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The cell surface of Toxoplasma gondii is rich in glycoconjugates which hold diverse and vital functions in the lytic cycle of this obligate intracellular parasite. Additionally, the cyst wall of bradyzoites, that shields the persistent form responsible for chronic infection from the immune system, is heavily glycosylated. Formation of glycoconjugates relies on activated sugar nucleotides, such as uridine diphosphate N -acetylglucosamine (UDP-GlcNAc). The glucosamine-phosphate- N -acetyltransferase (GNA1) generates N -acetylglucosamine-6-phosphate critical to produce UDP-GlcNAc. Here, we demonstrate that downregulation of T . gondii GNA1 results in a severe reduction of UDP-GlcNAc and a concomitant drop in glycosylphosphatidylinositols (GPIs), leading to impairment of the parasite’s ability to invade and replicate in the host cell. Surprisingly, attempts to rescue this defect through exogenous GlcNAc supplementation fail to completely restore these vital functions. In depth metabolomic analyses elucidate diverse causes underlying the failed rescue: utilization of GlcNAc is inefficient under glucose-replete conditions and fails to restore UDP-GlcNAc levels in GNA1-depleted parasites. In contrast, GlcNAc-supplementation under glucose-deplete conditions fully restores UDP-GlcNAc levels but fails to rescue the defects associated with GNA1 depletion. Our results underscore the importance of glucosamine-6-phosphate acetylation in governing T . gondii replication and invasion and highlight the potential of the evolutionary divergent GNA1 in Apicomplexa as a target for the development of much-needed new therapeutic strategies.

Boreal forest soils function as a terrestrial net sink in the global carbon cycle. The prevailing dogma has focused on aboveground plant litter as a principal source of soil organic matter. Using ¹⁴C bomb-carbon modeling, we show that 50 to 70% of stored carbon in a chronosequence of boreal forested islands derives from roots and root-associated microorganisms. Fungal biomarkers indicate impaired degradation and preservation of fungal residues in late successional forests. Furthermore, 454 pyrosequencing of molecular barcodes, in conjunction with stable isotope analyses, highlights root-associated fungi as important regulators of ecosystem carbon dynamics. Our results suggest an alternative mechanism for the accumulation of organic matter in boreal forests during succession in the long-term absence of disturbance.

P-glycoprotein (P-gp) is a crucial drug efflux transporter in the gastrointestinal tract, reducing drug uptake and expelling harmful xenobiotics to prevent pathological changes. Current P-gp enhancers primarily increase P-gp expression, requiring 1–3 days, thus missing the critical rescue window for acute poisoning. This study identifies glucosamine (GlcN) as a potent P-gp activator that swiftly enhances drug efflux, significantly reducing drug absorption without altering P-gp expression levels. GlcN directly binds to P-gp, boosting its transport efficiency. Only GlcN with a polymerization degree below 5 can activate P-gp, whereas higher polymerized chitooligosaccharides enhance drug absorption. Additionally, GlcN activation of P-gp has significant implications for cellular metabolism by expelling xenobiotics and metabolic by-products, maintaining cellular homeostasis. Our findings suggest GlcN’s potential as an effective antidote for paraquat poisoning and offer a detoxification strategy. This research provides a foundational understanding for developing improved detoxification agents and metabolic modulators. Wu et al. show that glucosamine acts as a P-glycoprotein activator, rapidly enhancing intestinal drug efflux and reducing drug absorption. The authors propose this as a detox strategy to improve survival outcomes following paraquat exposure.

Heparan sulfate (HS) is degraded in lysosome by a series of glycosidases. Before the glycosidases can act, the terminal glucosamine of HS must be acetylated by the integral lysosomal membrane enzyme heparan-α-glucosaminide N -acetyltransferase (HGSNAT). Mutations of HGSNAT cause HS accumulation and consequently mucopolysaccharidosis IIIC, a devastating lysosomal storage disease characterized by progressive neurological deterioration and early death where no treatment is available. HGSNAT catalyzes a unique transmembrane acetylation reaction where the acetyl group of cytosolic acetyl-CoA is transported across the lysosomal membrane and attached to HS in one reaction. However, the reaction mechanism remains elusive. Here we report six cryo-EM structures of HGSNAT along the reaction pathway. These structures reveal a dimer arrangement and a unique structural fold, which enables the elucidation of the reaction mechanism. We find that a central pore within each monomer traverses the membrane and controls access of cytosolic acetyl-CoA to the active site at its luminal mouth where glucosamine binds. A histidine-aspartic acid catalytic dyad catalyzes the transfer reaction via a ternary complex mechanism. Furthermore, the structures allow the mapping of disease-causing variants and reveal their potential impact on the function, thus creating a framework to guide structure-based drug discovery efforts. HGSNAT is a critical lysosomal membrane enzyme involved in the devastating lysosomal storage disease Sanfilippo syndrome. Here, Zhao et. al. reveal the mechanism for transmembrane acetylation catalyzed by HGSNAT with a series of cryo-EM structures.

