Sequential changes in glycolipid expression during human B cell differentiation: enzymatic bases

We have previously reported that human B cell differentiation is accompanied by sequential changes in glycosphingolipid expression. Pre-B cells contain lacto-series type II chain-based glycolipids and GM3 ganglioside; mature/activated B cells do not synthesize lacto-series compounds but express GM3 and globo-series glycolipids (Gb3 and Gb4); terminally differentiated B cells, in addition to these compounds, also contain GM2 ganglioside. At the cell surface, Gb3, Gb4 and GM2 constitute stage-specific antigens. To elucidate the biosynthetic mechanism leading to these modifications we have compared activities of the glycosyltransferases involved in the core structure assembly and the first elongation steps of neo-lacto, ganglio- and globo-series glycolipids. These glycosyltransferase activities have been measured in B cell lines and normal B lymphocytes at various stages of differentiation. We first determined the optimal requirements of the four glycosyltransferases which wynthesize Lc3, GM3, Gb4 and GM2 glycolipids in B lymphocytes and then tested these enzymes and the Gb3 synthetase in the selected B cells. The following results were obtained: ß1 → 3N-Acetylglucosaminyltransferase (Lc3 synthetase) has a high activity in pro- and pre-B cells whereas it is undetectable in more differentiated cells; α2 → 3 sialyltransferase (GM3 synthetase) is activated from the pre-B cell stage to the terminally differentiated myeloma cells; α → 4 galactosyltransferase (Gb3 synthetase) is only detected in cells representing the late stages of B cell differentiation; ß1 → 3N-Acetylgalactosaminyltransferase (Gb4 synthetase) is only found in some lymphoblastoid cell lines, representative of activated B cells whereas the ß1 → 4 N-Acetylgalactosaminyltransferase (GM2 synthetase) has a high activity in these lymphoblastoid cell lines and in terminally differentiated myeloma cells. These results suggest that the sequential shifts in the three major glycosphingolipid series observed during B cell differentiation are mostly due to sequential activations of the corresponding glycosyltransferases.     

Stable expression of a cDNA encoding a human beta 1 --> 3galactosyltransferase responsible for lacto-series type 1 core chain synthesis in non-expressing cells: variation in the nature of cell surface antigens expressed.

Transient expression of a human colonic adenocarcinoma Colo 205 cell derived cDNA in cell lines which ordinarily express only neolacto-series glycolipids has resulted in the expression of a beta 1 --> 3galactosyltransferase gene responsible for synthesis of glycolipids based upon the lacto-series type 1 core chain. Calcium phosphate transfected cells were panned on anti-IgM coated plates after initial treatment with a combination of monoclonal antibodies specific for type 1 chain terminal structures (TE-3) and a very broadly specific antibody reactive with multiple type 1 chain derivatives (TE-2). Adherent cells after panning were capable of efficiently transferring Gal in beta 1 --> 3-linkage to the acceptor glycolipid Lc3. Using these reagents, clones of stably transfected human colonic adenocarcinoma HCT-15 cells were produced and isolated. Parental HCT-15 cells do not express type 1 chain based antigens. The nature of the type 1 chain based antigens produced in each of these clones was analyzed by solid phase antibody binding assays. Three types of behavior were observed. Formation of type 1 terminal structures that were either exclusively sialylated or fucosylated, or a mixture of sialylated and fucosylated determinants occurred. In contrast, no difference in type 2 antigen expression between any clone and the parental cells was observed. These data suggest that coordination of subsequent reactions capable of modifying type 1 chain structures is not the same in all clones. The relationship of these results to aspects of cellular regulation of carbohydrate biosynthesis is discussed.     

