RESUMEN

EDEM3 encodes a protein that converts Man8GlcNAc2 isomer B to Man7-5GlcNAc2. It is involved in the endoplasmic reticulum-associated degradation pathway, responsible for the recognition of misfolded proteins that will be targeted and translocated to the cytosol and degraded by the proteasome. In this study, through a combination of exome sequencing and gene matching, we have identified seven independent families with 11 individuals with bi-allelic protein-truncating variants and one individual with a compound heterozygous missense variant in EDEM3. The affected individuals present with an inherited congenital disorder of glycosylation (CDG) consisting of neurodevelopmental delay and variable facial dysmorphisms. Experiments in human fibroblast cell lines, human plasma, and mouse plasma and brain tissue demonstrated decreased trimming of Man8GlcNAc2 isomer B to Man7GlcNAc2, consistent with loss of EDEM3 enzymatic activity. In human cells, Man5GlcNAc2 to Man4GlcNAc2 conversion is also diminished with an increase of Glc1Man5GlcNAc2. Furthermore, analysis of the unfolded protein response showed a reduced increase in EIF2AK3 (PERK) expression upon stimulation with tunicamycin as compared to controls, suggesting an impaired unfolded protein response. The aberrant plasma N-glycan profile provides a quick, clinically available test for validating variants of uncertain significance that may be identified by molecular genetic testing. We propose to call this deficiency EDEM3-CDG.

Asunto(s)

Proteínas de Unión al Calcio/genética , Trastornos Congénitos de Glicosilación/genética , Retículo Endoplásmico/genética , alfa-Manosidasa/genética , Adolescente , Alelos , Proteínas de Unión al Calcio/deficiencia , Línea Celular , Niño , Preescolar , Trastornos Congénitos de Glicosilación/sangre , Discapacidades del Desarrollo/genética , Femenino , Glicoproteínas/sangre , Glicosilación , Humanos , Lactante , Discapacidad Intelectual/genética , Masculino , Mutación , Linaje , Polisacáridos/sangre , Deficiencias en la Proteostasis/genética , alfa-Manosidasa/deficiencia

RESUMEN

N-glycosylation has remained mostly unexplored in Piroplasmida, an order of tick-transmitted pathogens of veterinary and medical relevance. Analysis of 11 piroplasmid genomes revealed three distinct scenarios regarding N-glycosylation: Babesia sensu stricto (s.s.) species add one or two N-acetylglucosamine (NAcGlc) molecules to proteins; Theileria equi and Cytauxzoon felis add (NAcGlc)2-mannose, while B. microti and Theileria s.s. synthesize dolichol-P-P-NAcGlc and dolichol-P-P-(NAcGlc)2 without subsequent transfer to proteins. All piroplasmids possess the gene complement needed for the synthesis of the N-glycosylation substrates, dolichol-P and sugar nucleotides. The oligosaccharyl transferase of Babesia species, T. equi and C. felis, is predicted to be composed of only two subunits, STT3 and Ost1. Occurrence of short N-glycans in B. bovis merozoites was experimentally demonstrated by fluorescence microscopy using a NAcGlc-specific lectin. In vitro growth of B. bovis was significantly impaired by tunicamycin, an inhibitor of N-glycosylation, indicating a relevant role for N-glycosylation in this pathogen. Finally, genes coding for N-glycosylation enzymes and substrate biosynthesis are transcribed in B. bovis blood and tick stages, suggesting that this pathway is biologically relevant throughout the parasite life cycle. Elucidation of the role/s exerted by N-glycans will increase our understanding of these successful parasites, for which improved control measures are needed.

RESUMEN

Changes in protein levels in different components of the apical junctional complex occur in colorectal cancer (CRC). Claudin­3 is one of the main constituents of tight junctions, and its overexpression can increase the paracellular flux of macromolecules, as well as the malignant potential of CRC cells. The aim of this study was to investigate the molecular mechanisms involved in the regulation of claudin­3 and its prognostic value in CRC. In silico evaluation in each of the CRC consensus molecular subtypes (CMSs) revealed that high expression levels of CLDN3 (gene encoding claudin­3) in CMS2 and CMS3 worsened the patients' long­term survival, whereas a decrease in claudin­3 levels concomitant with a reduction in phosphorylation levels of epidermal growth factor receptor (EGFR) and insulin­like growth factor 1 receptor (IGF1R) could be achieved by inhibiting N­glycan biosynthesis in CRC cells. We also observed that specific inactivation of these receptor tyrosine kinases (RTKs) led to a decrease in claudin­3 levels, and this regulation seems to be mediated by phospholipase C (PLC) and signal transducer and activator of transcription 3 (STAT3) in CRC cells. RTKs are modulated by their N­linked glycans, and inhibition of N­glycan biosynthesis decreased the claudin­3 levels; therefore, we evaluated the correlation between N­glycogenes and CLDN3 expression levels in each of the CRC molecular subtypes. The CMS1 (MSI immune) subtype concomitantly exhibited low expression levels of CLDN3 and N­glycogenes (MGAT5, ST6GAL1, and B3GNT8), whereas CMS2 (canonical) exhibited high gene expression levels of CLDN3 and N­glycogenes (ST6GAL1 and B3GNT8). A robust positive correlation was also observed between CLDN3 and B3GNT8 expression levels in all CMSs. These results support the hypothesis of a mechanism integrating RTK signaling and N­glycosylation for the regulation of claudin­3 levels in CRC, and they suggest that CLDN3 expression can be used to predict the prognosis of patients identified as CMS2 or CMS3.