Nearly 5% of the glucose-6-phosphate (Glc6P) in cells is diverted into the hexosamine biosynthetic pathway (HBP) to synthesize glucosamine-6-phosphate (GlcN6P) and uridine diphosphate N-acetyl-glucosamine-6-phosphate (UDP-GlcN6P). Fructose-6-phosphate (Fru6P) is a common intermediary between glycolysis and the HBP. Changes in HBP regulation cause abnormal protein N-glycosylation and O-linked-N-acetylglucosamine modification (O-GlcNAcylation), affecting protein function and modifying cellular responses to signals. The HBP enzymes glucosamine-6-phosphate deaminases 1 and 2 (GNPDA1 and 2) turn GlcN6P back into Fru6P and ammonium, and have been implicated in cancer and metabolic diseases. Despite the plentiful literature on this topic, the mechanisms involved are just beginning to be studied. In this review, we summarize, for the first time, the current knowledge regarding the possible roles of the isoenzymes of both GNPDAs in the pathogenesis and development of metabolic diseases and cancer from a molecular point of view, highlighting their importance not only in supplying carbon from glycolysis, but also in ammonia metabolism.

The biological properties of Heparan sulfate (HS) polysaccharides essentially rely on their ability to bind and modulate a multitude of protein ligands. These interactions involve internal oligosaccharide sequences defined by their sulfation patterns. Amongst these, the 6- O -sulfation of HS contributes significantly to the polysaccharide structural diversity and is critically involved in the binding of many proteins. HS 6- O -sulfation is catalyzed by 6- O -sulfotransferases (6OSTs) during biosynthesis, and it is further modified by the post-synthetic action of 6- O -endosulfatases (Sulfs), two enzyme families that remain poorly characterized. The aim of the present review is to summarize the contribution of 6- O -sulfates in HS structure/function relationships and to discuss the present knowledge on the complex mechanisms regulating HS 6- O -sulfation.

Antiproliferative effects of glucosamine, a glucose derivative with a similar structure to glucose, have been discovered, but the molecular mechanisms are not yet fully understood. Since glucosamine and glucose not only have similar structures but also are catalyzed by the same enzyme, hexokinase (HK), the present study delved into determining whether the antiproliferative effect of glucosamine involved the inhibition of glycolysis by competition with glucose. Whole-genome screening analysis showed that a number of the gene pathways controlled by glucosamine were directly and indirectly involved in glycolysis. In vitro experiments revealed that as more glucose was added, the antiproliferative effect of glucosamine decreased. Also, it was found that glucosamine was transported into cells mainly through glucose transporter (GLUT) 2 which was responsible for the antiproliferative effects of glucosamine. In addition, the present study found that cancer cell lines with low expression level of HKII show high sensitivity to glucosamine and a HK inhibitor, 3-bromopyruvate, enhanced the antiproliferative effect of glucosamine. Under hypoxic conditions, activated hypoxia-inducible factor 1α (HIF-1α) inducing glucose uptake and glycolysis hampered glucosamine-induced cell death and HIF1A knockdown or HK inhibitors restored the antiproliferative effects of glucosamine. These findings demonstrated that glucosamine is an efficient glycolysis inhibitor and that GLUT2 and HKII play important roles as biomarkers for determining sensitivity to glucosamine. Moreover, the results suggested that the antiproliferative effect of glucosamine may be more efficient when administered in combination with other glycolytic agents or inhibitors targeting HIF-1α.