Complex <Emphasis Type="Italic">N</Emphasis>-glycans or core 1-derived <Emphasis Type="Italic">O</Emphasis>-glycans are not required for the expression of stage-specific antigens SSEA-1, SSEA-3, SSEA-4, or Le<Superscript>Y</Superscript> in the preimplantation mouse embryo

The glycan epitopes termed stage-specific embryonic antigens (SSEA) occur on glycoproteins and glycolipids in mammals. However, it is not known whether these epitopes are attached to N- or O-glycans on glycoproteins and/or on glycolipids in the developing mouse embryo. In this paper the expression of the antigens SSEA-1, SSEA-3, SSEA-4 and Le^Y was examined on ovulated eggs, early embryos and blastocysts lacking either complex and hybrid N-glycans or core-1 derived O-glycans. In all cases, antigen expression determined by fluorescence microscopy of bound monoclonal antibodies to embryos at the stage of development of maximal expression was similar in mutant and control embryos. Thus, none of these developmental antigens are expressed solely on either complex N- or core 1-derived O-glycans attached to glycoproteins in the preimplantation mouse embryo. Furthermore, neither of these classes of glycan is essential for the expression of SSEA-1, SSEA-3, SSEA-4 or Le^Y on mouse embryos.     

Protein levels of genes encoded on chromosome 21 in fetal Down Syndrome brain (Part V): Overexpression of phosphatidyl-inositol-glycan class P protein (DSCR5)

Summary. Down Syndrome (DS, trisomy 21) is the most common genetic cause of mental retardation. The completed sequencing of genes encoded on chromosome 21 provides excellent basic information, however the molecular mechanisms leading to the phenotype of DS remain to be elucidated. Although overexpression of chromosome 21 encoded genes has been documented information at the protein expression level is mandatory as it is the proteins that carry out function. We therefore decided to evaluated expression level of seven proteins whose genes are encoded on chromosome 21: DSCR4, DSCR5, DSCR6; KIR4.2, GIRK2, KCNE1 and KCNE2 in fetal cortex brain of DS and controls at the early second trimester of pregnancy by Western blotting. -actin and neuron specific enolase (NSE) were used to normalise cell loss and neuronal loss. DSCR5 (PIG-P), a component of glycosylphosphatidylinositol-N-acetylglucosaminyltransferase (GPI-GnT), was overexpressed about twofold, even when levels were normalised with NSE. DSCR6 was overexpressed in addition but when normalised versus NSE, levels were comparable to controls. DSCR4 was not detectable in fetal brain. Potassium channels KIR4.2 and GIRK2 were comparable between DS and controls, whereas KCNE1 and KCNE2 were not detectable. Quantification of these proteins encoded on chromosome 21 revealed that not all gene products of the DS critical region are overexpressed in DS brain early in life, indicating that the DS phenotype cannot be simply explained by the gene dosage effect hypothesis. Overexpression of PIG-P (DSCR5) may lead to or represent impaired glycosylphosphatidylinositol-N-acetylglucosaminyltransferase mediated posttranslational modifications and subsequent anchoring of proteins to the plasma membrane.     

Expression of the β-subunit isoforms of the Na,K-ATpase in rat embryo tissues,inner ear and choroid plexus

Summary— We report evidence of the apical localization of the two Na, K-ATPase β-subunit isoforms in cells of the inner ear and of the choroid plexus of the rat. To this end, we generated isoform-specific antisera against the human Na, K-ATPase β1 and β2 subunits. These polyclonal rabbit antisera were raised against truncated β-isoform proteins that were made in E coli with pET expression vectors. Deglycosylation of the native antigen with N-endoglycosidase F shows four bands in the β1 isoform and five bands in the β2 iso-form immunoblots. In E15 rat embryos, the β1 isoform was detected in brain, heart and kidney and the β2 isoform only in brain. While β-subunit mRNA expression (Watts AG, Sanchéz-Watts G, Emanuel JR, Levenson R 1991 Proc Natl Acad Sci USA 88, 7425–7429), and immunoblotting and enzymatic activity have been determined (Zlokovic BV, Mackic JB, Wang L, McComb JG, McDonough A 1993 J Biol Chem 268, 8019–8025), very little is known about the specific localization of each β-isoform in the epithelia of choroid plexus and inner ear. Immunocytochemical preparations of 15-day-old whole rat embryos and adult rat brain showed an enhanced staining for the β1 and β2 isoforms in the apical membrane of the ampullary crests of the inner ear's semicircular ducts and in the cuboidal cells of the choroid plexus     