Asunto(s)

Antígenos CD/genética , Claudina-3/genética , Neoplasias Colorrectales/genética , N-Acetilglucosaminiltransferasas/genética , Sialiltransferasas/genética , Neoplasias Colorrectales/patología , Supervivencia sin Enfermedad , Receptores ErbB/genética , Femenino , Regulación Neoplásica de la Expresión Génica/genética , Glicosilación , Humanos , Masculino , Persona de Mediana Edad , Pronóstico , Receptor IGF Tipo 1/genética , Factor de Transcripción STAT3/genética , Transducción de Señal/genética

RESUMEN

Bifidobacteria represent one of the first colonizers of human gut microbiota, providing to this ecosystem better health and nutrition. To maintain a mutualistic relationship, they have enzymes to degrade and use complex carbohydrates non-digestible by their hosts. To succeed in the densely populated gut environment, they evolved molecular strategies that remain poorly understood. Herein, we report a novel mechanism found in probiotic Bifidobacteria for the depolymerization of the ubiquitous 2-acetamido-2-deoxy-4-O-(ß-d-mannopyranosyl)-d-glucopyranose (Man-ß-1,4-GlcNAc), a disaccharide that composes the universal core of eukaryotic N-glycans. In contrast to Bacteroidetes, these Bifidobacteria have a specialist and strain-specific ß-mannosidase that contains three distinctive structural elements conferring high selectivity for Man-ß-1,4-GlcNAc: a lid that undergoes conformational changes upon substrate binding, a tryptophan residue swapped between the two dimeric subunits to accommodate the GlcNAc moiety, and a Rossmann fold subdomain strategically located near to the active site pocket. These key structural elements for Man-ß-1,4-GlcNAc specificity are highly conserved in Bifidobacterium species adapted to the gut of a wide range of social animals, including bee, pig, rabbit, and human. Together, our findings uncover an unprecedented molecular strategy employed by Bifidobacteria to selectively uptake carbohydrates from N-glycans in social hosts.

Asunto(s)

Bifidobacterium/metabolismo , Microbioma Gastrointestinal/fisiología , Tracto Gastrointestinal/microbiología , Polisacáridos/metabolismo , beta-Manosidasa/metabolismo , Animales , Dominio Catalítico , Ecosistema , Humanos , Triptófano/metabolismo

RESUMEN

New therapeutic strategies against leishmaniasis are desirable, since the treatment against disease presents problems, such as the toxicity, high cost and/or parasite resistance. As consequence, new antileishmanial compounds are necessary to be identified, as presenting high activity against Leishmania, but low toxicity in mammalian hosts. In the present study, a Leishmania proteome mining strategy was developed, in order to select new drug targets with low homology to human proteins, but that are considered relevant for the parasite' survival. Results showed a hypothetical protein, which was functionally annotated as a glucosidase-like protein, as presenting such characteristics. This protein was associated with the metabolic network of the N-Glycan biosynthesis pathway in Leishmania, and two specific inhibitors - acarbose and miglitol - were predicted to be potential targets against it. In this context, miglitol [1-(2-Hydroxyethyl)-2-(hydroxymethyl)piperidine-3,4,5-triol] was tested against stationary promastigotes and axenic amastigotes of the Leishmania amazonensis and L. infantum species, and results showed high values of antileishmanial inhibition against both parasite species. Miglitol showed also efficacy in the treatment of Leishmania-infected macrophages; thus denoting its potential use as an antileishmanial candidate. In conclusion, this work presents a new drug target identified by a proteome mining strategy associated with bioinformatics tools, and suggested its use as a possible candidate to be applied in the treatment against disease.