•Uptake of GlcN in mice brains was measured by CEST MRI.•Anaesthetics affect GlcN uptake in the brain, confounding metabolic imaging findings.•Midazolam enables low-level isoflurane and facilitates GlcN uptake in mice brains.•The findings have broader implications and may apply to other glucose analogs. Anesthesia is often required during magnetic resonance imaging (MRI) examinations in animal studies. Anesthetic drugs differ in their capacity to interfere with homeostatic mechanisms responsible for glucose metabolism in the brain, which may create a constraint in the study design. Recent studies suggest that the chemical exchange saturation transfer (CEST) MRI scanning technique can detect localized metabolic changes in rodent brains induced by the uptake of glucose or its analogs; however, most of these studies do not account for the impact of anesthesia type on the brain metabolism. Herein, we aimed to evaluate the effect of reduced isoflurane levels on the preclinical imaging of glucosamine (GlcN) uptake in healthy mouse brains to establish optimal conditions for future brain imaging studies using the CEST MRI technique. The commonly used anesthesia protocol for longitudinal MRI examinations using 1.5% isoflurane level was compared to that using a mixture of low isoflurane (0.8%) level combined with midazolam (2 mg/kg, SC). Magnetization transfer ratio asymmetry (MTRasym) and area under the curve (AUC) analyses were used to characterize GlcN signals in the brain. The results indicated that mice injected with GlcN and anesthetized with 1.5% isoflurane exhibited low and insignificant changes in the MTRasym and AUC signals in the frontal cortex, whereas mice administered with 0.8% isoflurane combined with midazolam demonstrated a significant increase in these signals in the frontal cortex. This study highlights the diverse GlcN metabolic changes observed in mouse brains under variable levels of isoflurane anesthesia using the CEST MRI method. The results suggest that it is feasible to maintain anesthesia with low-dose isoflurane by integrating midazolam, which may enable the investigation of GlcN uptake in the brain. Thus, reducing isoflurane levels may support studies into mouse brain metabolism using the CEST MRI method and should be considered in future studies.

De novo uridine-diphosphate-N-acetylglucosamine (UDP-GlcNAc) biosynthesis requires glucose, glutamine, acetyl-CoA and uridine, however GlcNAc salvaged from glycoconjugate turnover and dietary sources also makes a significant contribution to the intracellular pool. Herein we ask whether dietary GlcNAc regulates nutrient transport and intermediate metabolism in C57BL/6 mice by increasing UDP-GlcNAc and in turn Golgi N-glycan branching. GlcNAc added to the drinking water showed a dose-dependent increase in growth of young mice, while in mature adult mice fat and body-weight increased without affecting calorie-intake, activity, energy expenditure, or the microbiome. Oral GlcNAc increased hepatic UDP-GlcNAc and N-glycan branching on hepatic glycoproteins. Glucose homeostasis, hepatic glycogen, lipid metabolism and response to fasting were altered with GlcNAc treatment. In cultured cells GlcNAc enhanced uptake of glucose, glutamine and fatty-acids, and enhanced lipid synthesis, while inhibition of Golgi N-glycan branching blocked GlcNAc-dependent lipid accumulation. The N-acetylglucosaminyltransferase enzymes of the N-glycan branching pathway (Mgat1,2,4,5) display multistep ultrasensitivity to UDP-GlcNAc, as well as branching-dependent compensation. Indeed, oral GlcNAc rescued fat accumulation in lean Mgat5 −/− mice and in cultured Mgat5 −/− hepatocytes, consistent with N-glycan branching compensation. Our results suggest GlcNAc reprograms cellular metabolism by enhancing nutrient uptake and lipid storage through the UDP-GlcNAc supply to N-glycan branching pathway.

Cerebral amyloid angiopathy (CAA) is a common feature of Alzheimer’s disease in which amyloid-β (Aβ) deposits in cerebral and leptomeningeal vessel walls, predisposing vessels to micro- and macro-hemorrhages. The vessel walls contain distinct proteins and heparan sulfate (HS), yet how vascular proteins and HS jointly associate with Aβ is unknown. We conducted the first multi-omics study to systematically characterize the proteins as well as the HS abundance, sulfation level, and disaccharide composition of leptomeninges from 23 moderate to severe CAA cases and controls. We then analyzed the associations between Aβ and other proteins, HS, and apolipoprotein E genotype. We found an increase in a minor HS disaccharide containing unsubstituted glucosamine, as well as 6-O sulfated disaccharides; Aβ40 levels positively correlated with unsubstituted glucosamine. There was also an increase in extracellular proteins derived from brain parenchyma or plasma, including olfactomedin-like protein 3, fibrinogen, serum amyloid protein, apolipoprotein E, and secreted frizzled related protein-3. Our findings of vascular HS and protein alterations specific to CAA-affected leptomeningeal vessels provide molecular insight into the extracellular remodeling that co-occurs with Aβ deposits and may indicate a basis for antemortem diagnostic assay development and therapeutic strategies to impede Aβ-HS interactions.