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Phosphacan, a chondroitin sulfate proteoglycan, is a repulsive cue of cerebellar granule cells. This study aims to explore the molecular mechanism. The glycosylphosphatidylinositol‐anchored neural adhesion molecule TAG‐1 is a binding partner of phosphacan, suggesting that the repulsive effect of phosphacan is possibly because of its interaction with TAG‐1. The repulsive effect was greatly reduced on primary cerebellar granule cells of TAG‐1‐deficient mice. Surface plasmon resonance analysis confirmed the direct interaction of TAG‐1 with chondroitin sulfate C. On postnatal days 1, 4, 7, 11, 15, and 20 and in adulthood, phosphacan was present in the molecular layer and internal granular layer, but not in the external granular layer. In contrast, transient TAG‐1 expression was observed exclusively within the premigratory zone of the external granular layer on postnatal days 1, 4, 7, and 11. Boyden chamber cell migration assay demonstrated that phosphacan exerted its repulsive effect on the spontaneous and brain‐derived neurotrophic factor (BDNF)‐induced migration of cerebellar granule cells. The BDNF‐induced migration was inhibited by MK‐2206, an Akt inhibitor. The pre‐treatment with a raft‐disrupting agent, methyl‐β‐cyclodextrin, also inhibited the BDNF‐induced migration, suggesting that lipid rafts are involved in the migration of cerebellar granule cells. In primary cerebellar granule cells obtained on postnatal day 7 and cultured for 7 days, the ganglioside GD3 and TAG‐1 preferentially localized in the cell body, whereas the ganglioside GD1b and NB‐3 localized in not only the cell body but also neurites. Pre‐treatment with the anti‐GD3 antibody R24, but not the anti‐GD1b antibody GGR12, inhibited the spontaneous and BDNF‐induced migration, and attenuated BDNF‐induced Akt activation. These findings suggest that phosphacan is responsible for the repulsion of TAG‐1‐expressing cerebellar granule cells via GD3 rafts to attenuate BDNF‐induced migration signaling. [ABSTRACT FROM AUTHOR]

The article explains the developments, properties and applications of a modified [alpha]-N-acetylgalactosaminidase (NAGA) produced in Chinese hamster ovary cells. The produced compound is shown to be extremely useful in the development of the enzyme replacement theory (ERT) that can be employed as a treatment of the Fabry disease.

Protein kinase C-delta (PKCδ) has a caspase-3 recognition sequence in its structure, suggesting its involvement in apoptosis. In addition, PKCδ was recently reported to function as an anti-cancer factor. The generation of a PKCδ knockout mouse model indicated that PKCδ plays a role in B cell homeostasis. However, the Pkcrd gene, which is regulated through complex transcription, produces multiple proteins via alternative splicing. Since gene mutations can result in the loss of function of molecular species required for each tissue, in the present study, conditional PKCδ knockout mice lacking PKCδI, II, IV, V, VI, and VII were generated to enable tissue-specific deletion of PKCδ using a suitable Cre mouse. We generated PKCδ-null mice that lacked whole-body expression of PKCδ. PKCδ+/- parental mice gave birth to only 3.4% PKCδ-/- offsprings that deviated significantly from the expected Mendelian ratio (χ2(2) = 101.7, P < 0.001). Examination of mice on embryonic day 11.5 (E11.5) showed the proportion of PKCδ-/- mice implanted in the uterus in accordance with Mendelian rules; however, approximately 70% of the fetuses did not survive at E11.5. PKCδ-/- mice that survived until adulthood showed enlarged spleens, with some having cardiac and pulmonary abnormalities. Our findings suggest that the lack of PKCδ may have harmful effects on fetal development, and heart and lung functions after birth. Furthermore, our study provides a reference for future studies on PKCδ deficient mice that would elucidate the effects of the multiple protein variants in mice and decipher the roles of PKCδ in various diseases. [ABSTRACT FROM AUTHOR]

Efficient catalytic arsa‐Wittig reactions have been developed by using 1‐phenylarsolane as a catalyst. A wide array of aldehydes was converted to the corresponding olefins in high yields with moderate to excellent E stereoselectivity in the presence of a catalytic amount of 1‐phenylarsolane. Moreover, density functional theory calculations were carried out to afford insight into the E/Z selectivity. [ABSTRACT FROM AUTHOR]

Recombinant [beta]-hexosaminidase A (HexA) was produced in the methylotrophic yeast Ogataea minuta to prepare a large amount of HexA for treatment based enzyme replacement therapy (ERT). The problem of antigenicity due to differences in N-glycan structures between mammalian and yeast glycoproteins was resolved by using [alpha]-1,6-mannosyltransferase deficient yeast as the host.

