteins inside the cell. Here, we showed that ribophorin I also functions to regulate the subcellular localization of malectin under various conditions. Even though malectin does not contain an ER-retention signal motif, we found that endogenous malectin mainly localizes in the ER, which is disrupted upon suppression of ribophorin I, leading to its movement from ER to Golgi. In contrast, under ER-stress conditions, malectin mainly localizes in the Golgi, which is restored to ER localization by overexpression of ribophorin I. These results indicate that the subcellular localization of malectin is accurately regulated by the expression level of ribophorin I, which will provide further insights into the understanding of the function of malectin.
Asunto(s)
Estrés del Retículo Endoplásmico , Lectinas/metabolismo , Proteínas de la Membrana/metabolismo , Retículo Endoplásmico/metabolismo , Células HeLa , Humanos , Transporte de ProteínasRESUMEN
Ischemia reperfusion (IR) injury is known as a major issue in cardiac transplantation and various pathogenesis are involved in myocardial IR injury. Here, we show that tumor necrosis factor receptor-associated factor 3 (TRAF3) was increased in hearts of mice with cardiac IR injury and in cardiomyocytes incubated with lipopolysaccharide (LPS) and H2O2. Reducing TRAF3 expression in vivo markedly reduced the infacrted area, attenuated the histological changes, improved cardiac dysfunction and injury in mice subjected to IR injury. Functional study further indicated that TRAF3 knockdown inhibited apoptosis in murine hearts of mice with cardiac IR injury and in LPS and H2O2-cotreated cardiomyocytes, as evidenced by the decreased expression of cleaved Caspase-3 and poly (ADP-ribose) polymerases (PARP). In addition, inflammatory response and oxidative stress observed in hearts of mice with IR operation were significantly alleviated by TRAF3 knockdown through inhibiting nuclear factor-κB (NF-κB) and xanthine oxidase (XO) signaling pathways, and similar results were detected in LPS and H2O2-cotreated cardiomyocytes in vitro. Moreover, the loss of TRAF3 also restrained the phosphorylated c-Jun N-terminal protein kinase (JNK) activation following cardiac IR injury. Importantly, blocking JNK activation, as TRAF3 knockdown, greatly reduced apoptosis, inflammation and reactive oxygen species (ROS) production in LPS and H2O2-cotreated cardiomyocytes. In contrast, TRAF3 knockdown-reduced apoptosis, inflammatory response and oxidative stress were significantly rescued by promoting JNK activity in LPS and H2O2-cotreated cardiomyocytes. In summary, the results of our study indicated that repressing TRAF3 expression could be served as essential therapeutic target for protection against cardiac IR injury through restraining JNK-meditated apoptosis, inflammation and the production of ROS.
Asunto(s)
Daño por Reperfusión Miocárdica/prevención & control , Miocitos Cardíacos/efectos de los fármacos , Factor 3 Asociado a Receptor de TNF/antagonistas & inhibidores , Animales , Peróxido de Hidrógeno , Inflamación/tratamiento farmacológico , Lipopolisacáridos , Sistema de Señalización de MAP Quinasas , Ratones , Daño por Reperfusión Miocárdica/tratamiento farmacológico , Miocitos Cardíacos/metabolismo , Estrés Oxidativo/efectos de los fármacos , Especies Reactivas de Oxígeno/metabolismo , Factor 3 Asociado a Receptor de TNF/metabolismoRESUMEN
Three pairs of new isopentenyl dibenzo[b,e]oxepinone enantiomers, (+)-(5S)-arugosin K (1a), (-)-(5R)-arugosin K (1b), (+)-(5S)-arugosin L (2a), (-)-(5R)-arugosin L (2b), (+)-(5S)-arugosin M (3a), (-)-(5R)-arugosin M (3b), and a new isopentenyl dibenzo[b,e]oxepinone, arugosin N (4), were isolated from a wetland soil-derived fungus Talaromyces flavus, along with two known biosynthetically-related compounds 5 and 6. Among them, arugosin N (4) and 1,6,10-trihydroxy-8-methyl-2-(3-methyl-2-butenyl)-dibenz[b,e]oxepin-11(6H)-one (CAS: 160585-91-1, 5) were obtained as the tautomeric mixtures. The structures of isolated compounds were determined by detailed spectroscopic analysis. In addition, the absolute configurations of these three pairs of new enantiomers were determined by quantum chemical ECD calculations.
