Ever-increasing evidence confirms the role of O-GlcNAcase (OGA) in mediating cell growth and development as well as pathology and underscores the importance of developing sensitive and selective chemical tools for the study of OGA biology. Here, based on our previously developed protein-encapsulation strategy, we designed and synthesized a series of fluorogenic probes based on resorufin, and assembled their composites with human serum albumin (HSA) for the selective detection of endogenous OGA activity in live cells. We show that host-guest self-assembly with HSA significantly enhances the OGA sensitivity of the probes in aqueous solution and cells. The structure of the complex of a glycoprobe and HSA was resolved by small-angle X-ray scattering. We demonstrate that the replacement of the acetyl group in GlcNAc with a propionyl group results in selectivity for OGA over hexosaminidases (HEX) that unselectively hydrolyze hexosamines. This allows us to differentiate between two cell lines with different endogenous OGA and HEX expression levels, and selectively detect OGA activity in live cells.

Aims/hypothesisType 2 diabetes is a chronic metabolic disorder characterised by insulin resistance and sustained hyperglycaemia, and is a major cause of blindness, kidney failure, heart attacks and stroke. Our team has recently identified hexosaminidase A (HEXA) as an endocrine factor secreted by the liver that regulates sphingolipid metabolism in skeletal muscle. Specifically, HEXA converts GM2 to GM3 gangliosides within cell-surface lipid rafts. Remodelling of ganglioside composition by HEXA enhances IGF1 signalling in skeletal muscle, increasing muscle glucose uptake and improving blood glucose control.MethodsWe produced a long-acting HEXA-FC fusion protein (murine HEXA and the fragment crystallisable [FC] region from IgG1) and evaluated the effects of chronic bi-weekly HEXA-FC administration (1 mg/kg body weight) on glycaemic control in C57BL/6 mice with diet-induced obesity and insulin resistance and the db/db mouse model of severe type 2 diabetes. Outcome measures included glucose and insulin tolerance, including a stable isotope-labelled GTT and assessment of tissue-specific glucose disposal, as well as proteomics analysis to define changes in skeletal muscle metabolism.ResultsChronic administration of a long-acting recombinant HEXA-FC fusion protein led to improvements in random blood glucose, fasting blood glucose and glucose tolerance, driven by increased glucose disposal into skeletal muscle, effects that were associated with enhancement of IGF1 signalling in muscle.Conclusions/interpretationGiven that skeletal muscle is a primary site of insulin resistance in individuals with type 2 diabetes, HEXA-FC protein therapy may open new avenues for therapeutic advancement in type 2 diabetes.Graphical

The prevalence of metabolic dysfunction-associated steatohepatitis (MASH) is increasing at an alarming rate. To date, only one therapy has been provisionally approved for the treatment of MASH and liver fibrosis, and novel strategies are urgently needed. In addition, the frequent coexistence of MASH and type 2 diabetes has further intensified interest in devising comprehensive therapies to simultaneously tackle both diseases. We have recently shown that increasing hepatic and/or circulating levels of hexosaminidase A (HEXA), a lysosomal enzyme that remodels GM2 to GM3 gangliosides within lipid rafts, offers therapeutic benefits for metabolic dysfunction-associated steatotic liver disease (MASLD) and type 2 diabetes. Taking advantage of the MUP-uPA mouse model of MASH, including both wild-type (WT) mice with mild MASH and MUP-uPA mice with severe MASH and fibrosis, we show that biweekly treatment with a long-lasting HEXA-FC analog improves features of MASLD, including hepatic steatosis and hepatocyte ballooning, in mice with mild MASH, as well as glycemic control across both mouse models. Mechanistically, HEXA-FC enhances hepatic fatty acid oxidation and peripheral glucose disposal while not impacting endogenous glucose production. Together, these outcomes suggest that while HEXA-FC treatment may offer therapeutic benefits in mild MASH and insulin resistance, it is ineffective against severe MASH and liver fibrosis.NEW & NOTEWORTHY The prevalence of metabolic dysfunction-associated steatohepatitis (MASH) and type 2 diabetes is increasing. Here, we show that chronic FC-HEXA recombinant protein treatment reduces hepatic lipid accumulation and improves blood glucose control in mice with mild MASH and insulin resistance.