Dibenzo[b,f]arsepins possessing severely distorted cores compared to those of other heteropins were synthesized. These derivatives exhibited dual photoluminescence in the green‐to‐red region (500–700 nm) and the near‐ultraviolet region (<380 nm), which could be attributed to the planarization of the arsepin core in the lowest singlet excited (S1) state. The computational approach for the assessment of the aromatic indices revealed that the dibenzoarsepins studied show aromaticity (8π system) in the S1 states in line with Baird's rule. The lone pair electrons of the arsenic atoms play a crucial role in the aromaticity in the S1 states. [ABSTRACT FROM AUTHOR]

Abstract: 2,3,4,5‐Tetraaryl‐1‐phenylarsoles were synthesized by utilizing safely generated diiodophenylarsine and zirconacyclopentadienes. The obtained peraryl arsoles showed aggregation‐induced emission (AIE), where intense emission was observed in the solid states (quantum yields up to 0.61), whereas the corresponding solutions were very weakly emissive. The optical and electronic properties were examined by experimental and computational methods. It was elucidated that the aryl groups at the 2,5‐positions affected the frontier orbitals and the aromaticity of the arsole core. On the other hand, those at the 1,3,5‐positions were perpendicular to the luminophore and effective for a restriction of aggregation‐caused quenching. Because the lone pair of the arsenic atom has a sufficient coordination ability due to the low aromaticity of the arsole moiety, a gold(I) chloride complex of 1,2,3,4,5‐pentaphenylarsole was synthesized. The complex formation caused a blue shift of the emission from the bare ligand. Interestingly, the complex showed luminescent mechanochromism; grinding the crystals with a blue emission (λem=445 nm) gave amorphous samples with a greenish–blue emission (λem=496 nm). [ABSTRACT FROM AUTHOR]

The electronic structures of dithienoarsoles, which were prepared through a facile and safe synthetic procedure, were tuned in multiple ways: structural modification of the π-conjugated system and reactions of the arsenic atom. Suzuki–Miyaura coupling reaction proceeded without coordination to the catalyst or oxidation during the reaction, though the lone pair of the arsenic atom was not protected. As a result, the emission color was successfully controlled. Moreover, oxidation and coordination of the arsenic atom effectively changed the electronic structures, resulting in emission color change. Theoretical calculations revealed that the LUMO levels were significantly lowered by oxidation and coordination. High air-stability and moderate coordination ability of a trivalent arsenic atom enabled various such transformations for emission color tuning. [ABSTRACT FROM AUTHOR]

Fabry disease is an X-linked genetic disorder caused by defects in the α-galactosidase A (GLA) gene, and heterogeneous mutations lead to quantitative and/or qualitative defects in GLA protein in male patients with Fabry disease. Random X-chromosomal inactivation modifies the clinical and biochemical features of female patients with Fabry disease. Functional polymorphisms have been frequently reported in recent times, and these increase the difficulty of understanding the pathogenetic basis of the disease. To date, GLA protein level has been measured using an enzyme-linked immunosorbent assay (ELISA). However, ELISA is not highly sensitive due to the high background noise. In this paper, we introduce a novel application of the immuno-polymerase chain reaction (PCR) method (termed Multiple Simultaneous Tag [MUSTag]) for measurement of the GLA protein level in blood samples. We compared the sensitivities of the MUSTag method with plates or magnetic beads with those of ELISA for recombinant human GLA and found that the apparent maximal sensitivity was higher for the former than for the latter. We then measured the GLA concentrations in serum and plasma from male patients with classic Fabry disease (Male Fabry), females with Fabry disease (Female Fabry), male subjects harboring the functional polymorphism p.E66Q (E66Q), and control (Control) subjects. Our results revealed that compared to the MUSTag plate and ELISA, the MUSTag beads assay afforded a clearer estimation of the GLA protein levels in the serum and plasma with minimal or no background noise, although all the methods could differentiate between the Male Fabry, E66Q, and Control groups. The Female Fabry group showed characteristic heterogeneity, which was consistent with the X-linked inheritance. This novel method is expected to be useful for the sensitive determination of GLA level in blood and elucidation of the pathogenetic basis of Fabry disease. [ABSTRACT FROM AUTHOR]

