Structure of a truncated human GlcNAc-1-phosphotransferase variant reveals the basis for its hyperactivity.

Mutations that cause loss of function of GlcNAc-1-phosphotransferase (PTase) lead to the lysosomal storage disorder mucolipidosis II. PTase is the key enzyme of the mannose 6-phosphate (M6P) targeting system that is responsible for tagging lysosomal hydrolases with the M6P moiety for their delivery to the lysosome. We had previously generated a truncated hyperactive form of PTase termed S1S3 which was shown to notably increase the phosphorylation level of secreted lysosomal enzymes and enhance their uptake by cells. Here, we report the 3.4 Å cryo-EM structure of soluble S1S3 lacking both transmembrane domains and cytosolic tails. The structure reveals a high degree of conservation of the catalytic core to full-length PTase. In this dimeric structure, the EF-hand of one protomer is observed interacting with the conserved region four of the other. In addition, we present a high-quality EM 3D map of the UDP-GlcNAc bound form of the full-length soluble protein showing the key molecular interactions between the nucleotide sugar donor and side chain amino acids of the protein. Finally, although the domain organization of S1S3 is very similar to that of the Drosophila melanogaster (fruit fly) PTase homolog, we establish that the latter does not act on lysosomal hydrolases.

Human GNPTAB stuttering mutations engineered into mice cause vocalization deficits and astrocyte pathology in the corpus callosum.

Stuttering is a common neurodevelopmental disorder that has been associated with mutations in genes involved in intracellular trafficking. However, the cellular mechanisms leading to stuttering remain unknown. Engineering a mutation in N-acetylglucosamine-1-phosphate transferase subunits α and ß (GNPTAB) found in humans who stutter into the mouse Gnptab gene resulted in deficits in the flow of ultrasonic vocalizations similar to speech deficits of humans who stutter. Here we show that other human stuttering mutations introduced into this mouse gene, Gnptab Ser321Gly and Ala455Ser, produce the same vocalization deficit in 8-day-old pup isolation calls and do not affect other nonvocal behaviors. Immunohistochemistry showed a marked decrease in staining of astrocytes, particularly in the corpus callosum of the Gnptab Ser321Gly homozygote mice compared to wild-type littermates, while the staining of cerebellar Purkinje cells, oligodendrocytes, microglial cells, and dopaminergic neurons was not significantly different. Diffusion tensor imaging also detected deficits in the corpus callosum of the Gnptab Ser321Gly mice. Using a range of cell type-specific Cre-drivers and a Gnptab conditional knockout line, we found that only astrocyte-specific Gnptab-deficient mice displayed a similar vocalization deficit. These data suggest that vocalization defects in mice carrying human stuttering mutations in Gnptab derive from abnormalities in astrocytes, particularly in the corpus callosum, and provide support for hypotheses that focus on deficits in interhemispheric communication in stuttering.

Differential Clearance of Aß Species from the Brain by Brain Lymphatic Endothelial Cells in Zebrafish.

The failure of amyloid beta (Aß) clearance is a major cause of Alzheimer's disease, and the brain lymphatic systems play a crucial role in clearing toxic proteins. Recently, brain lymphatic endothelial cells (BLECs), a non-lumenized lymphatic cell in the vertebrate brain, was identified, but Aß clearance via this novel cell is not fully understood. We established an in vivo zebrafish model using fluorescently labeled Aß42 to investigate the role of BLECs in Aß clearance. We discovered the efficient clearance of monomeric Aß42 (mAß42) compared to oligomeric Aß42 (oAß42), which was illustrated by the selective uptake of mAß42 by BLECs and peripheral transport. The genetic depletion, pharmacological inhibition via the blocking of the mannose receptor, or the laser ablation of BLECs resulted in the defective clearance of mAß42. The treatment with an Aß disaggregating agent facilitated the internalization of oAß42 into BLECs and improved the peripheral transport. Our findings reveal a new role of BLECs in the differential clearance of mAß42 from the brain and provide a novel therapeutic strategy based on promoting Aß clearance.