A New Amylose Derivative for the Preparation of Protein–Carbohydrate Conjugates

N,N-Dimethylformamidase (DMFase) from Alcaligenes sp. strain KUFA-1, a bacterium that can grow on N,N-dimethylformamide (DMF) as the sole carbon and nitrogen source, catalyzes the first step of the DMF degradation. The DMFase gene dmfA1A2 was cloned in Escherichia coli, and its nucleotides were sequenced. The deduced amino acid sequence of the enzyme consisted of two α- and two β-subunits with 132 and 762 amino acids, respectively, and had little similarity to sequences in protein databases, including various amidases. The protein may be a new kind of amidase. DMFase activity was detected in E. coli cells transformed with an expression plasmid of the cloned DMFase gene. The properties of recombinant DMFase purified from E. coli were identical to those of Alcaligenes DMFase.     

Gene transfer in bovine blastocysts using replication-defective retroviral vectors packaged with gibbon ape leukemia virus envelopes

With this work we demonstrate that murine leukemia virus (MLV)-based replication-defective retroviral vectors encapsidated with Gibbon ape leukemia virus (GaLV) envelopes are significantly more infectious to bovine embryonic trachea (EBTr) cells than vectors encapsidated with murine xenotropic envelope proteins. In a test of internal promoter activity in an MLV retroviral vector, the rat β-actin promoter was shown to be better than the herpes simplex virus type 1 thymidine kinase (TK) and human cytomegalovirus (CMV) immediate early promoters for the expression of an E. coli β-galactosidase marker gene in bovine target cells. By co-culture of bovine blastocysts and virus-producing cells, or by culture of embryos in the medium harvested from virus-producing cells, we transferred the E. coli β-galactosidase gene into trophoblasts and also into inner cell mass (ICM) cells of a bovine embryo through the infection of the MLV-based replication-defective retroviruses encapsidated with GaLV envelope proteins. The infection was confirmed by the expression of the E. coli β-galactosidase gene under a β-actin internal promoter. In addition, co-culture of ICM cells with virus-producing cells resulted in differentiation of ICM cells into embryoid bodies expressing the marker genes. © 1993 Wiley-Liss, Inc.     

BGAL1 depletion boosts the level of β‐galactosylation of N‐ and O‐glycans in N. benthamiana

Glyco‐design of proteins is a powerful tool in fundamental studies of structure–function relationship and in obtaining profiles optimized for efficacy of therapeutic glycoproteins. Plants, particularly Nicotiana benthamiana, are attractive hosts to produce recombinant glycoproteins, and recent advances in glyco‐engineering facilitate customized N‐glycosylation of plant‐derived glycoproteins. However, with exception of monoclonal antibodies, homogenous human‐like β1,4‐galactosylation is very hard to achieve in recombinant glycoproteins. Despite significant efforts to optimize the expression of β1,4‐galactosyltransferase, many plant‐derived glycoproteins still exhibit incomplete processed N‐glycans with heterogeneous terminal galactosylation. The most obvious suspects to be involved in trimming terminal galactose residues are β‐galactosidases (BGALs) from the glycosyl hydrolase family GH35. To elucidate the so far uncharacterized mechanisms leading to the trimming of terminal galactose residues from glycans of secreted proteins, we studied a N. benthamiana BGAL known to be active in the apoplast (NbBGAL1). Here, we determined the NbBGAL1 subcellular localization, substrate specificity and in planta biological activity. We show that NbBGAL1 can remove β1,4‐ and β1,3‐galactose residues on both N‐ and O‐glycans. Transient BGAL1 down‐regulation by RNA interference (RNAi) and BGAL1 depletion by genome editing drastically reduce β‐galactosidase activity in N. benthamiana and increase the amounts of fully galactosylated complex N‐glycans on several plant‐produced glycoproteins. Altogether, our data demonstrate that NbBGAL1 acts on galactosylated complex N‐glycans of plant‐produced glycoproteins.     