To find a new biomarker of Tay-Sachs disease and Sandhoff disease. The lyso-GM2 ganglioside (lyso-GM2) levels in the brain and plasma in Sandhoff mice were measured by means of high performance liquid chromatography and the effect of a modified hexosaminidase (Hex) B exhibiting Hex A-like activity was examined. Then, the lyso-GM2 concentrations in human plasma samples were determined. The lyso-GM2 levels in the brain and plasma in Sandhoff mice were apparently increased compared with those in wild-type mice, and they decreased on intracerebroventricular administration of the modified Hex B. The lyso-GM2 levels in plasma of patients with Tay-Sachs disease and Sandhoff disease were increased, and the increase in lyso-GM2 was associated with a decrease in Hex A activity. Lyso-GM2 is expected to be a potential biomarker of Tay-Sachs disease and Sandhoff disease. [ABSTRACT FROM AUTHOR]

Abstract: Fabry disease is a genetic disease caused by a deficiency of α-galactosidase A (GLA), which leads to systemic accumulation of glycolipids, predominantly globotriaosylceramide (Gb3). With the introduction and spread of enzyme replacement therapy (ERT) with recombinant GLAs for this disease, a useful biomarker for assessing the response to ERT is strongly required. We measured the tissue level of lyso-globotriaosylsphingosine (lyso-Gb3) in Fabry mice by means of high performance liquid chromatography, and compared it with the Gb3 level. The results revealed a marked increase in the lyso-Gb3 level in most tissues of Fabry mice, and which decreased after the administration of a recombinant GLA as in the case of Gb3, which is usually used as a biomarker of Fabry disease. The response was more impressive for lyso-Gb3 compared with for Gb3, especially in kidney tissues, in which a defect significantly influences the morbidity and mortality in patients with this disease. The plasma level of lyso-Gb3 also decreased after the injection of the enzyme, and it was well related to the degradation of tissue lyso-Gb3. Thus, lyso-Gb3 is expected to be a useful new biomarker for assessing the response to ERT for Fabry disease. [Copyright &y& Elsevier]

A modified α-N-acetylgalactosaminidase (NAGA) with α-galactosidase A (GLA)-like substrate specificity was designed on the basis of structural studies and was produced in Chinese hamster ovary cells. The enzyme acquired the ability to catalyze the degradation of 4-methylumbellifeiyl-α-D-galactopyranoside. It retained the original NAGA's stability in plasma and N-glycans containing many mannose 6-phosphate (M6P) residues, which are advantageous for uptake by cells via M6P receptors. There was no immunological cross-reactivity between the modified NAGA and GLA, and the modified NAGA did not react to serum from a patient with Fabry disease recurrently treated with a recombinant GLA. The enzyme cleaved globotriaosylceramide (Gb3) accumulated in cultured fibroblasts from a patient with Fabry disease. Furthermore, like recombinant GLA proteins presently used for enzyme replacement therapy (ERT) for Fabry disease, the enzyme intravenously injected into Fabry model mice prevented Gb3 storage in the liver, kidneys, and heart and improved the pathological changes in these organs. Because this modified NAGA is hardly expected to cause an allergic reaction in Fabry disease patients, it is highly promising as a new and safe enzyme for ERT for Fabry disease. [ABSTRACT FROM AUTHOR]