Asunto(s)

Antiprotozoarios/química , Biología Computacional , Minería de Datos , Descubrimiento de Drogas , Leishmania/metabolismo , Proteoma , Proteómica , Animales , Antiprotozoarios/farmacología , Biología Computacional/métodos , Femenino , Humanos , Leishmania/efectos de los fármacos , Leishmania/genética , Leishmaniasis Cutánea/tratamiento farmacológico , Leishmaniasis Cutánea/parasitología , Macrófagos/efectos de los fármacos , Macrófagos/parasitología , Ratones , Modelos Moleculares , Anotación de Secuencia Molecular , Conformación Proteica , Proteómica/métodos , Relación Estructura-Actividad

RESUMEN

A strain of embryonic human kidney cells (HEK293) was transiently co-transfected with the expression vectors coding for the α- and ß-subunits of human thyroid-stimulating hormone (hTSH), and, for the first time, a human cell-derived recombinant hTSH was synthesized and extensively characterized. The purification strategy involving two steps provided an overall yield of 55% and a purity level > 90%. The purified material (hTSH-HEK) was analyzed and compared to a CHO-derived recombinant preparation (hTSH-CHO) and to a pituitary-derived (hTSH-Pit) preparation. The three preparations showed an equivalent purity (> 95%) with a hTSH-HEK molecular mass 2.1% lower than that of hTSH-CHO and 2.7% higher than that of hTSH-Pit. Remarkable differences were found in the carbohydrate moiety, the lowest sialic acid content and highest fucose content being observed in hTSH-HEK. In vivo biological activity was confirmed for the three preparations, the hTSH-HEK bioactivity being 39 and 16% lower than those of hTSH-CHO and hTSH-Pit, respectively. The hTSH-HEK circulatory half-life (t 1/2) was also shorter than those of hTSH-CHO (1.5-fold) and hTSH-Pit (1.2-fold). According to these findings, HEK-293-derived hTSH can be considered to be useful for clinical applications, in view as well of its human origin and particular carbohydrate composition.

Asunto(s)

Carbohidratos/análisis , Células Epiteliales/metabolismo , Glicoproteínas/biosíntesis , Tirotropina/biosíntesis , Animales , Células CHO , Cricetinae , Cricetulus , Fucosa/análisis , Glicosilación , Células HEK293 , Semivida , Humanos , Ácido N-Acetilneuramínico/análisis , Proteínas Recombinantes/biosíntesis , Espectrometría de Masa por Láser de Matriz Asistida de Ionización Desorción , Transfección

RESUMEN

Paracoccidioidomycosis is the most prevalent systemic mycosis in Latin America. It is caused by the temperature-dependent dimorphic fungus Paracoccidioides brasiliensis. The P. brasiliensis cell wall is a dynamic outer structure, composed of a network of glycoproteins and polysaccharides, such as chitin, glucan and N-glycosylated proteins. These glycoproteins can interact with the host to affect infection rates, and are known to perform other functions. We inhibited N-linked glycosylation using tunicamycin (TM), and then evaluated the expression of P. brasiliensis genes related to cell wall remodeling. Our results suggest that cell wall synthesis related genes, such as ß-1,3-glucanosyltransferase (PbGEL3), 1,3-ß-D-glucan synthase (PbFKS1), and α-1,4-amylase (PbAMY), as well as cell wall degrading related genes, such as N-acetyl-ß-D-glucosaminidase (PbNAG1), α-1,3-glucanase (PbAGN), and ß-1,3-glucanase (PbBGN1 and PbBGN2), have their expression increased by the N-glycosylation inhibition, as detected by qRT-PCR. The observed increases in gene expression levels reveal possible compensatory mechanisms for diminished enzyme activity due to the lack of glycosylation caused by TM.

RESUMEN

The use of plants as heterologous hosts is one of the most promising technologies for manufacturing valuable recombinant proteins. Plant seeds, in particular, constitute ideal production platforms for long-term applications requiring a steady supply of starting material, as they combine the general advantages of plants as bioreactors with the possibility of biomass storage for long periods in a relatively small volume, thus allowing manufacturers to decouple upstream and downstream processing. In the present work we have used transgenic tobacco seeds to produce large amounts of a functionally active mouse monoclonal antibody against the Hepatitis B Virus surface antigen, fused to a KDEL endoplasmic reticulum retrieval motif, under control of regulatory sequences from common bean (Phaseolus vulgaris) seed storage proteins. The antibody accumulated to levels of 6.5 mg/g of seed in the T3 generation, and was purified by Protein A affinity chromatography combined with SEC-HPLC. N-glycan analysis indicated that, despite the KDEL signal, the seed-derived plantibody bore both high-mannose and complex-type sugars that indicate partial passage through the Golgi compartment, although its performance in the immunoaffinity purification of HBsAg was unaffected. An analysis discussing the industrial feasibility of replacing the currently used tobacco leaf-derived plantibody with this seed-derived variant is also presented.