Disease-causing missense mutations within the N-terminal transmembrane domain of GlcNAc-1-phosphotransferase impair endoplasmic reticulum translocation or Golgi retention.

Transport of newly synthesized lysosomal enzymes to the lysosome requires tagging of these enzymes with the mannose 6-phosphate moiety by UDP-GlcNAc:lysosomal enzyme N-acetylglucosamine-1-phosphotransferase (GlcNAc-1-phosphotransferase), encoded by two genes, GNPTAB and GNPTG. GNPTAB encodes the α and ß subunits, which are initially synthesized as a single precursor that is cleaved by Site-1 protease in the Golgi. Mutations in this gene cause the lysosomal storage disorders mucolipidosis II (MLII) and mucolipidosis III αß (MLIII αß). Two recent studies have reported the first patient mutations within the N-terminal transmembrane domain (TMD) of the α subunit of GlcNAc-1-phosphotransferase that cause either MLII or MLIII αß. Here, we demonstrate that two of the MLII missense mutations, c.80T>A (p.Val27Asp) and c.83T>A (p.Val28Asp), prevent the cotranslational insertion of the nascent GlcNAc-1-phosphotransferase polypeptide chain into the endoplasmic reticulum. The remaining four mutations, one of which is associated with MLII, c.100G>C (p.Ala34Pro), and the other three with MLIII αß, c.70T>G (p.Phe24Val), c.77G>A (p.Gly26Asp), and c.107A>C (p.Glu36Pro), impair retention of the catalytically active enzyme in the Golgi with concomitant mistargeting to endosomes/lysosomes. Our results uncover the basis for the disease phenotypes of these patient mutations and establish the N-terminal TMD of GlcNAc-1-phosphotransferase as an important determinant of Golgi localization.

An Antibody-Immobilized Silica Inverse Opal Nanostructure for Label-Free Optical Biosensors.

Three-dimensional SiO2-based inverse opal (SiO2-IO) nanostructures were prepared for use as biosensors. SiO2-IO was fabricated by vertical deposition and calcination processes. Antibodies were immobilized on the surface of SiO2-IO using 3-aminopropyl trimethoxysilane (APTMS), a succinimidyl-[(N-maleimidopropionamido)-tetraethyleneglycol] ester (NHS-PEG4-maleimide) cross-linker, and protein G. The highly accessible surface and porous structure of SiO2-IO were beneficial for capturing influenza viruses on the antibody-immobilized surfaces. Moreover, as the binding leads to the redshift of the reflectance peak, the influenza virus could be detected by simply monitoring the change in the reflectance spectrum without labeling. SiO2-IO showed high sensitivity in the range of 10³-105 plaque forming unit (PFU) and high specificity to the influenza A (H1N1) virus. Due to its structural and optical properties, SiO2-IO is a promising material for the detection of the influenza virus. Our study provides a generalized sensing platform for biohazards as various sensing strategies can be employed through the surface functionalization of three-dimensional nanostructures.

Multiple Domains of GlcNAc-1-phosphotransferase Mediate Recognition of Lysosomal Enzymes.

The Golgi enzyme UDP-GlcNAc:lysosomal enzymeN-acetylglucosamine-1-phosphotransferase (GlcNAc-1-phosphotransferase), an α2ß2γ2hexamer, mediates the initial step in the addition of the mannose 6-phosphate targeting signal on newly synthesized lysosomal enzymes. This tag serves to direct the lysosomal enzymes to lysosomes. A key property of GlcNAc-1-phosphotransferase is its unique ability to distinguish the 60 or so lysosomal enzymes from the numerous non-lysosomal glycoproteins with identical Asn-linked glycans. In this study, we demonstrate that the two Notch repeat modules and the DNA methyltransferase-associated protein interaction domain of the α subunit are key components of this recognition process. Importantly, different combinations of these domains are involved in binding to individual lysosomal enzymes. This study also identifies the γ-binding site on the α subunit and demonstrates that in the majority of instances the mannose 6-phosphate receptor homology domain of the γ subunit is required for optimal phosphorylation. These findings serve to explain how GlcNAc-1-phosphotransferase recognizes a large number of proteins that lack a common structural motif.