Mammalian sulfoglycolipids are comprised of two major classes of compounds, sulfatide (SO3-3Gal-ceramide) and seminolipid (SO3-3Gal-alkylacylglycerol). Sulfatide is present in relatively high levels in myelin, and seminolipid is present in testis. The sulfation of these sulfoglycolipids is catalyzed by a common enzyme, cerebroside sulfotransferase (CST). Disruption of the Cst gene in mice revealed that sulfatide and seminolipid are essential for, respectively, myelin formation and spermatogenesis. The present study describes the generation of a recombinant single-chain variable fragment (scFv) antibody against sulfoglycolipid, for use in the functional analysis of sulfoglycolipids in living cells. A positive hybridoma producing anti-sulfoglycolipid IgG3, referred to as DI8, was initially obtained by immunizing CST-null mice with an isolated sulfatide. The DI8 monoclonal antibody was found to bind specifically to sulfoglycolipids with the terminal 3-O–sulfated galactose structure, as evidenced by ELISA and thin-layer chromatogram-immunostaining. The antibody stained seminolipid on the cell surface of spermatogenic cells of wild-type testis, but it did not react with any cells in the seminiferous tubules of CST-null testis. Total RNA was extracted from this hybridoma, and cDNAs that encode the variable regions of the heavy and light chains of IgG3 were obtained by RT-PCR. These DNA fragments were linked through a DNA linker coding (Gly4Ser)3, and the recombinant scFv fragment was then inserted into a phagemid vector pCANTAB 5E. The scFv antibody that was displayed at the tip of the M13 phage in the form of a g3p fusion protein bound to sulfatide. Furthermore, a soluble form of the scFv antibody was also found to bind to the sulfoglycolipids in ELISA. [ABSTRACT FROM PUBLISHER]

P3 is a mouse monoclonal antibody (mAb) that binds to several NeuGc‐containing gangliosides. It also reacts with antigens expressed in human breast tumors (Vázquez et al. (1995) Hybridoma, 14, 551–556). In this work, the binding specificity of P3 has been characterized in more detail using a panel of glycolipids that included several disialylated gangliosides and several chemical derivatives of NeuGc‐GM3. The carboxyl group and the nitrogen function of sialic acid were found to play important roles in the antibody binding, whereas the glycerol tail appears to be nonrelevant. Molecular modeling was used to analyze the binding data, including the finding that P3 selectively recognizes the internal NeuGc in GD3. For this purpose, conformational studies of GD3 were performed using molecular dynamics. It was concluded that sialic acid binds the P3 antibody through its upper face (the one on which the carboxyl group is exposed) and the C4‐C5 side of the sugar ring, whereas none or very little contact between the galactose residue and the protein is evident. Conformational analysis of GD3 revealed that, despite the large flexibility of the NeuGcα8NeuGc linkage, the P3 binding epitope on the external sialic acid is not well exposed for any of the possible conformations this linkage can adopt, whereas the internal sialic acid presents the epitope in a proper way for several of these conformations. As a final result, a coherent picture of the epitope that fits the wide binding data was obtained. [ABSTRACT FROM PUBLISHER]

We previously described the differential distribution of major gangliosides (GM1, GD1a, GD1b, GT1b and GQ1b) in adult rat brain detected by specific antibodies (Kotani,M., Kawashima,I., Ozawa,I., Terashima,T. and Tai,T. , 3, 137–146, 1993). We report here the distribution of minor gangliosides in the adult rat brain by an immunofluorescence technique with mouse monoclonal antibodies (MAbs). Ten MAbs (GMR6, GMB28, GMR11, GMR19, GMR2, GMR7, GGR51, AMR10, NGR54 and NGR53) that specifically recognize GM3, GM2, GT1a, GD3, -Acdisialoganglioside, GD2, GM1b, GM4, IVNeuAcα-nLcCer and IVNeuAcα-nLcCer, respectively, were used. Our study revealed that there is a cell type-specific expression of minor gangliosides, as well as major gangliosides, in the rat brain. In the cerebellar cortex, GM3 was expressed intensely in the white matter and slightly in the granular layer. GD3 was present in both the granular layer and the white matter, but not in the Purkinje cell layer or in the molecular layer. An -Ac-disialoganglioside, which was suggested to be -Ac-LD1, was detected exclusively in both the molecular layer and Purkinje cell layer. The presence of GD2 was restricted to the granular layer. GM4 was associated with some astrocytes, but not with myelin or oligodendrocytes. GM2, GT1a, GM1b, IVNeuAcα-nLcCer and IVNeuAcαnLcCer gangliosides were not clearly detected in the cerebellar cortex. In other regions, such as cerebral cortex, hippocampal formation and spinal cord, the expression of the gangliosides was also highly localized to a specific cell type and layer. [ABSTRACT FROM PUBLISHER]

