Mucin-type glycosylation as a regulatory factor of amyloid precursor protein processing.

Mucin-type O-glycosylation is found not only in mucus proteins, but also in a number of cell membrane and secretory proteins. Several recent studies demonstrate that site-specific O-GalNAc glycosylation plays an important role in regulating protein functions by modulating proteolytic processing. Proteolysis of the amyloid precursor protein (APP) is physiologically important, since cleavages at ß and γ positions generate amyloid ß (Aß), a major component in the brain of patients with Alzheimer's disease. Akasaka-Manya et al. (Excess APP O-glycosylation by GalNAc-T6 decreases Aß production. J Biochem 2017;161:99-111) showed a specific glycosylation at a site proximal to the ß-secretase cleavage site and decreased productions of Aß1-40 and Aß1-42 in HEK293T cells transfected with a particular mucin-type glycan initiation enzyme, GalNAc-T6, indicating a novel pharmaceutical strategy to inhibit the production of Aß through the upregulation of mucin-type O-glycosylation.

Sialyltransferase ST3Gal IV deletion protects against temporal lobe epilepsy.

Temporal lobe epilepsy (TLE) often becomes refractory, and patients with TLE show a high incidence of psychiatric symptoms, including anxiety and depression. Therefore, it is necessary to identify molecules that were previously unknown to contribute to epilepsy and its associated disorders. We previously found that the sialyltransferase ST3Gal IV is up-regulated within the neural circuits through which amygdala-kindling stimulation propagates epileptic seizures. In contrast, this study demonstrated that kindling stimulation failed to evoke epileptic seizures in ST3Gal IV-deficient mice. Furthermore, approximately 80% of these mice failed to show tonic-clonic seizures with stimulation, whereas all littermate wild-type mice showed tonic-clonic seizures. This indicates that the loss of ST3Gal IV does not cause TLE in mice. Meanwhile, ST3Gal IV-deficient mice exhibited decreased acclimation in the open field test, increased immobility in the forced swim test, enhanced freezing during delay auditory fear conditioning, and sleep disturbances. Thus, the loss of ST3Gal IV modulates anxiety-related behaviors. These findings indicate that ST3Gal IV is a key molecule in the mechanisms underlying anxiety - a side effect of TLE - and may therefore also be an effective target for treating epilepsy, acting through the same circuits.

Identification and expression analysis of zebrafish polypeptide α-N-acetylgalactosaminyltransferase Y-subfamily genes during embryonic development.

Mucin-type glycosylation is one of the most common posttranslational modifications of secretory and membrane proteins and has diverse physiological functions. The initial biosynthesis of mucin-type carbohydrates is catalyzed by UDP-GalNAc: polypeptide α-N-acetylgalactosaminyltransferases (GalNAc-Ts) encoded by GALNT genes. Among these, GalNAc-T8, -T9, -T17, and -T18 form a characteristic subfamily called "Y-subfamily" and have no or very low in vitro transferase activities when assayed with typical mucin peptides as acceptor substrates. Although the Y-subfamily isozymes have been reported to be possibly involved in various diseases, their in vivo functions have not been reported. Here, we isolated zebrafish Y-subfamily galnt genes, and determined their spatial and temporal expressions during the early development of zebrafish. Our study demonstrated that all the Y-subfamily isozymes were well conserved in zebrafish with GalNAc-T18 having two orthologs, galnt18a and galnt18b, and with the other three isozymes each having a corresponding ortholog, galnt8, galnt9, and galnt17. The galnt8 was expressed in the cephalic mesoderm and hatching gland during early developmental stages, and differently expressed in the head, somatic muscles, and liver in the later stages. The other three orthologs also exhibited the characteristic expression patterns, although their expressions were generally strong in the nervous systems. In addition to the expression in the brain, galnt17 and galnt18a were expressed in the somitic muscles, and galnt18a and galnt18b in the notochord. These expression patterns may contribute to the functional analysis of the Y-subfamily, whose physiological roles still remain to be elucidated.

A rapid and efficient method for neuronal induction of the P19 embryonic carcinoma cell line.