Transcriptomic analysis following polystyrene nanoplastic stress in the Pacific white shrimp, Litopenaeus vannamei

Abstract Sandhoff disease (SD) is an ultra-rare lysosomal storage disorder (LSD), which affects ~ 1/1000, 000 live birth. SD is caused by genetic deficiency of beta (β) Hexosaminidase and resulting excess central nervous system (CNS) synthesis of GM2 ganglioside (GM2) and its impact on neuron death. The exact mechanisms underlying such GM2-driven neuron death are unknown in SD. Glucosylceramide (GC) induced complement 5a (C5a) and its C5aR1 activation causes tissue inflammation in experimental and clinical Gaucher disease. Additionally, C5a-C5aR1 axis was found to sparks CNS inflammation and neurodegeneration in several brain diseases including, intracerebral hemorrhage, traumatic brain injury, myasthenia gravis, amyotrophic lateral sclerosis, neuromyelitis optica spectrum, Alzheimer, and Huntington’s diseases. Here, we determined the increased brain levels of C5a and C5aR1 in a beta (β) hexosaminidase inhibitor (HABI)-induced experimental mouse model of SD, when compared to vehicle (PBS) injected WT control mice. Also, HABI-induced experimental mouse model of SD showed increased microglial cells activation, massive brain generation of pro-inflammatory cytokines and loss of neurons, when compared to vehicle injected WT mice. To assess the relevance of C5a-C5aR1 axis activation for brain inflammation in SD, we targeted β hexosaminidase with HAB-I in WT and C5aR1−/− mice. Strikingly, as compared to HABI-injected WT mice, HABI-injected C5aR1−/− mice caused marked reduction in microglial cell activation, brain production of pro-inflammatory cytokines, and the loss of neurons. These data suggest that the C5a-C5aR1 axis is a critical driver of neuroinflammation and neurodegeneration in SD. No Funding

Liver-Secreted Hexosaminidase A Regulates Insulin-Like Growth Factor Signaling and Glucose Transport in Skeletal Muscle.

Nematode galectin binds IgE and modulates mast cell activity

Aberrant glycosylation has been associated with several processes of tumorigenesis from cell signaling, migration and invasion, to immune regulation and metastasis formation. The biosynthesis of glycoconjugates is regulated through concerted and finely tuned enzymatic reactions. This includes the levels and activity of glycosyltransferases and glycosidases, nucleotide sugar metabolism, substrate availability, epigenetic condition, and cellular functional state. Glioblastoma (GBM) is the most aggressive brain tumor, frequently occurring in adults with overall survival not surpassing 17months after diagnosis. GBM has been classified by the World Health Organization (WHO) as a grade 4 astrocytoma and stratified into G-CIMP, proneural, classical, and mesenchymal subtypes. Several biomolecular features associated with GBM aggressiveness have been elucidated; however, more studies are needed to elucidate the role of glycosylation in GBM pathology, looking at their potential as cancer targets. Here, we focus on the alteration of genes involved in protein N- and O-linked glycosylation in GBM. Specifically, the mRNA levels of glycogenes were analyzed using astrocytoma-TCGA-RNAseq datasets from public repositories. A total of 68 genes were differentially regulated in the most aggressive, mesenchymal subtype of GBM compared to the proneural and classical subtypes, and the expression of these genes was compared to normal brain tissues. Among them, we focused on 38 genes coding for proteins that belong to: 1) asparagine glycosylation (ALG); 2) glycosyltransferases (B3T, B4T); 3) fucosyltransferase (FUT); 4) acetylgalactosaminyltransferases (GALNT); 5) hexosaminidase (HEX); 6) mannosidase (MAN); 7) acetylglucosaminyltransferase (MGAT); 8) sialidase or neuraminidase (NEU); 9) solute carrier 35 family (SLC); and 10) sialyltransferase (ST). The differential expression of some genes was already reported in several solid tumors; however, several of them were found to be dysregulated in GBM for the first time. These data represent an important starting point to perform further orthogonal and functional validations to pinpoint the role of these glycogenes in GBM as diagnostic and therapeutic targets.