Asunto(s)
Oxepinas , Microbiología del Suelo , Talaromyces/química , Oxepinas/química , Oxepinas/aislamiento & purificación , HumedalesRESUMEN
We previously demonstrated that malectin associates with ribophorin I, which is a subunit of oligosaccharyltransferase in the endoplasmic reticulum (ER). When malectin and ribophorin I are overexpressed in the ER, secretion of an α1-antitrypsin (AT) variant whose folding is defective, termed null Hong Kong (AT(NHK)), is decreased. To confirm whether the interaction between malectin and ribophorin I is involved in the decreased secretion of misfolded glycoproteins, we constructed an expression vector encoding truncated malectin, which could not associate with ribophorin I and had an Lys-Asp-Glu-Leu ER-retention sequence at its C-terminus. Expression of wild-type malectin abrogated AT(NHK) secretion, whereas expression of truncated malectin did not affect AT(NHK) secretion. Both wild-type and truncated malectin bound to AT(NHK), and the level of AT(NHK) was similar in cells expressing wild-type malectin and those expressing truncated malectin. Furthermore, we previously showed that decreased secretion of misfolded AT(NHK) by malectin overexpression is restored by treatment with a proteasome inhibitor. These results clearly demonstrate that the association of malectin with ribophorin I is required to capture misfolded glycoproteins and direct them to the degradation pathway.
Asunto(s)
Lectinas/metabolismo , Proteínas de la Membrana/metabolismo , alfa 1-Antitripsina/metabolismo , Células HeLa , Humanos , Lectinas/genética , Proteínas de la Membrana/química , Proteínas de la Membrana/genética , Mutación , Oligopéptidos/química , Oligopéptidos/metabolismo , Pliegue de Proteína , Mapas de Interacción de Proteínas , Señales de Clasificación de Proteína , Proteolisis , Deficiencias en la Proteostasis/genética , Deficiencias en la Proteostasis/metabolismo , alfa 1-Antitripsina/química , alfa 1-Antitripsina/genéticaRESUMEN
Malectin is an endoplasmic reticulum-resident lectin, which recognizes di-glucosylated Glc(2)Man(9)GlcNAc(2) (G2M9) N-glycans on newly synthesized glycoproteins. We previously demonstrated that malectin preferentially associates with misfolded glycoproteins and inhibits their secretion (Chen, Y., Hu, D., Yabe, R., Tateno, H., Qin, S. Y., Matsumoto, N., Hirabayashi, J., and Yamamoto, K. (2011) Mol. Biol. Cell 22, 3559-3570). The sugar binding activity of malectin is required for this process. However, because G2M9 N-glycans are generated at the very early stage of processing and are typically found on both misfolded glycoproteins and glycoproteins undergoing folding, other mechanisms must underlie the preference of malectin for misfolded glycoproteins. Here, we searched for proteins that were co-immunoprecipitated with malectin, and we found that malectin formed a stable complex with an endoplasmic reticulum-resident transmembrane protein, ribophorin I. Co-expression of malectin and ribophorin I significantly enhanced the association between malectin and a folding-defective α1-antitrypsin variant (null Hong Kong) and reduced its secretion; however, secretion of wild-type α1-antitrypsin was not affected. The enhanced association and reduced secretion were counteracted by siRNA-mediated down-regulation of ribophorin I. Also, a reporter assay revealed that ribophorin I preferentially interacted with misfolded ribonuclease A but not with the native form, suggesting that ribophorin I may function as a chaperone that recognizes misfolded proteins inside cells. These results provide the first evidence of the mechanism by which malectin preferentially associates with misfolded glycoproteins.
