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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.

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Mucopolysaccharidosis type IIIB (MPS IIIB) is an autosomal inherited disease caused by mutations in gene encoding the lysosomal enzyme N-acetyl-alpha-glucosaminidase (NAGLU). These mutations result in reduced NAGLU activity, preventing it from catalyzing the hydrolysis of the glycosaminoglycan heparan sulfate (HS). There are currently no approved treatments for MPS IIIB. A novel approach in the treatment of lysosomal storage diseases is the use of pharmacological chaperones (PC). In this study, we used a drug repurposing approach to identify and characterize novel potential PCs for NAGLU enzyme. We modeled the interaction of natural and artificial substrates within the active cavity of NAGLU (orthosteric site) and predicted potential allosteric sites. We performed a virtual screening for both the orthosteric and the predicted allosteric site against a curated database of human tested molecules. Considering the binding affinity and predicted blood-brain barrier permeability and gastrointestinal absorption, we selected atovaquone and piperaquine as orthosteric and allosteric PCs. The PCs were evaluated by their capacity to bind NAGLU and the ability to restore the enzymatic activity in human MPS IIIB fibroblasts These results represent novel PCs described for MPS IIIB and demonstrate the potential to develop novel therapeutic alternatives for this and other protein deficiency diseases.

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Introduction: Although breast milk is the ideal food source for newborns during the first six months of life, a high percentage of children receive infant formulas. There is evidence that specific diet habits may influence individual metabolic profile. Therefore, in newborns, such profile can be influenced by the use of infantile formulas given the composition differences that display compared to human milk. Up to now, there are no reports in the literature that address this issue. Objectives: this work aims to compare the metabolic profile of full-term newborns that were feed with either breast milk (n = 32) or infantile formulas (n = 21). Methods: Metabolic profile was established based on urine analysis through gas chromatography-mass spectrometry (GC-MS) and nuclear magnetic resonance (H-NMR). Results: our results evidenced a more gluconeogenic profile in breast-fed infants characterized by elevation of Kreb's cycle intermediaries like fumaric, succinic and ketoglutaric acids compared to infants receiving infant formula. In addition, infant formula fed infants presented urinary excretion of metabolites derived from specific compounds present in this type of diet that were not observed in breast-fed infants, for instance D-glucitol, and 4-deoxytetronic. Moreover, in infant formula fed infants there was excretion of basal levels of metabolites of clinical relevance like 3-hydroxy-3-methyl-glutaric, 2-methyl-3-keto-valeric and 3,4-dihydroxybutyric. Conclusion: These results show the importance of understanding the metabolic impact of diet in newborn population in normal and pathological contexts.

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Chlamydia psittaci is a highly zoonotic bacteria distributed worldwide; it is responsible for psittacosis, one of the most important infectious diseases affecting the Psittacidae, mostly parrots. This work was aimed at determining C. psittaci prevalence and genotype in 177 parrots confiscated in Colombia; cloacal swab (166) and faecal (177) samples were analysed from birds confiscated and housed in a Temporary Wildlife Reception Centre (Centro de Reception de Fauna Temporal). Conventional PCR was run on the samples for amplifying the MOMP gene and then the ompA gene. The C. psittaci genotype A was found in 81.3 % (144/177) of the birds analysed. Cloacal swabs accounted for 129/166 (77.7 %) positive samples and faecal matter for 53/177 (29.9 %), 38 birds proving positive for both types of sample; there was an 8.15 times greater probability of detection for cloacal swabs compared to faecal swabs (p < 0.05). Clinical examination findings were correlated with the animals' positivity for cloacal swabs, faecal matter or both, finding a statistically significant relationship with low respiratory rate (p < 0.05) and broken plumage for cloacal swab sample results (p < 0.1). Even though 85 % seroprevalence has previously been reported in Colombia using indirect ELISA, this study reports for the first time C. psittaci genotype A endemicity in psittacines in captivity in Colombia using molecular techniques, considering the zoonotic risk involved in having these birds as pets.