Asunto(s)

Antígenos de Superficie de la Hepatitis B/inmunología , Nicotiana/embriología , Planticuerpos/inmunología , Semillas/inmunología , Cromatografía Líquida de Alta Presión , Cromatografía Liquida/métodos , Electroforesis en Gel de Poliacrilamida , Antígenos de Superficie de la Hepatitis B/aislamiento & purificación

RESUMEN

ST3Gal-II, a type II transmembrane protein, is the main mammalian sialyltransferase responsible for GD1a and GT1b ganglioside biosynthesis in brain. It contains two putative N-glycosylation sites (Asn(92) and Asn(211)). Whereas Asn(92) is only conserved in mammalian species, Asn(211) is highly conserved in mammals, birds and fish. The present study explores the occupancy and relevance for intracellular trafficking and enzyme activity of these potential N-glycosylations in human ST3Gal-II. We found that ST3Gal-II distributes along the Golgi complex, mainly in proximal compartments. By pharmacological, biochemical and site-directed mutagenesis, we observed that ST3Gal-II is mostly N-glycosylated at Asn(211) and that this co-translational modification is critical for its exit from the endoplasmic reticulum and proper Golgi localization. The individual N-glycosylation sites had different effects on ST3Gal-II enzymatic activity. Whereas the N-glycan at position Asn(211) seems to negatively influence the activity of the enzyme using both glycolipid and glycoprotein as acceptor substrates, the single N-glycan mutant at Asn(92) had only a moderate effect. Lastly, we demonstrated that the N-terminal ST3Gal-II domain containing the cytosolic, transmembrane and stem region (amino acids 1-51) is able to drive a protein reporter out of the endoplasmic reticulum and to retain it in the Golgi complex. This suggests that the C-terminal domain of ST3Gal-II depends on N-glycosylation to attain an optimum conformation for proper exit from the endoplasmic reticulum, but it does not represent an absolute requirement for Golgi complex retention of the enzyme.

Asunto(s)

Retículo Endoplásmico/enzimología , Aparato de Golgi/enzimología , Sialiltransferasas/metabolismo , Animales , Asparagina/genética , Asparagina/metabolismo , Células CHO , Cricetinae , Cricetulus , Retículo Endoplásmico/genética , Evolución Molecular , Glicosilación , Aparato de Golgi/genética , Humanos , Estructura Terciaria de Proteína , Transporte de Proteínas/fisiología , Sialiltransferasas/genética , beta-Galactosida alfa-2,3-Sialiltransferasa

RESUMEN

The cell wall of Paracoccidioides brasiliensis, which consists of a network of polysaccharides and glycoproteins, is essential for fungal pathogenesis. We have previously reported that N-glycosylation of proteins such as N-acetyl-ß-D-glucosaminidase is required for the growth and morphogenesis of P. brasiliensis. In the present study, we investigated the influence of tunycamicin (TM)-mediated inhibition of N-linked glycosylation on α- and ß-(1,3)-glucanases and on α-(1,4)-amylase in P. brasiliensis yeast and mycelium cells. The addition of 15 µg/ml TM to the fungal cultures did not interfere with either α- or ß-(1,3)-glucanase production and secretion. Moreover, incubation with TM did not alter α- and ß-(1,3)-glucanase activity in yeast and mycelium cell extracts. In contrast, α-(1,4)-amylase activity was significantly reduced in underglycosylated yeast and mycelium extracts after exposure to TM. In spite of its importance for fungal growth and morphogenesis, N-glycosylation was not required for glucanase activities. This is surprising because these activities are directed to wall components that are crucial for fungal morphogenesis. On the other hand, N-glycans were essential for α-(1,4)-amylase activity involved in the production of malto-oligosaccharides that act as primer molecules for the biosynthesis of α-(1,3)-glucan. Our results suggest that reduced fungal α-(1,4)-amylase activity affects cell wall composition and may account for the impaired growth of underglycosylated yeast and mycelium cells.

Asunto(s)

Antiinfecciosos/farmacología , Glucano 1,3-beta-Glucosidasa/metabolismo , Glicósido Hidrolasas/metabolismo , Glicosilación/efectos de los fármacos , Paracoccidioides/crecimiento & desarrollo , Tunicamicina/farmacología , alfa-Amilasas/metabolismo , Paracoccidioides/citología , Paracoccidioides/efectos de los fármacos , Paracoccidioides/enzimología

RESUMEN

Human malignant malaria is caused by Plasmodium falciparum and accounts for almost 900,000 deaths per year, the majority of which are children and pregnant women in developing countries. There has been significant effort to understand the biology of P. falciparum and its interactions with the host. However, these studies are hindered because several aspects of parasite biology remain controversial, such as N- and O-glycosylation. This review describes work that has been done to elucidate protein glycosylation in P. falciparum and it focuses on describing biochemical evidence for N- and O-glycosylation. Although there has been significant work in this field, these aspects of parasite biochemistry need to be explored further.

Asunto(s)

Procesamiento Proteico-Postraduccional , Plasmodium falciparum , Proteínas Protozoarias , Glicosilación