RESUMO

Candidemia is an opportunistic mycosis with high morbidity and mortality rates. Even though Candida albicans is the main causative agent, other Candida species, such as Candida tropicalis, are relevant etiological agents of candidiasis and candidemia. Compared with C. albicans, there is currently limited information about C. tropicalis' biological aspects, including those related to the cell wall and the interaction with the host. Currently, it is known that its cell wall contains O-linked mannans, and the contribution of these structures to cell fitness has previously been addressed using cells subjected to chemical treatments or in mutants where O-linked mannans and other wall components are affected. Here, we generated a C. tropicalis pmt2∆ null mutant, which was affected in the first step of the O-linked mannosylation pathway. The null mutant was viable, contrasting with C. albicans where this gene is essential. The phenotypical characterization showed that O-linked mannans were required for filamentation; proper cell wall integrity and organization; biofilm formation; protein secretion; and adhesion to extracellular matrix components, in particular to fibronectin; and type I and type II collagen. When interacting with human innate immune cells, it was found that this cell wall structure is dispensable for cytokine production, but mutant cells were more phagocytosed by monocyte-derived macrophages. Furthermore, the null mutant cells showed virulence attenuation in Galleria mellonella larvae. Thus, O-linked mannans are minor components of the cell wall that are involved in different aspects of C. tropicalis' biology.

RESUMO

Mucopolysaccharidosis IVA (MPS IVA) is a lysosomal storage disease caused by mutations in the gene encoding the lysosomal enzyme N-acetylgalactosamine-6-sulfate sulfatase (GALNS), resulting in the accumulation of keratan sulfate (KS) and chondroitin-6-sulfate (C6S). Previously, it was reported the production of an active human recombinant GALNS (rGALNS) in E. coli BL21(DE3). However, this recombinant enzyme was not taken up by HEK293 cells or MPS IVA skin fibroblasts. Here, we leveraged a glyco-engineered E. coli strain to produce a recombinant human GALNS bearing the eukaryotic trimannosyl core N-glycan, Man3GlcNAc2 (rGALNSoptGly). The N-glycosylated GALNS was produced at 100 mL and 1.65 L scales, purified and characterized with respect to pH stability, enzyme kinetic parameters, cell uptake, and KS clearance. The results showed that the addition of trimannosyl core N-glycans enhanced both protein stability and substrate affinity. rGALNSoptGly was capture through a mannose receptor-mediated process. This enzyme was delivered to the lysosome, where it reduced KS storage in human MPS IVA fibroblasts. This study demonstrates the potential of a glyco-engineered E. coli for producing a fully functional GALNS enzyme. It may offer an economic approach for the biosynthesis of a therapeutic glycoprotein that could prove useful for MPS IVA treatment. This strategy could be extended to other lysosomal enzymes that rely on the presence of mannose N-glycans for cell uptake.

RESUMO

N-glycosylation is the most common protein modification in the eukaryotic secretory pathway. It involves the attachment a high mannose glycan to Asn residues in the context of Asn-X-Ser/Thr/Cys, a motif known as N-glycosylation sequon. This process is mediated by STT3A and STT3B, the catalytic subunits of the oligosaccharyltransferase complexes. STT3A forms part of complexes associated with the SEC61 translocon and functions co-translationally. Vacant sequons have another opportunity for glycosylation by complexes carrying STT3B. Local sequence information plays an important role in determining N-glycosylation efficiency, but non-local factors can also have a significant impact. For instance, certain proteins associated with human genetic diseases exhibit abnormal N-glycosylation levels despite having wild-type acceptor sites. Here, we investigated the effect of protein stability on this process. To this end, we generated a family of 40 N-glycan acceptors based on superfolder GFP, and we measured their efficiency in HEK293 cells and in two derived cell lines lacking STT3B or STT3A. Sequon occupancy was highly dependent on protein stability, improving as the thermodynamic stability of the acceptor proteins decreases. This effect is mainly due to the activity of the STT3B-based OST complex. These findings can be integrated into a simple kinetic model that distinguishes local information within sequons from global information of the acceptor proteins.