Simple and rapid detection of bacteria using a nuclease-responsive DNA probe.

We demonstrate simple and rapid bacterial detection using a nuclease-responsive DNA probe. The probe consisting of a fluorescent dye and a quencher at the 5' and 3' termini, respectively, was designed to be cleaved by nucleases such as endonucleases, exonucleases, and DNases, which are released from bacteria using an optimized lysis buffer. The fluorescence signal of the cleaved DNA probe correlates with the number of Gram-negative Escherichia coli and Gram-positive Staphylococcus aureus, and the detection limit was 103 CFU for E. coli and 104 CFU for S. aureus. Moreover, this method is specific for live bacteria and takes just one minute to get the signal including sample collection. These features make the present bacterial detection method a powerful on-site bacterial contamination assay which is simple, rapid, and quantitative.

Structure and function of the DUF2233 domain in bacteria and in the human mannose 6-phosphate uncovering enzyme.

DUF2233, a domain of unknown function (DUF), is present in many bacterial and several viral proteins and was also identified in the mammalian transmembrane glycoprotein N-acetylglucosamine-1-phosphodiester α-N-acetylglucosaminidase ("uncovering enzyme" (UCE)). We report the crystal structure of BACOVA_00430, a 315-residue protein from the human gut bacterium Bacteroides ovatus that is the first structural representative of the DUF2233 protein family. A notable feature of this structure is the presence of a surface cavity that is populated by residues that are highly conserved across the entire family. The crystal structure was used to model the luminal portion of human UCE (hUCE), which is involved in targeting of lysosomal enzymes. Mutational analysis of several residues in a highly conserved surface cavity of hUCE revealed that they are essential for function. The bacterial enzyme (BACOVA_00430) has ∼1% of the catalytic activity of hUCE toward the substrate GlcNAc-P-mannose, the precursor of the Man-6-P lysosomal targeting signal. GlcNAc-1-P is a poor substrate for both enzymes. We conclude that, for at least a subset of proteins in this family, DUF2233 functions as a phosphodiester glycosidase.

Maternal nanoplastic ingestion induces an increase in offspring body weight through altered lipid species and microbiota.

The rapidly increasing prevalence of obesity and overweight, especially in children and adolescents, has become a serious societal issue. Although various genetic and environmental risk factors for pediatric obesity and overweight have been identified, the problem has not been solved. In this study, we examined whether environmental nanoplastic (NP) pollutants can act as environmental obesogens using mouse models exposed to NPs derived from polystyrene and polypropylene, which are abundant in the environment. We found abnormal weight gain in the progeny until 6 weeks of age following the oral administration of NPs to the mother during gestation and lactation. Through a series of experiments involving multi-omic analyses, we have demonstrated that NP-induced weight gain is caused by alterations in the lipid composition (lysophosphatidylcholine/phosphatidylcholine ratio) of maternal breast milk and he gut microbiota distribution of the progeny. These data indicate that environmental NPs can act as obesogens in childhood.

Combined effects of microplastics and benz[a]anthracene on cardiotoxicity in zebrafish (Danio rerio) larvae: Size matters.

The size of microplastics (MPs) plays an important role in combined toxic effects including synergistic or antagonistic effects. However, the influence of the size of MPs on the combined toxicity of contaminants remains unclear. In this study, we employed a zebrafish model to investigate the effects of MP size on the combined toxicity of benz[a]anthracene (BaA), a representative polyaromatic hydrocarbon, using three different sizes of polystyrene MPs (PSMPs) (0.2, 1.0, and 10 µm). Treatment of all groups did not result in any mortality of the zebrafish larvae. However, small-sized PSMPs (0.2 µm) enhanced the toxic effect of BaA in larvae such as cardiac defect and disruption of vessel formation. Medium-sized PSMPs (1.0 µm) were boundary in terms of the combined toxic effect; however, large-sized PSMPs (10 µm) alleviated the cardiotoxicity of BaA, including cardiac defect, ROS levels, and cell death. The combined effects showed a correlation with the body burden of MPs and BaA in larvae according to particle size (in the order of 0.2 µm > 1.0 µm > 10 µm). The synergistic effects occurred likely because the small PSMPs facilitated the body burden of BaA, induced excessive ROS by Ahr-mediated activity, and caused cell death in the heart, resulting in increased heart defects in the larvae. In contrast, large PSMPs abated the combined toxic effect through decreased body burden, whereas medium PSMPs form a boundary in combined effects. Therefore, the combined toxic effects of MPs are dependent on their size, which plays an important role in the transport and accumulation of environmental pollutants.