BACKGROUND: P19 mouse embryonic carcinoma cells are conventionally induced to differentiate into neural cells by suspension culture in the presence of retinoic acid to form cell aggregates, followed by adhesion culture in a poly-l-lysine-coated dish. Drawbacks of this procedure include it taking more than 10 days to obtain mature neurons, and non-neuronal proliferating cells occupying the majority of the cell population with time. NEW METHOD: Here, we show a novel method for the rapid and efficient neurogenesis of P19 cells, without aggregate formation in a suspension culture. The new approach is based on an adherent serum-free culture in a laminin-coated dish in the presence of FGF8, a γ-secretase inhibitor, and cytosine arabinoside. RESULTS: The new method efficiently induced P19 cells to differentiate into neurons within 4 days, and subsequently into mature neurons that were responsive to several neurotransmitters, giving spontaneous neuronal network activity within 6 days. COMPARISON WITH EXISTING METHOD: The novel method accelerated neuritogenesis and enhanced population of neuron selectively compared to the conventional method. Proliferating non-neuronal cells were eliminated by adding cytosine arabinoside during neuronal maturation. CONCLUSIONS: The method is useful for studying neuronal differentiation or activities.

GTDC2 modifies O-mannosylated α-dystroglycan in the endoplasmic reticulum to generate N-acetyl glucosamine epitopes reactive with CTD110.6 antibody.

Hypoglycosylation is a common characteristic of dystroglycanopathy, which is a group of congenital muscular dystrophies. More than ten genes have been implicated in α-dystroglycanopathies that are associated with the defect in the O-mannosylation pathway. One such gene is GTDC2, which was recently reported to encode O-mannose ß-1,4-N-acetylglucosaminyltransferase. Here we show that GTDC2 generates CTD110.6 antibody-reactive N-acetylglucosamine (GlcNAc) epitopes on the O-mannosylated α-dystroglycan (α-DG). Using the antibody, we show that mutations of GTDC2 identified in Walker-Warburg syndrome and alanine-substitution of conserved residues between GTDC2 and EGF domain O-GlcNAc transferase resulted in decreased glycosylation. Moreover, GTDC2-modified GlcNAc epitopes are localized in the endoplasmic reticulum (ER). These data suggested that GTDC2 is a novel glycosyltransferase catalyzing GlcNAcylation of O-mannosylated α-DG in the ER. CTD110.6 antibody may be useful to detect a specific form of GlcNAcylated O-mannose and to analyze defective O-glycosylation in α-dystroglycanopathies.

A putative polypeptide N-acetylgalactosaminyltransferase/Williams-Beuren syndrome chromosome region 17 (WBSCR17) regulates lamellipodium formation and macropinocytosis.

We previously identified a novel polypeptide N-acetylgalactosaminyltransferase (GalNAc-T) gene, which is designated Williams-Beuren syndrome chromosome region 17 (WBSCR17) because it is located in the chromosomal flanking region of the Williams-Beuren syndrome deletion. Recent genome-scale analysis of HEK293T cells treated with a high concentration of N-acetylglucosamine (GlcNAc) demonstrated that WBSCR17 was one of the up-regulated genes possibly involved in endocytosis (Lau, K. S., Khan, S., and Dennis, J. W. (2008) Genome-scale identification of UDP-GlcNAc-dependent pathways. Proteomics 8, 3294-3302). To assess its roles, we first expressed recombinant WBSCR17 in COS7 cells and demonstrated that it was N-glycosylated and localized mainly in the Golgi apparatus, as is the case for the other GalNAc-Ts. Assay of recombinant WBSCR17 expressed in insect cells showed very low activity toward typical mucin peptide substrates. We then suppressed the expression of endogenous WBSCR17 in HEK293T cells using siRNAs and observed phenotypic changes of the knockdown cells with reduced lamellipodium formation, altered O-glycan profiles, and unusual accumulation of glycoconjugates in the late endosomes/lysosomes. Analyses of endocytic pathways revealed that macropinocytosis, but neither clathrin- nor caveolin-dependent endocytosis, was elevated in the knockdown cells. This was further supported by the findings that the overexpression of recombinant WBSCR17 stimulated lamellipodium formation, altered O-glycosylation, and inhibited macropinocytosis. WBSCR17 therefore plays important roles in lamellipodium formation and the regulation of macropinocytosis as well as lysosomes. Our study suggests that a subset of O-glycosylation produced by WBSCR17 controls dynamic membrane trafficking, probably between the cell surface and the late endosomes through macropinocytosis, in response to the nutrient concentration as exemplified by environmental GlcNAc.

Expression and function of glycogen synthase kinase-3 in human hair follicles.