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Fructo-oligosaccharides (FOS) are one of the most well-studied and commercialized prebiotics. FOS can be obtained either by controlled hydrolysis of inulin or by sucrose transfructosylation. FOS produced from sucrose are typically classified as short-chain FOS (scFOS), of which the best known are 1-kestotriose (GF2), 1,1-kestotetraose (GF3), and 1,1,1-kestopentaose (GF4), produced by fructosyltransferases (FTases) or ß-fructofuranosidases. In previous work, FOS production was studied using the Aspergillus oryzae N74 strain, its ftase gene was heterologously expressed in Komagataella phaffii (Pichia pastoris), and the enzyme's tertiary structure modeled. More recently, residues that may be involved in protein-substrate interactions were predicted. In this study, the aim was to experimentally validate previous in silico results by independently producing recombinant wild-type A. oryzae N74 FTase and three single-point mutations in Komagataella phaffii (Pichia pastoris). The R163A mutation virtually abolished the transfructosylating activity, indicating a requirement for the positively charged arginine residue in the catalytic domain D. In contrast, transfructosylating activity was improved by introducing the mutations V242E or F254H, with V242E resulting in higher production of GF2 without affecting that of GF3. Interestingly, initial sucrose concentration, reaction temperature and the presence of metal cofactors did not affect the enhanced activity of mutant V242E. Overall, these results shed light on the mechanism of transfructosylation of the FTase from A. oryzae and expand considerations regarding the design of biotechnological processes for specific FOS production.

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The utility of low-resolution 1H-NMR analysis for the identification of biomarkers provided evidence for rapid biochemical diagnoses of organic acidemia and aminoacidopathy. 1H-NMR, with a sensitivity expected for a field strength of 400 MHz at 64 scans was used to establish the metabolomic urine sample profiles of an infant population diagnosed with small molecule Inborn Errors of Metabolism (smIEM) compared to unaffected individuals. A qualitative differentiation of the 1H-NMR spectral profiles of urine samples obtained from individuals affected by different organic acidemias and aminoacidopathies was achieved in combination with GC-MS. The smIEM disorders investigated in this study included phenylalanine metabolism; isovaleric, propionic, 3-methylglutaconicm and glutaric type I acidemia; and deficiencies in medium chain acyl-coenzyme and holocarboxylase synthase. The observed metabolites were comparable and similar to those reported in the literature, as well as to those detected with higher-resolution NMR. In this study, diagnostic marker metabolites were identified for the smIEM disorders. In some cases, changes in metabolite profiles differentiated post-treatments and follow-ups while allowing for the establishment of different clinical states of a biochemical disorder. In addition, for the first time, a 1H-NMR-based biomarker profile was established for holocarboxylase synthase deficiency spectrum.

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Metachromatic leukodystrophy (MLD) is a human neurodegenerative disorder characterized by progressive damage on the myelin band in the nervous system. MLD is caused by the impaired function of the lysosomal enzyme Arylsulphatase A (ARSA). The physiopathology mechanisms and the biochemical consequences in the brain of ARSA deficiency are not entirely understood. In recent years, the use of genome-scale metabolic (GEM) models has been explored as a tool for the study of the biochemical alterations in MLD. Previously, we modeled the metabolic consequences of different lysosomal storage diseases using single GEMs. In the case of MLD, using a glia GEM, we previously predicted that the metabolism of glycosphingolipids and neurotransmitters was altered. The results also suggested that mitochondrial metabolism and amino acid transport were the main reactions affected. In this study, we extended the modeling of the metabolic consequences of ARSA deficiency through the integration of neuron and glial cell metabolic models. Cell-specific models were generated from Recon2, and these were used to create a neuron-glial bi-cellular model. We propose a workflow for the integration of this type of model and its subsequent study. The results predicted the impairment pathways involved in the transport of amino acids, lipids metabolism, and catabolism of purines and pyrimidines. The use of this neuron-glial GEM metabolic reconstruction allowed to improve the prediction capacity of the metabolic consequences of ARSA deficiency, which might pave the way for the modeling of the biochemical alterations of other inborn errors of metabolism with central nervous system involvement.