A Simple and Rapid Methicillin-Resistant Staphylococcus aureus (MRSA) Screening Test Using a Mannose-Binding Lectin (MBL)-Conjugated Gold Nanoparticle Probe.

Rapid diagnosis of methicillin-resistant Staphylococcus aureus (MRSA) is essential for guiding clinical treatment and preventing the spread of MRSA infections. Herein, we present a simple and rapid MRSA screening test based on the aggregation effect of mannose-binding lectin (MBL)-conjugated gold nanoparticles (AuNP), called the MRSA probe. Recombinant MBL protein is a member of the lectin family and part of the innate immune system. It can recognize wall teichoic acid (WTA) on the membrane of MRSA more specifically than that of methicillin-sensitive Staphylococcus aureus (MSSA) under optimized salt conditions. Thus, the MRSA probe can selectively bind to MRSA, and the aggregation of the probes on the surface of the target bacteria can be detected and analyzed by the naked eye within 5 min. To demonstrate the suitability of the method for real-world application, we tested 40 clinical S. aureus isolates (including 20 MRSA specimens) and recorded a sensitivity of 100%. In conclusion, the MRSA probe-based screening test with its excellent sensitivity has the potential for successful application in the microbiology laboratory.

Three-dimensional label-free visualization of the interactions of PM2.5 with macrophages and epithelial cells using optical diffraction tomography.

Particulate matter ≤ 2.5 µm (PM2.5) poses health risks related to various diseases and infections. However, the interactions between PM2.5 and cells such as uptake and cell responses have not been fully investigated despite advances in bioimaging techniques, because the heterogeneous morphology and composition of PM2.5 make it challenging to employ labeling techniques, such as fluorescence. In this work, we visualized the interaction between PM2.5 and cells using optical diffraction tomography (ODT), which provides quantitative phase images by refractive index distribution. Through ODT analysis, the interactions of PM2.5 with macrophages and epithelial cells, such as intracellular dynamics, uptake, and cellular behavior, were successfully visualized without labeling techniques. ODT analysis clearly shows the behavior of phagocytic macrophages and nonphagocytic epithelial cells for PM2.5. Moreover, ODT analysis could quantitatively compare the accumulation of PM2.5 inside the cells. PM2.5 uptake by macrophages increased substantially over time, but uptake by epithelial cells increased only marginally. Our findings indicate that ODT analysis is a promising alternative approach to visually and quantitatively understanding the interaction of PM2.5 with cells. Therefore, we expect ODT analysis to be employed to investigate the interactions of materials and cells that are difficult to label.

Differential particle and ion kinetics of silver nanoparticles in the lungs and biotransformation to insoluble silver sulfide.

The measurement of nanoparticles (NPs) in a biological matrix is essential in various toxicity studies. However, the current knowledge has limitations in differentiating particulate and ionic forms and further identification of their biotransformation. Herein, we evaluate the biotransformation and differential lung clearance kinetics of particulate and ionic forms using PEGylated silver NPs (AgNP-PEGs; 47.51 nm) and PEGylated gold NPs (AuNP-PEGs; 11.76 nm). At 0, 3, and 6 h and 1, 3, 7, and 14 days after a single pharyngeal aspiration in mice at 25 µg/mouse, half of the lung is digested by proteinase K (PK) to separate particulates and ions, and the other half is subjected to the acid digestion method for comparison. The quantitative and qualitative evaluation of lung clearance kinetics suggests that AgNP-PEGs are quickly dissolved and transformed into insoluble silver sulfide (Ag2S), which shows a fast-clearing early phase (0 -6 h; particle T1/2: 4.8 h) and slow-clearing late phase (1 -14 days; particle T1/2: 13.20 days). In contrast, AuNP-PEGs were scarcely cleared or biotransformed in the lungs for 14 days. The lung clearance kinetics of AgNPs and biotransformation shown in this study can be informed by the PK digestion method and cannot be obtained using the acid digestion method.