Beta-catenin is involved in the hair follicle morphogenesis and stem cell differentiation, and inhibition of glycogen synthase kinase-3 (GSK-3) increases beta-catenin concentration in the cytoplasm. To examine the effects of GSK-3 inhibition on the hair follicle epithelium, we first examined the expression of GSK-3 in plucked human hair follicles by RT-PCR and found GSK-3 expression in hair follicles. Western blotting with a GSK-3beta-specific antibody, Y174, also demonstrated GSK-3beta expression in the follicles. Moreover, GSK-3beta immunostaining with Y174 showed that GSK-3beta colocalized with hair follicle bulge markers. Contrary to GSK-3beta, GSK-3 alpha was widely expressed throughout the follicles when immunostained with a specific antibody, EP793Y. We then investigated the influence of GSK-3 inhibition. A GSK-3 inhibitor, BIO, promoted the growth of human outer root sheath cells, which could be cultured for up to four passages. The BIO-treated cells exhibited smaller and more undifferentiated morphology than control cells. Moreover, in organ culture of plucked human hair, outer root sheath cells in the middle of a hair follicle proliferated when cultured with BIO. These results indicate that GSK-3beta is expressed in hair bulge stem cells and BIO promotes the growth of ORS cells, possibly by regulating the GSK-3 signaling pathway.

Inhibition of glycogen synthase kinase-3 enhances the expression of alkaline phosphatase and insulin-like growth factor-1 in human primary dermal papilla cell culture and maintains mouse hair bulbs in organ culture.

Dermal papilla (DP) at the hair follicle base is important for hair growth. Recent studies demonstrated that mouse vibrissa DP cells can be cultured in the presence of fibroblast growth factor-2 (FGF-2), but lose expression of versican and their follicle-inducing activity during the culture, and that activation of the Wnt signal, which is inhibited by glycogen synthase kinase-3 (GSK-3), in the DP cells promotes hair growth activity. We therefore investigated the influence of a GSK-3 inhibitor, (2'Z,3'E)-6-bromoindirubin-3'-oxime (BIO), on the growth of human DP cells and mouse vibrissa follicles in culture. We first demonstrated that, similarly to mouse DP cells, human DP cells were able to be cultured up to 15 passages in the presence of FGF-2, and lost the expression of alkaline phosphatase (ALP). When human DP cells later than ten passages were treated with BIO, the expression of ALP as well as insulin-like growth factor-1 (IGF-1), another DP marker, was significantly elevated. Nuclear and perinuclear translocation of beta-catenin was also observed. We then cultured mouse vibrissa follicles. In the presence of BIO, the follicles could be maintained for at least 3 days without detectable regression of the hair bulbs. The morphology and ALP expression were well preserved. BIO successfully retrieved the expression of DP marker molecules, such as ALP and IGF-1 in cultured human DP cells, and maintained mouse hair bulbs. Thus, treatment with BIO may be useful to prepare DP cells with hair follicle-inducing activity.

Function of conserved aromatic residues in the Gal/GalNAc-glycosyltransferase motif of UDP-GalNAc:polypeptide N-acetylgalactosaminyltransferase 1.

UDP-GalNAc:polypeptide N-acetylgalactosaminyltransferases (GalNAc transferases), which initiate mucin-type O-glycan biosynthesis, have broad acceptor substrate specificities, and it is still unclear how they recognize peptides with different sequences. To increase our understanding of the catalytic mechanism of GalNAc-T1, one of the most ubiquitous isozymes, we studied the effect of substituting six conserved aromatic residues in the highly conserved Gal/GalNAc-glycosyltransferase motif with leucine on the catalytic properties of the enzyme. Our results indicate that substitutions of Trp302 and Phe325 have little impact on enzyme function and that substitutions of Phe303 and Tyr309 could be made with only limited impact on the interaction(s) with donor and/or acceptor substrates. By contrast, Trp328 and Trp316 are essential residues for enzyme functions, as substitution with leucine, at either site, led to complete inactivation of the enzymes. The roles of these tryptophan residues were further analyzed by evaluating the impact of substitutions with additional amino acids. All evaluated substitutions at Trp328 resulted in enzymes that were completely inactive, suggesting that the invariant Trp328 is essential for enzymatic activity. Trp316 mutant enzymes with nonaromatic replacements were again completely inactive, whereas two mutant enzymes containing a different aromatic amino acid, at position 316, showed low catalytic activity. Somewhat surprisingly, a kinetic analysis revealed that these two amino acid substitutions had a moderate impact on the enzyme's affinity for the donor substrate. By contrast, the drastically reduced affinity of the Trp316 mutant enzymes for the acceptor substrates suggests that Trp316 is important for this interaction.

Cloning and expression of a brain-specific putative UDP-GalNAc: polypeptide N-acetylgalactosaminyltransferase gene.