We investigated the localization of major gangliosides in adult rat brain by an immunofluorescence technique with mouse monoclonal antibodies (MAbs). Five MAbs (GMB16, GMR17, GGR12, GMR5 and GMR13) that specifically recognize gangliosides GM1, GD1a, GD1b, GT1b and GQ1b, respectively, were used. We have found that there is a cell type-specific expression of the ganglioside in the rat central nervous system. In cerebellar cortex, GM1 was expressed in myelin and some glial cells. GD1a was detected exclusively in the molecular layer. GD1b and GQ1b were present restrictedly on the granular layer; GD1b was detected on the surface of the granular cell bodies, whereas GQ1b was present in the cerebellar glomerulus. GT1b was distributed intensely in both the molecular layer and the granular layer. In cerebral cortex, GM1 was detected in some glial cells. Dense staining was limited to the white matter. GD1a was distributed in layers I, II/III and Va, and the upper part of layer VI, whereas GQ1b was localized in layers IV and Vb, and the lower part of layer VI. GD1b was detected beneath layer III. GT1b appeared to be distributed throughout all layers. In other regions, such as hippocampal formation and spinal cord, the expression of the ganglioside was also highly localized to a specific cell type and layer. [ABSTRACT FROM PUBLISHER]

Sphingolipid biosynthesis in synchronized HeLa cells was studied by pulse labeling with [14C]Ser or [14C]Gal and a simple TLC method. The major HeLa cell sphingolipids are ceramide (Cer), sphingomyelin, glucosylceramide (GlcCer), lactosylceramide (LacCer), globotriaosylceramide (Gb3Cer), Nacetylneuraminosylgangliotriaosylceramide (GM2) and sialylparagloboside (GM1-GlcNAc). The sphingolipid biosynthetic profiles of HeLa cells in the G1, G1/S boundary, S and G2 phases were similar, but significant changes occurred during M phase, when incorporation of radioactivity into sphingomyelin, Gb3Cer and a mixture of GM2 and GM1-GlcNAc decreased, and those of Cer and GlcCer increased. These data indicate that transfer of phosphocholine and galactose to Cer and GlcCer, respectively, decreased in mitotic cells, resulting in accumulation of Cer and GlcCer. Analysis of LacCer synthase activity revealed that GlcCer accumulation was not due to reduced activity of this enzyme. The results suggest that Cer and GlcCer accumulation in mitotic cells resulted from suppression of sphingomyelin and LacCer synthesis, probably caused by vesiculation of membranous organelles, such as the endoplasmic reticulum and Golgi apparatus. [ABSTRACT FROM AUTHOR]

In Fabry disease, large amounts of globotriaosylceramide (Gb3) and related glycosphingolipids accumulate in organs due to a deficiency of α-galactosidase A (GLA) activity. Enzyme replacement therapy (ERT) with recombinant GLA is now available, and it has been reported that ERT is beneficial for patients with Fabry disease, especially those who start treatment at an early stage of the disease. However, it seems that the efficacy of ERT differs with each organ, and Gb3 accumulated in the kidneys shows resistance to ERT when it is started at a late stage. In this study, we examined the differences in cleavage of Gb3 isoforms, and lyso-Gb3 and its analogues in the kidneys, liver, and heart in young Fabry mice subjected to ERT. The results revealed that recurrent administration of recombinant GLA had prominent effects in terms of degradation of Gb3 and its derivatives accumulated in the organs. However, particular Gb3 isoforms, i.e., Gb3 (C20:0) and Gb3 (C24OH), accumulated in the kidneys largely escaped from degradation. Such Gb3 isoforms may gradually accumulate in the kidneys from a young age, which results in a reduction in the efficacy of ERT for Fabry disease. [ABSTRACT FROM AUTHOR]