Hexosaminidase A (HexA), a heterodimer consisting of HEXA and HEXB, converts the ganglioside sphingolipid GM2 to GM3 by removing a terminal N-acetyl-d-galactosamine. HexA enzyme deficiency in humans leads to GM2 accumulation in cells, particularly in neurons, and is associated with neurodegeneration. While HexA and sphingolipid metabolism have been extensively investigated in the context of neuronal lipid metabolism, little is known about the metabolic impact of HexA and ganglioside degradation in other tissues. Here, we focussed on the role of HexA in the liver, which is a major regulator of systemic lipid metabolism. We find that hepatic Hexa expression is induced by lipid availability and increased in the presence of hepatic steatosis, which is associated with increased hepatic GM3 content. To assess the impact of HEXA on hepatic lipid metabolism, we used an adeno-associated virus to overexpress HEXA in the livers of high-fat diet fed mice. HEXA overexpression was associated with increased hepatic GM3 content and increased expression of enzymes involved in the degradation of glycated sphingolipids, ultimately driving sphingomyelin accumulation in the liver. In addition, HEXA overexpression led to substantial proteome remodeling in cell surface lipid rafts, which was associated with increased VLDL processing and secretion, hypertriglyceridemia and ectopic lipid accumulation in peripheral tissues. This study established an important role of HEXA in modulating hepatic sphingolipid and lipoprotein metabolism.

Chitin and its derived products have great potential in various industries, such as biomedical, pharmaceutical, food, among others, due to its biodegradability, biocompatibility, null toxicity, and versatility. At present, these compounds are obtained by extraction with physical, chemical, or enzymatic processes. The fungus Colletotrichum lindemuthianum DSM 12250 which produces the enzyme chitin deacetylase (ClCDA), is proposed as an alternative for the biotransformation of fungal chitin. Chitin with a % N-Acetylation of 63.6% was obtained from the dry mycelium of Aspergillus niger that, based on analysis of Fourier transform infrared spectroscopy (FT-IR) and Differential Scanning Calorimetry (DSC), is structurally similar to commercial chitin. During fermentation in submerged culture, chitin presence was not found to generate inhibition in microbial growth or the activity of the ClCDA. The maximum enzymatic activity was 0.910 ± 0.002 U/mL and deacetylation of 20.9% were achieved. This was possible due to the enzymatic machinery of the fungus that includes CDA, endo-chitinases, and N-acetyl hexosaminidases. These compounds contribute to decreasing the substrate complexity, obtaining chitosan with a DDA of 57.3%. Submerged culture fermentation of Colletotrichum lindemuthianum DSM 12250 with fungal chitin as substrate represents a potential alternative for integrating different steps in the extraction of chitin-derived products while contributing to a cleaner and more efficient production.

Mutations in CLN5 cause a subtype of neuronal ceroid lipofuscinosis (NCL) called CLN5 disease. While the precise role of CLN5 in NCL pathogenesis is not known, recent work revealed that the protein has glycoside hydrolase activity. Previous work on the Dictyostelium discoideum homolog of human CLN5, Cln5, revealed its secretion during the early stages of development and its role in regulating cell adhesion and cAMP-mediated chemotaxis. Here, we used Dictyostelium to examine the effect of cln5-deficiency on various growth and developmental processes during the life cycle. During growth, cln5– cells displayed reduced cell proliferation, cytokinesis, viability, and folic acid-mediated chemotaxis. In addition, the growth of cln5– cells was severely impaired in nutrient-limiting media. Based on these findings, we assessed autophagic flux in growth-phase cells and observed that loss of cln5 increased the number of autophagosomes suggesting that the basal level of autophagy was increased in cln5– cells. Similarly, loss of cln5 increased the amounts of ubiquitin-positive proteins. During the early stages of multicellular development, the aggregation of cln5– cells was delayed and loss of the autophagy genes, atg1 and atg9, reduced the extracellular amount of Cln5. We also observed an increased amount of intracellular Cln5 in cells lacking the Dictyostelium homolog of the human glycoside hydrolase, hexosaminidase A (HEXA), further supporting the glycoside hydrolase activity of Cln5. This observation was also supported by our finding that CLN5 and HEXA expression are highly correlated in human tissues. Following mound formation, cln5– development was precocious and loss of cln5 affected spore morphology, germination, and viability. When cln5– cells were developed in the presence of the autophagy inhibitor ammonium chloride, the formation of multicellular structures was impaired, and the size of cln5– slugs was reduced relative to WT slugs. These results, coupled with the aberrant autophagic flux observed in cln5– cells during growth, support a role for Cln5 in autophagy during the Dictyostelium life cycle. In total, this study highlights the multifaceted role of Cln5 in Dictyostelium and provides insight into the pathological mechanisms that may underlie CLN5 disease.