Asunto(s)
Retículo Endoplásmico/metabolismo , Glicoproteínas/metabolismo , Lectinas/metabolismo , Proteínas de la Membrana/metabolismo , Secuencia de Aminoácidos , Western Blotting , Glicoproteínas/química , Glicoproteínas/genética , Células HEK293 , Humanos , Inmunoprecipitación , Lectinas/genética , Proteínas de la Membrana/genética , Datos de Secuencia Molecular , Mutación , Polisacáridos/metabolismo , Unión Proteica , Pliegue de Proteína , Interferencia de ARN , Espectrometría de Fluorescencia , Espectrometría de Masa por Láser de Matriz Asistida de Ionización Desorción , alfa 1-Antitripsina/química , alfa 1-Antitripsina/genética , alfa 1-Antitripsina/metabolismoRESUMEN
From the fungus Trichoderma sp., we isolated seven novel 18-residue peptaibols, neoatroviridins E-K (1-7), and six new 14-residue peptaibols, harzianins NPDG J-O (8-13). Additionally, four previously characterized 18-residue peptaibols neoatroviridins A-D (14-17) were also identified. The structural configurations of the newly identified peptaibols (1-13) were determined by comprehensive nuclear magnetic resonance (NMR) and high-resolution electrospray ionization tandem mass spectrometry (HR-ESI-MS/MS) data. Their absolute configurations were further determined using Marfey's method. Notably, compounds 12 and 13 represent the first 14-residue peptaibols containing an acidic amino acid residue. In antimicrobial assessments, all 18-residue peptaibols (1-7, 14-17) exhibited moderate inhibitory activities against Staphylococcus aureus 209P, with minimum inhibitory concentration (MIC) values ranging from 8-32 µg·mL-1. Moreover, compound 9 exhibited moderate inhibitory effect on Candida albicans FIM709, with a MIC value of 16 µg·mL-1.
Asunto(s)
Antiinfecciosos , Trichoderma , Peptaiboles/farmacología , Peptaiboles/química , Trichoderma/química , Trichoderma/metabolismo , Espectrometría de Masas en Tándem/métodos , Antiinfecciosos/farmacología , Espectrometría de Masa por Ionización de Electrospray/métodosRESUMEN
Newly synthesized glycoproteins destined for secretion are transported from the endoplasmic reticulum (ER), through the Golgi and toward the cell surface. In this secretion pathway, several intracellular ER- or Golgi-resident transmembrane proteins serve as cargo receptors. ER-Golgi intermediate compartment (ERGIC)-53, VIP36 and VIPL, which have an L-type lectin domain within the luminal portion, participate in the vectorial transport of glycoproteins via sugar-protein interactions. To understand the nature of these receptors, monoclonal antibodies were generated against human ERGIC-53, VIP36 and VIPL using 293T cells expressing these receptors on cell surfaces. These cells were used to immunize rats and for screening antibody-producing clones. Flow cytometric analysis and immunoprecipitation studies showed that the obtained monoclonal antibodies bound specifically to the corresponding cargo receptors. Immunostaining of HeLa cells using the monoclonal antibodies showed that the localization of ERGIC-53 changed from relatively broad distribution in both the ER and the Golgi under normal conditions to a compact distribution in the Golgi under ER stress conditions. This redistribution was also observed by the overexpression of ERGIC-53 and abrogated by co-expression with VIPL but not VIP36. Real-time polymerase chain reaction revealed that ERGIC-53 along with several chaperone proteins was up-regulated after tunicamycin treatment; however, the expression of VIPL was unchanged. Furthermore, ERGIC-53 co-precipitated with VIPL but not VIP36, indicating that ERGIC-53 may interact with VIPL in the ER, which may regulate the localization of ERGIC-53 inside cells. Taken together, these observations provide new insights into the regulation of these cargo receptors and the quality control of glycoproteins within cells.