Assuntos

RESUMO

Follicle-stimulating hormone (FSH) is an important protein used for bovine ovarian hyperstimulation in multiple ovulation and embryo transfer technology (MOET). Several attempts to produce bovine FSH (bFSH) in recombinant systems have been reported, nonetheless, up to date, the most commonly used products are partially purified preparations derived from porcine or ovine (pFSH or oFSH) pituitaries. Here we describe the development of a biotechnology process to produce a novel, hyperglycosylated, long-acting recombinant bFSH (LA-rbFSH) by fusing copies of a highly O-glycosylated peptide. LA-rbFSH and a nonmodified version (rbFSH) were produced in suspension CHO cell cultures and purified by IMAC with high purity levels (>99%). LA-rbFSH presented a higher glycosylation degree and sialic acid content than rbFSH. It also demonstrated a notable improvement in pharmacokinetic properties after administration to rats, including a higher concentration in plasma and a significant (seven-fold) reduction in apparent clearance (CLapp). In addition, the in vivo specific bioactivity of LA-rbFSH in rats was 2.4-fold higher compared to rbFSH. These results postulate this new molecule as an attractive substitute for commercially available porcine pituitary-derived products.

Assuntos

RESUMO

Exoglycosidase enzymes hydrolyze the N-glycosylations of cell wall enzymes, releasing N-glycans that act as signal molecules and promote fruit ripening. Vesicular exoglycosidase α-mannosidase enzymes of the GH38 family (EC 3.2.1.24; α-man) hydrolyze N-glycans in non-reduced termini. Strawberry fruit (Fragaria × ananassa) is characterized by rapid softening as a result of cell wall modifications during the fruit ripening process. Enzymes acting on cell wall polysaccharides explain the changes in fruit firmness, but α-man has not yet been described in F. × ananassa, meaning that the indirect effects of N-glycan removal on its fruit ripening process are unknown. The present study identified 10 GH38 α-man sequences in the F. × ananassa genome with characteristic conserved domains and key residues. A phylogenetic tree built with the neighbor-joining method and three groups of α-man established, of which group I was classified into three subgroups and group III contained only Poaceae spp. sequences. The real-time qPCR results demonstrated that FaMAN genes decreased during fruit ripening, a trend mirrored by the total enzyme activity from the white to ripe stages. The analysis of the promoter regions of these FaMAN genes was enriched with ripening and phytohormone response elements, and contained cis-regulatory elements related to stress responses to low temperature, drought, defense, and salt stress. This study discusses the relevance of α-man in fruit ripening and how it can be a useful target to prolong fruit shelf life.

Assuntos

RESUMO

This report outlines the case of a child affected by a type of congenital disorder of glycosylation (CDG) known as ALG2-CDG (OMIM 607906), presenting as a congenital myasthenic syndrome (CMS) caused by variants identified in ALG2, which encodes an α1,3-mannosyltransferase (EC 2.4.1.132) involved in the early steps of N-glycosylation. To date, fourteen cases of ALG2-CDG have been documented worldwide. From birth, the child experienced perinatal asphyxia, muscular weakness, feeding difficulties linked to an absence of the sucking reflex, congenital hip dislocation, and hypotonia. Over time, additional complications emerged, such as inspiratory stridor, gastroesophageal reflux, low intake, recurrent seizures, respiratory infections, an inability to maintain the head upright, and a global developmental delay. Whole genome sequencing (WGS) revealed the presence of two ALG2 variants in compound heterozygosity: a novel variant c.1055_1056delinsTGA p.(Ser352Leufs*3) and a variant of uncertain significance (VUS) c.964C>A p.(Pro322Thr). Additional studies, including determination of carbohydrate-deficient transferrin (CDT) revealed a mild type I CDG pattern and the presence of an abnormal transferrin glycoform containing a linear heptasaccharide consisting of one sialic acid, one galactose, one N-acetyl-glucosamine, two mannoses and two N-acetylglucosamines (NeuAc-Gal-GlcNAc-Man2-GlcNAc2), ALG2-CDG diagnostic biomarker, confirming the pathogenicity of these variants.