BackgroundTay–Sachs disease (TSD) is a lysosomal storage disease caused by mutations in the HEXA gene that encodes the HexosaminidaseA (HEXA) enzyme. As HEXA normally functions to degrade the protein GM2‐ganglioside in lysosomes, decreased levels of HEXAcauses an accumulation of the protein and leads to neurological toxicity. Typical clinical manifestations of TSD include neurodevelopmental regression, muscle weakness, hypotonia, hyperreflexia, ataxia, seizures, and other neurological symptoms. It is quite rare in Asian populations, wherein only two cases have been reported in Korea to date.MethodsClinical records, radiological assessments, and laboratory findings, such as plasma hexosaminidase assay and HEXA analysis, were extracted from the medical records of three (1 male and 2 female) independent Korean children with infantile form of Tay–Sachs disease.ResultsAll three children presented with neurodevelopmental regression and strabismus at around 8 months of age. Presence of cherry‐red spots in the macula led to conduction of biochemical and genetic studies for TSD confirmation. The plasma hexosaminidase assay revealed decreased HEXA activity and low to normal total hexosaminidase activity. Similarly, genetic analysis revealed 4 variants from 6 alleles, including 2 previously reported and 2 novel variants, in the HEXA gene.ConclusionWe presented three Korean children, who were recently diagnosed with infantile‐type TSDvia enzyme assay and genetic analysis. Furthermore, results showed that fundus examination can be helpful for early diagnosis of children with neurodevelopmental regression.

Biochemical characterization of a GH19 chitinase from Streptomyces alfalfae and its applications in crystalline chitin conversion and biocontrol

Background: The article aimed to assess the activity of the hexosaminidase (HEX) and its HEX A and HEX B isoenzymes in persons who suddenly died due to ethanol poisoning and explain the cause of their death. Methods: The research involved two groups of the deceased group A—22 people (20 males, 2 females; the average age 46 years) who died due to alcohol intoxication (with the blood alcohol content of 4‰ and above in all biological materials at the time of death—blood, urine, cerebrospinal fluid, and vitreous humor), and group B—30 people (22 males, 8 females; the average age 54 years), who died suddenly due to other reasons than alcohol. Results: The highest activity of the HEX was found in the serum of A and B groups. A significantly lower activity of HEX, HEX A, and HEX B was observed in the urine of group A in comparison to the sober decedents. Conclusion: The lower activity of HEX and its isoenzymes in the dead’s urine due to ethanol poisoning may suggest its usefulness as a potential marker of harmful alcohol drinking. Damage done to the kidneys by ethanol poisoning may be one of the possible mechanisms leading to death. Kidneys may be damaged intravitally via the inflammatory agent. Thus, it is necessary to conduct further research to evaluate the diagnostic usefulness of exoglycosidases while determining the death mechanisms of people who lost their lives due to ethanol poisoning.

Presynaptic Dysfunction in Neurons Derived from Tay–Sachs iPSCs

Journal Digest

JScreen is a national public health initiative based out of Emory University that provides reproductive carrier screening through an online portal and follow-up genetic counseling services. In 2014, JScreen began reporting to patients variants of uncertain significance (VUSs) in the gene that causes Tay-Sachs disease (HEXA). Genetic counseling was provided to discuss the VUS and patients were offered hexosaminidase A (HEXA) blood enzyme testing to assist with VUS reclassification. To identify patient reactions and factors influencing their follow-up testing decisions after receiving these results, we conducted a retrospective quantitative study by administering online surveys to 62 patients with HEXA VUSs. Participants who pursued enzyme testing and those who did not both experienced low levels of distress when receiving the VUS results. Perceptions of HEXA carrier status after genetic counseling, decisional conflict levels, plans to have children in the near future, time available to pursue enzyme testing, and eligibility for research were significant factors influencing decision-making to pursue or not pursue enzyme testing. Genetic counseling played an important role in helping patients understand the VUS and follow-up testing options. When discussing VUSs with patients, it would be beneficial for genetic counselors to focus on the patient's perception of the VUS, anxiety related to the uncertainty of their results, and follow-up options, when available.