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GM2 gangliosidosis, Tay-Sachs and Sandhoff diseases, are lysosomal storage disorders characterized by the lysosomal accumulation of GM2 gangliosides. This accumulation is due to deficiency in the activity of the ß-hexosaminidases Hex-A or Hex-B, which are dimeric hydrolases formed by αß or ßß subunits, respectively. These disorders show similar clinical manifestations that range from mild systemic symptoms to neurological damage and premature death. There is still no effective therapy for GM2 gangliosidoses, but some therapeutic alternatives, as enzyme replacement therapy, have being evaluated. Previously, we reported the production of active human recombinant ß-hexosaminidases (rhHex-A and rhHex-B) in the methylotrophic yeast Pichia pastoris. In this study, we evaluated in vitro the cellular uptake, intracellular delivery to lysosome, and reduction of stored substrates. Both enzymes were taken-up via endocytic pathway mediated by mannose and mannose-6-phosphate receptors and delivered to lysosomes. Noteworthy, rhHex-A diminished the levels of stored lipids and lysosome mass in fibroblasts from Tay-Sachs patients. Overall, these results confirm the potential of P. pastoris as host to produce recombinant ß-hexosaminidases intended to be used in the treatment of GM2 gangliosidosis.

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Mucopolysaccharidosis IVA (MPS IVA) is a lysosomal storage disease caused by mutations in the gene encoding for the enzyme N-acetylgalactosamine-6-sulfate sulfatase (GALNS), leading to lysosomal accumulation of keratan sulfate (KS) and chondroitin-6-sulfate. In this study, we identified and characterized bromocriptine (BC) as a novel PC for MPS IVA. BC was identified through virtual screening and predicted to be docked within the active cavity of GALNS in a similar conformation to that observed for KS. BC interacted with similar residues to those predicted for natural GALNS substrates. In vitro inhibitory assay showed that BC at 50 µM reduced GALNS activity up to 30%. However, the activity of hrGALNS produced in HEK293 cells was increased up to 1.48-fold. BC increased GALNS activity and reduced lysosomal mass in MPS IVA fibroblasts in a mutation-dependent manner. Overall, these results show the potential of BC as a novel PC for MPS IVA and contribute to the consolidation of PCs as a potential therapy for this disease.

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Iduronate-2-sulfatase (IDS) is a lysosomal enzyme involved in the metabolism of the glycosaminoglycans heparan (HS) and dermatan (DS) sulfate. Mutations on IDS gene produce mucopolysaccharidosis II (MPS II), characterized by the lysosomal accumulation of HS and DS, leading to severe damage of the central nervous system (CNS) and other tissues. In this study, we used a neurochemistry and proteomic approaches to identify the brain distribution of IDS and its interacting proteins on wild-type mouse brain. IDS immunoreactivity showed a robust staining throughout the entire brain, suggesting an intracellular reactivity in nerve cells and astrocytes. By using affinity purification and mass spectrometry we identified 187 putative IDS partners-proteins, mainly hydrolases, cytoskeletal proteins, transporters, transferases, oxidoreductases, nucleic acid binding proteins, membrane traffic proteins, chaperons and enzyme modulators, among others. The interactions with some of these proteins were predicted by using bioinformatics tools and confirmed by co-immunoprecipitation analysis and Blue Native PAGE. In addition, we identified cytosolic IDS-complexes containing proteins from predicted highly connected nodes (hubs), with molecular functions including catalytic activity, redox balance, binding, transport, receptor activity and structural molecule activity. The proteins identified in this study would provide new insights about IDS physiological role into the CNS and its potential role in the brain-specific protein networks.