We isolated a rat cDNA clone and its human orthologue, which are most homologous to UDP-GalNAc: polypeptide N-acetylgalactosaminyltransferase 9, by homology-based PCR from brain. Nucleotide sequence analysis of these putative GalNAc-transferases (designated pt-GalNAc-T) showed that they contained structural features characteristic of the GalNAc-transferase family. It was also found that human pt-GalNAc-T was identical to the gene WBSCR17, which is reported to be in the critical region of patients with Williams-Beuren Syndrome, a neurodevelopmental disorder, and to be predominantly expressed in brain and heart. In order to investigate the expression of pt-GalNAc-T in brain in more detail, we first examined that of human pt-GalNAc-T by Northern blot analysis and found the expression of the 5.0-kb mRNA to be most abundant in cerebral cortex with somewhat less abundant in cellebellum. The expression of rat pt-GalNAc-T was investigated more extensively. The brain-specific expression of 2.0-kb and 5.0-kb transcripts was demonstrated by Northern blot analysis. In situ hybridization in the adult brain revealed high levels of expression in cerebellum, hippocampus, thalamus, and cerebral cortex. Moreover, observation at high magnification revealed the expression to be associated with neurons, but not with glial cells. Analysis of the rat embryos also demonstrated that rat pt-GalNAc-T was expressed in the nervous system, including in the diencephalons, cerebellar primordium, and dorsal root ganglion. However, recombinant human pt-GalNAc-T, which was expressed in insect cells, did not glycosylate several peptides derived from mammalian mucins, suggesting that it may have a strict substrate specificity. The brain-specific expression of pt-GalNAc-T suggested its involvement in brain development, through O-glycosylation of proteins in the neurons.

Cloning and characterization of a novel radish protein kinase which is homologous to fungal cot-I like and animal Ndr protein kinases.

According to the similarity of the amino acid sequences in their catalytic domains, eukaryotic protein kinases have been classified into the five main groups: 'AGC', 'CaMK', 'CMGC', 'PTK' and 'other'. The AGC group, represented by the cyclic nucleotide-dependent kinases (PKA and PKG), the calcium-phospholipid-dependent kinases (PKC) and the ribosomal S6 protein kinases, are poorly characterized in plants except for a few cases. In this study, in order to gain a better understanding of plant protein kinases in the AGC group, three cDNAs encoding novel protein kinases, RsNdr1 and RsNdr2a/b, were cloned from radish and characterized by molecular and biochemical methods. The deduced amino acid sequences of RsNdr1 and RsNdr2a/b contained all 12 conserved catalytic subdomains which are characteristic of the eukaryotic Ser/Thr protein kinases. A cell lysate from E. coli overexpressing RsNdr1 fusion protein had protein kinase activity toward a conventional protein substrate (myelin basic protein), whereas that from E. coli harboring a fusion plasmid encoding kinase-dead RsNdr1 or RsNdr2 did not show any protein kinase activity. A phylogenetic tree for 17 protein kinases from various organisms showed that the RsNdrs are more closely related to the protein kinases in a particular subgroup of the 'AGC' (fungal cot1-like and animal Ndr kinases) than to the authentic 'AGC' protein kinases, such as PKA, PKC or ribosomal S6 kinase.

Characterization of a novel polypeptide N-acetylgalactosaminyltransferase (dGalNAc-T3) from Drosophila.

Polypeptide N-acetylgalactosaminyltransferases (GalNAc-transferases) catalyze the initial reaction of mucin-type O-glycosylation. Here, we report the first biochemical characterization of one of the Drosophila GalNAc-transferases, dGalNAc-T3. This enzyme retains conserved motifs essential for the catalytic activity, but is a novel isozyme in that it has several inserted sequences in its lectin-like domain. Northern hybridization analysis of this isozyme identified a 2.5-kb mRNA in Drosophila larva. Biochemical characterization was carried out using the recombinant soluble dGalNAc-T3 expressed in COS7 cells. dGalNAc-T3, which required Mn2+ for the activity, had a pH optimum ranging from pH 7.5 to 8.5, and glycosylated most effectively at 29-33 degrees C. Its Km for UDP-GalNAc was 10.7 microM, which is as low as that of mammalian isozymes. dGalNAc-T3 glycosylated the peptides containing a sequence of XTPXP or TTAAP most efficiently. The enzyme was irreversibly inhibited by p-chloromercuriphenylsulphonic acid, indicating the presence of essential Cys residues for the activity.