Objective To investigate the value of gene sequencing and protein conformation in the diagnosis process by reporting a case of erythrocyte hexokinase deficiency and reviewing relevant literature. Methods One patient with erythrocyte hexokinase deficiency and his families in the pediatrics of Sun Yat-sen Memorial Hospital of Sun Yat-sen University on May 23, 2016 were selected as the research objects. Clinical data was collected from the initial records by retrospective analysis. Pathogenic gene was detected by target sequence capture and next generation high-throughput sequencing, and the effects of mutant genes on hexokinase function were studied by observing changes in the 3-dimensional structure of proteins. Results The patient exhibited chronic hemolytic anemia after birth. The high-throughput second-generation gene sequencing result of the DNA sample of the child showed that a de novo mutation in the HK1 gene located on chromosome 10, the substitution of cytidine at site 34 of exon1 by thymine : NM_033496: exon1: c. C34T: p. Arg12X, which was a nonsense mutation. Bioinformatics analysis and protein function simulation confirmed that the mutation of the gene can lead to the premature termination of the synthesis of the peptide chain. The short peptide synthesized after the mutation does not contain the binding group and catalytic group of the erythrocyte hexokinase, resulting in the loss of enzyme activity. The patient was eventually diagnosed with erythrocyte hexokinase-deficient hemolytic anemia caused by the HK1 gene mutation. Conclusions In clinical practice, patients with chronic non-spherical erythrocyte hemolytic anemia should be alert to erythrocyte hexosaminidase deficiency disease. Those with conditions should be diagnosed by gene sequencing and protein configuration analysis. Key words: Hexokinase deficiency; Hemolytic anemia; Sequence analysis; Protein structure, quaternary; HK1 gene

Despite indoor mould being one of the most common problems in residential properties in the UK, there are not any widely accepted methodologies for its measurement. This paper focusses on this problem of measurement and reports on the findings from a rigorous testing scheme carried out to quantify air and surface mould concentrations and particle counts within 71 rooms from 64 properties in North London, some with and some without visible mould. The aim was to investigate the potential of passive and active air sampling strategies (sampling from still and actively mixed air, respectively) to explain visible mould, and understand how home/room characteristics correlate with the obtained readings. Airborne mould levels were quantified using an Andersen sampler (passively and actively), as well as by a chemical method based on the quantification of the N-acetylhexosaminidase (NAHA) activity (actively), which was also used to quantify surface mould. The mould levels were then correlated against physical characteristics of the tested homes/rooms, collected by means of survey sheets developed as part of this study. The findings did not reveal any independent variable governing all or most of the response variables, but a complex analysis suggested that whether it is a house or a flat could depict mould levels in the air and on the surfaces. It was also shown that a robust testing protocol should combine air and surface based methods, and an active air sampling strategy leads to a more accurate appraisal of airborne mould levels. Finally, the results showed that while there is some correlation between visible mould (and other moisture induced problems such as condensation) and measured air mould concentrations, lack of visible mould within a room does not necessarily mean low air mould concentrations, and thus one should not rely solely on visual inspection.

Cross-reactions between innate immunity, lysosomal function, and purinergic pathways may link signaling systems in cellular pathologies. We found activation of toll-like receptor 3 (TLR3) triggers lysosomal ATP release from both astrocytes and retinal pigmented epithelial (RPE) cells. ATP efflux was accompanied by lysosomal acid phosphatase and beta hexosaminidase release. Poly(I:C) alkalinized lysosomes, and lysosomal alkalization with bafilomycin or chloroquine triggered ATP release. Lysosomal rupture with glycyl-L-phenylalanine-2-naphthylamide (GPN) eliminated both ATP and acid phosphatase release. Secretory lysosome marker LAMP3 colocalized with VNUT, while MANT-ATP colocalized with LysoTracker. Unmodified membrane-impermeant 21-nt and “non-targeting” scrambled 21-nt siRNA triggered ATP and acid phosphatase release, while smaller 16-nt RNA was ineffective. Poly(I:C)-dependent ATP release was reduced by TBK-1 block and in TRPML1−/− cells, while TRPML activation with ML-SA1 was sufficient to release both ATP and acid phosphatase. The ability of poly(I:C) to raise cytoplasmic Ca2+ was abolished by removing extracellular ATP with apyrase, suggesting ATP release by poly(I:C) increased cellular signaling. Starvation but not rapamycin prevented lysosomal ATP release. In summary, stimulation of TLR3 triggers lysosomal alkalization and release of lysosomal ATP through activation of TRPML1; this links innate immunity to purinergic signaling via lysosomal physiology, and suggests even scrambled siRNA can influence these pathways.