Antioxidants prevent particulate matter-induced senescence of lung fibroblasts.

Particulate matter (PM) contributes to human diseases, particularly lung disease; however, the molecular mechanism of its action is yet to be determined. Herein, we found that prolonged PM exposure induced the cellular senescence of normal lung fibroblasts via a DNA damage-mediated response. This PM-induced senescence (PM-IS) was only observed in lung fibroblasts but not in A549 lung adenocarcinoma cells. Mechanistic analysis revealed that reactive oxygen species (ROS) activate the DNA damage response signaling axis, increasing p53 phosphorylation, ultimately leading to cellular senescence via an increase in p21 expression without affecting the p16-pRB pathway. A549 cells, instead, were resistant to PM-IS due to the PM-induced ROS production suppression. Water-soluble antioxidants, such as vitamin C and N-Acetyl Cysteine, were found to alleviate PM-IS by suppressing ROS production, implying that antioxidants are a promising therapeutic intervention for PM-mediated lung pathogenesis.

Analysis of mannose 6-phosphate uncovering enzyme mutations associated with persistent stuttering.

GlcNAc-1-phosphodiester-N-acetylglucosaminidase ("uncovering enzyme" (UCE); EC 3.1.4.45) is a Golgi enzyme that mediates the second step in the synthesis of the mannose 6-phosphate lysosomal targeting signal on acid hydrolases. Recently, three mutations (two missense and one deletion/frameshift) in the NAGPA gene that encodes UCE have been identified in individuals with persistent stuttering. We now demonstrate that each mutation leads to lower cellular UCE activity. The p.R328C mutation impairs folding in the endoplasmic reticulum, resulting in degradation of a significant portion by the proteasomal system. The p.H84Q mutation also impairs folding and, in addition, decreases the specific activity of the enzyme that folds sufficiently to traffic to the Golgi. The p.F513SfsX113 frameshift mutation adds 113 amino acids to the C terminus of the cytoplasmic tail of the protein, including a VWLL sequence that causes rapid degradation via the proteasomal system. These biochemical findings extend the genetic data implicating mutations in the NAGPA gene in the persistent stuttering phenotype.

Fluorescent Polypropylene Nanoplastics for Studying Uptake, Biodistribution, and Excretion in Zebrafish Embryos.

Nanoplastics (NPs) are emerging environmental pollutants and are a significant concern for human health. The small size of NPs allows them to accumulate within and adversely affect various tissues by penetrating the gastrointestinal barrier. However, most toxicity studies on NPs have been based on commercial polystyrene nanoparticles. Among plastics, polypropylene (PP) is one of the most widely used, and it is continuously micronized in the environment. Although PP has high potential for forming NPs by weathering, little is known about the biological effects of polypropylene nanoplastics (PPNPs) due to a lack of particle models. Here, we present a simple and high-yield method for PPNP production by nonsolvent-induced phase separation. The synthesized PPNPs were spherical in shape, with an average diameter of 562.15 ± 118.47 nm and a high yield of over 84%. These PPNPs were fluorescently labeled by the combined swelling-diffusion method to study their biodistribution after exposure to developing zebrafish embryos (ZFEs). We found that the fluorescent PPNPs were internalized by ingestion, distributed in the intestine of developing ZFEs, and eventually excreted. This study will aid evaluations of the potential risks of environmentally relevant plastics at the nanoscale.

A review on the impacts of nanomaterials on neuromodulation and neurological dysfunction using a zebrafish animal model.