Differential Expression of GABAA/Benzodiazepine Receptor Subunit mRNAs and Ligand Binding Sites in Mouse Cerebellar Neurons Following In Vivo Ethanol Administration: An Autoradiographic Analysis

Abstract: The γ-aminobutyric acid_A (GABA_A)/benzodiazepine (BZ) receptor is a pentamer composed of subunits belonging to several classes (α_1–6, β_1–4, γ_1–4, δ, and ρ₁ and ρ₂). In situ hybridization, radioligand autoradiography, and immunocytochemistry were used to examine GABA_A/BZ receptor α₁, α₆, β₂, β₃, and γ₂ subunit expression in murine Purkinje, granule, and deep cerebellar neurons after in vivo ethanol exposure. Chronic ethanol treatment resulted in decreased α₁ subunit mRNA expression in each cell type, whereas the expression of α₆ and γ₂ subunit mRNA levels increased; no changes were observed in the expression of β₂ and β₃ subunit mRNA. GABA and BZ agonist binding and antibody staining paralleled the changes in mRNA levels. Acute ethanol injection resulted in increased expression of α₁ and β₃ mRNAs, whereas levels of α₆, β₂, and γ₂ mRNAs remained stable. Our results indicate that, in cerebellar neurons, the expression of specific GABA_A/BZ receptor subunit mRNAs, polypeptides, and binding sites is independently regulated by in vivo administration of alcohol. The observed changes were not restricted to any one cerebellar cell type, because subunit expression in Purkinje, granule, and deep cerebellar cells was similarly affected.     

Activation of Unusual Mac-1+CD4− CD8− T Cells Bearing αβ Antigen Receptor in Murine Lungs during Infection with Mycobacterium bovis BCG: Regulation by Interferon-γ

We have recently (Kawakami et al, Immunol. Lett. 1995;46: 143) demonstrated that unusual Mac-1⁺CD4^?CD8^? T cells bearing αβ antigen receptor (Mac-1⁺ αβ T cells) reside in a considerable proportion in murine lungs. The present study was performed to examine the dynamics of accumulation of these cells in the lungs following intravenous administration of Mycobacterium bovis BCG (BCG). Mac-1⁺ αβ T cells accumulated rapidly 24 hr after infection, followed by a gradual increase over the observation period of 15 days. Furthermore, the expression of Ia, ICAM-1 and FcγR II/III on their surface intensified dramatically after BCG infection. The kinetics of enhancement of Ia expression was slower than that of ICAM-1, with the maximum level attained in one day in the latter molecule but in two weeks in the former. Neutralization of endogenous IFN-γ by specific mAb completely blocked the augmented expression of Ia on Mac-1⁺ αβ T cells after BCG infection, but did not have any significant effect on that of ICAM-1. In contrast, in vivo administration of IFN-γ enhanced the expression of ICAM-1 as well as that of Ia. Our results indicate that accumulation of Mac-1 αβ T cells within the lung is associated with a differential change in the expression of surface antigens, and suggest that these cells may play a role in the host defense against mycobacterial infection.     

Immunomodulatory Effects of Oat β-Glucan Administered Intragastrically or Parenterally on Mice Infected with Eimeria vermiformis

The immunostimulatory effect of intragastrically or parenterally administered β-(1→3; 1→4) glucan, extracted from oats (OβG), on disease resistance to Eimeria vermiformis was studied in C57BL/6 mice. Multiple administrations of OβG by intragastric or subcutaneous routes reduced fecal oocyst shedding compared to the non-treated control group. The administration of OβG by subcutaneous route resulted in higher levels of total serum immunoglobulins and antigen (sporozoite and merozoite)-specific immunoglobulins as compared with the non-treated group. To evaluate the effect of a single subcutaneous dose, groups of mice were treated with OβG 2 days before E. vermiformis infection, at the time of infection and at 2 or 6 days after infection. From day 11 post-infection the oocyst discharge was significantly diminished (P< 0.05-0.01) in the OβG-treated groups, except in those treated 6 days after infection, as compared to the non-treated control group. The proliferative responses to E. vermiformis sporozoite antigen of lymphocytes isolated from the spleen were significantly increased (P < 0.05) when OβG was administered 2 days before or at the time of E. vermiformis infection. Lymphocyte proliferative responses to merozoite antigen were not influenced by treatment. In conclusion, OβG appeared to up-regulate immune mechanisms and provide enhanced resistance against eimerian coccidiosis in mice.     