RESUMO

BACKGROUND: Candida albicans is one of the most prevalent fungi causing infections in the world. Mnt1 is a mannosyltransferase that participates in both the cell wall biogenesis and biofilm growth of C. albicans. While the cell wall performs crucial functions in pathogenesis, biofilm growth is correlated with sequestration of drugs by the extracellular matrix. Therefore, antifungals targeting CaMnt1 can compromise fungal development and potentially also render Candida susceptible to drug therapy. Despite its importance, CaMnt1 has not yet been purified to high standards and its biophysical properties are lacking. RESULTS: We describe a new protocol to obtain high yield of recombinant CaMnt1 in Komagataella phaffii using methanol induction. The purified protein's identity was confirmed by MALDI-TOF/TOF mass spectroscopy. The Far-UV circular dichroism (CD) spectra demonstrate that the secondary structure of CaMnt1 is compatible with a protein formed by α-helices and ß-sheets at pH 7.0. The fluorescence spectroscopy results show that the tertiary structure of CaMnt1 is pH-dependent, with a greater intensity of fluorescence emission at pH 7.0. Using our molecular modeling protocol, we depict for the first time the ternary complex of CaMnt1 bound to its two substrates, which has enabled the identification of residues involved in substrate specificity and catalytic reaction. Our results corroborate the hypothesis that Tyr209 stabilizes the formation of an oxocarbenium ion-like intermediate during nucleophilic attack of the acceptor sugar, opposing the double displacement mechanism proposed by other reports. CONCLUSIONS: The methodology presented here can substantially improve the yield of recombinant CaMnt1 expressed in flask-grown yeasts. In addition, the structural characterization of the fungal mannosyltransferase presents novelties that can be exploited for new antifungal drug's development.

RESUMO

Clusia is one of the most important genera of the Clusiaceae family, comprising up to 400 species. This review describes the identification of twenty-two flavonoids from Clusia species, which includes five flavonols (1-4 and 11), six flavones (5-10), one catechin (12), one flavanone (13), and nine biflavonoids (14-22). O- and C-glycosylation are frequently observed amongst these flavonoids. Furthermore, seven biphenyls (23-29) and nine xanthones (30-38) have been isolated from Clusia species. Biphenyls and xanthones show limited occurrence within the genus, but together with biosynthetic insights, they might offer important chemophenetics leads for the consolidation of the genus Clusia within the Clusiaceae family. Altogether, this work provides an overview of the chemistry of the genus Clusia in terms of flavonoids, biphenyls and xanthones, as well as it discusses biological activities and chemophenetics of the isolated compounds, when appropriate.

RESUMO

Glycosylation is the most common protein and lipid post-translational modification in humans. Congenital disorders of glycosylation (CDG) are characterized by both genetic and clinical heterogeneity, presenting multisystemic manifestations, and in most cases are autosomal recessive in inheritance. The PIGN gene is responsible for the addition of phosphoethanolamine to the first mannose in the glycosylphosphatidylinositol (GPI)-anchor biosynthesis pathway, a highly conserved process that enables proteins to bind to the cell surface membrane. Here, we report a family with two siblings pediatric cases with the exact same compound heterozygous variants in PIGN. The (c.776T > C) variant of uncertain significance (VUS) together with a known pathogenic variant (c.932T > G), resulting in clinical features compatible with PIGN-related conditions, more specific the CDG. This is the first time that PIGN variant c.776T > C is reported in literature in individuals with PIGN-congenital disorder of glycosylation (PIGN-CDG), and the current submission in ClinVar by Invitae® is specifically of our case. Detailed clinical information and molecular analyses are presented. Here, we show for the first time two affected siblings with one pathogenic variant (c.932T > G) and the c.776T > C VUS in trans. In honor of the family, we propose the name Bella-Noah Syndrome for disorder.