Nanomaterials have been widely employed from industrial to medical fields due to their small sizes and versatile characteristics. However, nanomaterials can also induce unexpected adverse effects on health. In particular, exposure of the nervous system to nanomaterials can cause serious neurological dysfunctions and neurodegenerative diseases. A number of studies have adopted various animal models to evaluate the neurotoxic effects of nanomaterials. Among them, zebrafish has become an attractive animal model for neurotoxicological studies due to several advantages, including the well-characterized nervous system, efficient genome editing, convenient generation of transgenic lines, high-resolution in vivo imaging, and an array of behavioral assays. In this review, we summarize recent studies on the neurotoxicological effects of nanomaterials, particularly engineered nanomaterials and nanoplastics, using zebrafish and discuss key findings with advantages and limitations of the zebrafish model in neurotoxicological studies.

Structure of the human GlcNAc-1-phosphotransferase αß subunits reveals regulatory mechanism for lysosomal enzyme glycan phosphorylation.

Vertebrates use the mannose 6-phosphate (M6P)-recognition system to deliver lysosomal hydrolases to lysosomes. Key to this pathway is N-acetylglucosamine (GlcNAc)-1-phosphotransferase (PTase) that selectively adds GlcNAc-phosphate (P) to mannose residues of hydrolases. Human PTase is an α2ß2γ2 heterohexamer with a catalytic core and several peripheral domains that recognize and bind substrates. Here we report a cryo-EM structure of the catalytic core of human PTase and the identification of a hockey stick-like motif that controls activation of the enzyme. Movement of this motif out of the catalytic pocket is associated with a rearrangement of part of the peripheral domains that unblocks hydrolase glycan access to the catalytic site, thereby activating PTase. We propose that PTase fluctuates between inactive and active states in solution, and selective substrate binding of a lysosomal hydrolase through its protein-binding determinant to PTase locks the enzyme in the active state to permit glycan phosphorylation. This mechanism would help ensure that only N-linked glycans of lysosomal enzymes are phosphorylated.

Maternal exposure to polystyrene nanoplastics causes brain abnormalities in progeny.

As global plastic production continues to grow, microplastics released from a massive quantity of plastic wastes have become a critical environmental concern. These microplastic particles are found in a wide range of living organisms in a diverse array of ecosystems. In this study, we investigated the biological effects of polystyrene nanoplastic (PSNP) on development of the central nervous system using cultured neural stem cells (NSCs) and mice exposed to PSNP during developmental stages. Our study demonstrates that maternal administration of PSNP during gestation and lactating periods altered the functioning of NSCs, neural cell compositions, and brain histology in progeny. Similarly, PSNP-induced molecular and functional defects were also observed in cultured NSCs in vitro. Finally, we show that the abnormal brain development caused by exposure to high concentrations of PSNP results in neurophysiological and cognitive deficits in a gender-specific manner. Our data demonstrate the possibility that exposure to high amounts of PSNP may increase the risk of neurodevelopmental defects.

Functions of the alpha, beta, and gamma subunits of UDP-GlcNAc:lysosomal enzyme N-acetylglucosamine-1-phosphotransferase.

UDP-GlcNAc:lysosomal enzyme N-acetylglucosamine-1-phosphotransferase is an alpha(2)beta(2)gamma(2) hexamer that mediates the first step in the synthesis of the mannose 6-phosphate recognition marker on lysosomal acid hydrolases. Using a multifaceted approach, including analysis of acid hydrolase phosphorylation in mice and fibroblasts lacking the gamma subunit along with kinetic studies of recombinant alpha(2)beta(2)gamma(2) and alpha(2)beta(2) forms of the transferase, we have explored the function of the alpha/beta and gamma subunits. The findings demonstrate that the alpha/beta subunits recognize the protein determinant of acid hydrolases in addition to mediating the catalytic function of the transferase. In mouse brain, the alpha/beta subunits phosphorylate about one-third of the acid hydrolases at close to wild-type levels but require the gamma subunit for optimal phosphorylation of the rest of the acid hydrolases. In addition to enhancing the activity of the alpha/beta subunits toward a subset of the acid hydrolases, the gamma subunit facilitates the addition of the second GlcNAc-P to high mannose oligosaccharides of these substrates. We postulate that the mannose 6-phosphate receptor homology domain of the gamma subunit binds and presents the high mannose glycans of the acceptor to the alpha/beta catalytic site in a favorable manner.