Stage-specific embryonic antigens (SSEA-3 and -4) are epitopes of a unique globo-series ganglioside isolated from human teratocarcinoma cells.

Two monoclonal antibodies (MC631 and MC813-70) raised against 4- to 8-cell stage mouse embryos and a human teratocarcinoma cell line, respectively, detect the stage-specific embryonic antigens, the previously defined SSEA-3 and SSEA-4, described herein. These antibodies were both reactive with a unique globo-series ganglioside with the structure shown below: (formula; see text) The antibodies were found to recognize sequential regions of this ganglioside, i.e., MC813-70 recognizes the terminal 'a' structure whereas antibody MC631 recognizes the internal 'b' structure. Thus, a set of two antibodies defines this unique embryonic antigen. During differentiation of human teratocarcinoma 2102Ep cells, the globo-series glycolipids defined by these antibodies decrease and the lacto-series glycolipids, reacting with the SSEA-1 antibody appear. This antigenic conversion suggests that a shift of glycolipid synthesis from globo-series to lacto-series glycolipids occurs during differentiation of human teratocarcinoma and perhaps of pre-implantation mouse embryos.     

Immunomodulatory Activities of Oat β-Glucan In Vitro and In Vivo

Previous studies have shown that β-glucans extracted from yeast or fungi potentiate immune responses. In the present study, the immunomodulatory activities of β-(1→3, 1→4)-glucan, derived from oats, were investigated. The ability of oat β-glucan (OβG) to stimulate IL-1 and TNF-α release from murine peritoneal macrophages and the murine macrophage cell line P338D1, was assessed. In vitro stimulation of macrophages with OβG resulted in the production of IL-1 in a dose and time-dependent manner, whereas only small amounts of TNF-α could be detected in the culture supernatants. OβG also induced the production of IL-2, IFN-γ and IL-4 secretion in a dose-dependent manner in cultured spleen cells. The intraperitoneal administration of OβG in mice resulted in the accumulation of leucocytes, predominantly macrophages, in the peritoneal cavity. Furthermore, OβG was tested for its ability to enhance non-specific resistance to a bacterial challenge in mice. Survival of mice challenged with Staphylococcus aureus was enhanced by a single intraperitoneal administration of 500 μg of OβG 3 days prior to bacterial challenge. In conclusion, these studies demonstrated that OβG possesses immunomodulatory activities capable of stimulating immune functions both in vitro and in vivo.     

Isolation and characterization of α‐(1→3)‐glucan‐degrading bacteria from the gut of Diaperis boleti feeding on Laetiporus sulphureus

Bacteria degrading α‐(1→3)‐glucan were sought in the gut of fungivorous insects feeding on fruiting bodies of a polypore fungus Laetiporus sulphureus, which are rich in this polymer. One isolate, from Diaperis boleti, was selected in an enrichment culture in the glucan‐containing medium. The bacterium was identified as Paenibacillus sp. based on the results of the ribosomal DNA analysis. The Paenibacillus showed enzyme activity of 4.97 mU/cm³ and effectively degraded fungal α‐(1→3)‐glucan, releasing nigerooligosaccharides and a trace amount of glucose. This strain is the first reported α‐(1→3)‐glucan‐degrading microorganism in the gut microbiome of insects inhabiting fruiting bodies of polypore fungi.     