Abstract: Enzyme enhancement therapy (EET) for Fabry disease involving imino sugars has been developed and attracted interest. It is thought that imino sugars act as pharmacological chaperones for wild-type and mutant α-galactosidases (GLAs) in cells, but the mechanisms underlying the molecular interactions between the imino sugars and the enzyme have not been clarified yet. We examined various kinds of imino sugars and found that galactostatin bisulfite (GBS) inhibited GLA in vitro and increased the enzyme activity in cultured Fabry fibroblasts as in the case of 1-deoxygalactonojirimycin (DGJ). Then, we analyzed the molecular interactions between the imino sugars and recombinant human GLA by means of isothermal titration calorimetry and surface plasmon resonance biosensor assays, and first determined the thermodynamic and binding-kinetics parameters of imino sugar and GLA complex formation. The results revealed that DGJ bound to the enzyme more strongly than GBS, the binding of DGJ to the enzyme protein being enthalpy-driven. In the case of GBS, the reaction was mainly enthalpy-driven, but there was a possibility that entropy-driven factors were involved in the binding. Structural analysis in silico revealed that both the chemicals fit into the active-site pocket and undergo hydrogen bonding with residues comprising the active-site pocket including the catalytic ones. The side chain of GBS was oriented towards the entrance of the active-site pocket, and thus it could be in contact with residues comprising the wall of the active-site pocket. Thermodynamic, kinetic and structural studies should provide us with a lot of information for improving EET for Fabry disease. [Copyright &y& Elsevier]

Abstract: Recently, male subjects harboring the c.196G>C nucleotide change which leads to the E66Q enzyme having low α-galactosidase A (GLA) activity have been identified at an unexpectedly high frequency on Japanese and Korean screening for Fabry disease involving dry blood spots and plasma/serum samples. Individuals with the E66Q enzyme have been suspected to have the later-onset Fabry disease phenotype leading to renal and cardiac disease. However, there has been no convincing evidence for this. To determine whether c.196G>C (E66Q) is disease-causing or not, we performed biochemical, pathological and structural studies. It was predicted that the E66Q amino acid substitution causes a small conformational change on the molecular surface of GLA, which leads to instability of the enzyme protein. However, biochemical studies revealed that subjects harboring the E66Q enzyme exhibited relatively high residual enzyme activity in white blood cells, and that there was no accumulation of globotriaosylceramide in cultured fibroblasts or an increased level of plasma globotriaosylsphingosine in these subjects. An electron microscopic examination did not reveal any pathological changes specific to Fabry disease in biopsied skin tissues from a male subject with the E66Q enzyme. These results strongly suggest that the c.196G>C is not a pathogenic mutation but is a functional polymorphism. [Copyright &y& Elsevier]

Human β-hexosaminidase A (HexA) is a heterodimeric glycoprotein composed of α- and β-subunits that degrades GM2 gangliosides in lysosomes. GM2 gangliosidosis is a lysosomal storage disease in which an inherited deficiency of HexA causes the accumulation of GM2 gangliosides. In order to prepare a large amount of HexA for a treatment based on enzyme replacement therapy (ERT), recombinant HexA was produced in the methylotrophic yeast Ogataea minuta instead of in mammalian cells, which are commonly used to produce recombinant enzymes for ERT. The problem of antigenicity due to differences in N-glycan structures between mammalian and yeast glycoproteins was potentially resolved by using α-l,6-mannosyltransferase-deficient (ochlΔ) yeast as the host. Genes encoding the α- and β-subunits of HexA were integrated into the yeast cell, and the heterodimer was expressed together with its isozymes HexS (αα) and HexB (ββ). A total of 57 mg of β-hexosaminidase isozymes, of which 13 mg was HexA (αβ), was produced per liter of medium. HexA was purified with immobilized metal affinity column for the His tag attached to the β-subunit. The purified HexA was treated with α-mannosidase to expose mannose-6-phosphate (M6P) residues on the N-glycans. The specific activities of HexA and M6P-exposed HexA (M6PHexA) for the artificial substrate 4MU-GIcNAc were 1.2 ± 0.1 and 1.7 ± 0.3 mmol/h/mg, respectively. The sodium dodecyl sulfate-polyacrylamide gel electrophoresis pattern suggested a C-terminal truncation in the {3-subunit of the recombinant protein. M6PHexA was incorporated dose dependently into GM2 gangliosidosis patient-derived fibroblasts via M6P receptors on the cell surface, and degradation of accumulated GM2 ganglioside was observed. [ABSTRACT FROM AUTHOR]