Asunto(s)
Estrés del Retículo Endoplásmico , Retículo Endoplásmico/metabolismo , Lectinas de Unión a Manosa/metabolismo , Proteínas de la Membrana/metabolismo , Animales , Anticuerpos Monoclonales/inmunología , Aparato de Golgi/metabolismo , Células HEK293 , Células HeLa , Humanos , Lectinas/genética , Lectinas/inmunología , Lectinas/metabolismo , Lectinas de Unión a Manosa/genética , Lectinas de Unión a Manosa/inmunología , Proteínas de la Membrana/genética , Proteínas de la Membrana/inmunología , Proteínas de Transporte de Membrana/genética , Proteínas de Transporte de Membrana/inmunología , Proteínas de Transporte de Membrana/metabolismo , Ratas , Ratas Endogámicas Lew , Estrés Fisiológico , Transcripción Genética , Tunicamicina/farmacología , Regulación hacia Arriba/efectos de los fármacosRESUMEN
Misfolded glycoproteins are translocated from the endoplasmic reticulum (ER) into the cytoplasm for proteasome-mediated degradation. OS-9 protein is thought to participate in ER-associated glycoprotein degradation (ERAD). The recombinant biotinylated mannose 6-phosphate receptor homology (MRH) domain of human OS-9 (OS-9(MRH)) together with six kinds of mutated OS-9(MRH) were prepared and mixed with R-phycoerythrin (PE)-labeled streptavidin to form tetramers (OS-9(MRH)-SA). The PE-labeled OS-9(MRH)-SA bound to HeLaS3 cells in a metal ion-independent manner through amino acid residues homologous to those participating in sugar binding of the cation-dependent mannose 6-phosphate receptor, and this binding was greatly increased by swainsonine, deoxymannojirimycin, or kifunensine treatment. N-Acetylglucosaminyltransferase I-deficient Lec1 cells, but not Lec2 or Lec8 cells, were also strongly bound by the tetramer. OS-9(MRH)-SA binding to the cells was strongly inhibited by Manalpha1,6(Manalpha1,3)Manalpha1,6(Manalpha1,3)Man and Manalpha1,6Man. To further determine the specificity of native ligands for OS-9(MRH), frontal affinity chromatography was performed using a wide variety of 92 different oligosaccharides. We found that several N-glycans containing terminal alpha1,6-linked mannose in the Manalpha1,6(Manalpha1,3)Manalpha1,6(Manalpha1,3)Man structure were good ligands for OS-9(MRH), having K(a) values of approximately 10(4) M(-1) and that trimming of either an alpha1,6-linked mannose from the C-arm or an alpha1,3-linked mannose from the B-arm abrogated binding to OS-9(MRH). An immunoprecipitation experiment demonstrated that the alpha1-antitrypsin variant null(Hong Kong), but not wild-type alpha1-antitrypsin, selectively interacted with OS-9 in the cells in a sugar-dependent manner. These results suggest that trimming of the outermost alpha1,2-linked mannose on the C-arm is a critical process for misfolded proteins to enter ERAD.
Asunto(s)
Retículo Endoplásmico/metabolismo , Lectinas/química , Lectinas/metabolismo , Proteínas de Neoplasias/química , Proteínas de Neoplasias/metabolismo , Animales , Sitios de Unión , Células CHO , Secuencia de Carbohidratos , Cricetinae , Cricetulus , Células HeLa , Humanos , Datos de Secuencia Molecular , Polisacáridos/química , Polisacáridos/metabolismoRESUMEN
Malectin is a membrane-anchored endoplasmic reticulum (ER)-resident lectin, which is first identified in Xenopus laevis in 2008 and highly conserved among animals. Malectin plays an important role in the quality control of glycoprotein in the ER through recognizing the Glc2Man9GlcNAc2 (G2M9) oligosaccharide chain on the newly synthesized glycoproteins. In this chapter, we will describe the preparation of recombinant soluble malectin and its tetramer, which might be developed as useful tools for detection of Glcα1-3Glc containing glycans on the cell surface.