Function of the lectin domain of polypeptide N-acetylgalactosaminyltransferase 1.

All UDP-GalNAc:polypeptide N-acetylgalactosaminyltransferases cloned to date contain a lectin domain at the C-terminus, consisting of three tandem repeat sequences (alpha,beta, and gamma). We previously reported that the alpha repeat of one of the most ubiquitous isozymes, GalNAc-T1, is a functional lectin that recognizes O-linked GalNAc residues on the acceptor polypeptides with multiple acceptor sites; the domain appears not to be involved in the glycosylation of acceptors with a single acceptor site. In this report, we studied the function of the beta and gamma repeats in the GalNAc-T1 lectin domain, by site-directed mutagenesis and analysis of the catalytic properties of mutant enzymes. We found that the beta repeat recognizes GalNAc and is involved in glycosylation of acceptors with multiple glycosylation sites. The gamma repeat, on the other hand, showed no significant GalNAc-binding activity. These results indicate that the lectin domain of GalNAc-T1 has at least two functional repeats, allowing the possibility of multivalent interactions with GalNAc residues on the acceptor polypeptide during glycosylation.

The lectin domain of UDP-GalNAc:polypeptide N-acetylgalactosaminyltransferase 1 is involved in O-glycosylation of a polypeptide with multiple acceptor sites.

Mucin type O-glycosylation begins with the transfer of GalNAc to serine and threonine residues on proteins by a family of UDP-GalNAc:polypeptide N-acetylgalactosaminlytransferases. These enzymes all contain a lectin-like (QXW)(3) repeat sequence at the C terminus that consists of three tandem repeats (alpha, beta, and gamma). The putative lectin domain of one of the most ubiquitous isozymes, GalNAc-T1, is reportedly not functional. In this report, we have reevaluated the role of the GalNAc-T1 lectin domain. Deletion of the lectin domain resulted in a complete loss of enzymatic activity. We also found that GalNAc-T1 has two activities distinguished by their sensitivities to inhibition with free GalNAc; one activity is sensitive, and the other is resistant. In our experiments, the former activity is represented by the O-glycosylation of apomucin, an acceptor that contains multiple glycosylation sites, and the latter is represented by synthetic peptides that contain a single glycosylation site. Site-directed mutagenesis of the lectin domain selectively reduced the former activity and identified Asp(444) in the alpha repeat as the most important site for GalNAc recognition. A further reduction of the GalNAc-inhibitable activity was observed when both Asp(444) and the corresponding aspartate residues in the beta and the gamma repeats were mutated. This suggests a cooperative involvement of each repeat unit in the glycosylation of polypeptides with multiple acceptor sites.

Identification of two cysteine residues involved in the binding of UDP-GalNAc to UDP-GalNAc:polypeptide N-acetylgalactosaminyltransferase 1 (GalNAc-T1).

Biosynthesis of mucin-type O-glycans is initiated by a family of UDP-GalNAc:polypeptide N-acetylgalactosaminyltransferases, which contain several conserved cysteine residues among the isozymes. We found that a cysteine-specific reagent, p-chloromercuriphenylsulfonic acid (PCMPS), irreversibly inhibited one of the isozymes (GalNAc-T1). Presence of either UDP-GalNAc or UDP during PCMPS treatment protected GalNAc-T1 from inactivation, to the same extent. This suggests that GalNAc-T1 contains free cysteine residues interacting with the UDP moiety of the sugar donor. For the functional analysis of the cysteine residues, several conserved cysteine residues in GalNAc-T1 were mutated individually to alanine. All of the mutations except one resulted in complete inactivation or a drastic decrease in the activity, of the enzyme. We identified only Cys212 and Cys214, among the conserved cysteine residues in GalNAc-T1, as free cysteine residues, by cysteine-specific labeling of GalNAc-T1. To investigate the role of these two cysteine residues, we generated cysteine to serine mutants (C212S and C214S). The serine mutants were more active than the corresponding alanine mutants (C212A and C214A). Kinetic analysis demonstrated that the affinity of the serine-mutants for UDP-GalNAc was decreased, as compared to the wild type enzyme. The affinity for the acceptor apomucin, on the other hand, was essentially unaffected. The functional importance of the introduced serine residues was further demonstrated by the inhibition of all serine mutant enzymes with diisopropyl fluorophosphate. In addition, the serine mutants were more resistant to modification by PCMPS. Our results indicate that Cys212 and Cys214 are sites of PCMPS modification, and that these cysteine residues are involved in the interaction with the UDP moiety of UDP-GalNAc.