Amyloid β‐induced astrogliosis is mediated by β1‐integrin via NADPH oxidase 2 in Alzheimer's disease

Astrogliosis is a hallmark of Alzheimer′s disease (AD) and may constitute a primary pathogenic component of that disorder. Elucidation of signaling cascades inducing astrogliosis should help characterizing the function of astrocytes and identifying novel molecular targets to modulate AD progression. Here, we describe a novel mechanism by which soluble amyloid‐β modulates β1‐integrin activity and triggers NADPH oxidase (NOX)‐dependent astrogliosis in vitro and in vivo. Amyloid‐β oligomers activate a PI3K/classical PKC/Rac1/NOX pathway which is initiated by β1‐integrin in cultured astrocytes. This mechanism promotes β1‐integrin maturation, upregulation of NOX2 and of the glial fibrillary acidic protein (GFAP) in astrocytes in vitro and in hippocampal astrocytes in vivo. Notably, immunochemical analysis of the hippocampi of a triple‐transgenic AD mouse model shows increased levels of GFAP, NOX2, and β1‐integrin in reactive astrocytes which correlates with the amyloid β‐oligomer load. Finally, analysis of these proteins in postmortem frontal cortex from different stages of AD (II to V/VI) and matched controls confirmed elevated expression of NOX2 and β1‐integrin in that cortical region and specifically in reactive astrocytes, which was most prominent at advanced AD stages. Importantly, protein levels of NOX2 and β1‐integrin were significantly associated with increased amyloid‐β load in human samples. These data strongly suggest that astrogliosis in AD is caused by direct interaction of amyloid β oligomers with β1‐integrin which in turn leads to enhancing β1‐integrin and NOX2 activity via NOX‐dependent mechanisms. These observations may be relevant to AD pathophysiology.     

Differential regulation of CaMKII inhibitor β protein expression after exposure to a novel context and during contextual fear memory formation

Understanding of the molecular basis of long‐term fear memory (fear LTM) formation provides targets in the treatment of emotional disorders. Ca²⁺/calmodulin‐dependent protein kinase II (CaMKII) is one of the key synaptic molecules involved in fear LTM formation. There are two endogenous inhibitor proteins of CaMKII, CaMKII Nα and Nβ, which can regulate CaMKII activity in vitro. However, the physiological role of these endogenous inhibitors is not known. Here, we have investigated whether CaMKII Nβ protein expression is regulated after contextual fear conditioning or exposure to a novel context. Using a novel CaMKII Nβ‐specific antibody, CaMKII Nβ expression was analysed in the naïve mouse brain as well as in the amygdala and hippocampus after conditioning and context exposure. We show that in naïve mouse forebrain CaMKII Nβ protein is expressed at its highest levels in olfactory bulb, prefrontal and piriform cortices, amygdala and thalamus. The protein is expressed both in dendrites and cell bodies. CaMKII Nβ expression is rapidly and transiently up‐regulated in the hippocampus after context exposure. In the amygdala, its expression is regulated only by contextual fear conditioning and not by exposure to a novel context. In conclusion, we show that CaMKII Nβ expression is differentially regulated by novelty and contextual fear conditioning, providing further insight into molecular basis of fear LTM.     

Immunochemistry of the Lewis-blood-group system: Partial characterization of Lea-, Leb-, and H-type 1 (LedH)-blood-group active glycosphingolipids from human plasma

Four different H-type 1 (Le^dH) blood-group-active glycosphingolipids (Le^dH-I–IV) have been isolated from the plasma of blood-group O Le(a?b?) secretors. The agglutination of O Le(a?b?) erythrocytes from secretors by 50 μl of 4 hemagglutinating units of caprine anti-Le^dH (anti-H-type 1) serum was inhibited by 0.02 μg of each of all four glycolipids. No Le^a or Le^b activities or reaction against Ulex europaeus lectin could be found. Le^dH-I, -II, -III, and -IV at 0.05, 0.01, 0.01, and 0.02 μg each are sufficient for incubation in order to convert 9 × 10⁷ O Le(a?b?) erythrocytes from nonsecretors into H-type 1 (Le^dH)-positive cells. Structural analysis of the H-type 1 glycolipids was performed in comparison to that of Le^a- and Le^b-blood-group-active glycolipids from human plasma isolated previously: Gas chromatography of peracetylated alditols revealed sugar composition. Combined gas chromatography-mass spectrometry established the glycosidic linkages. Together with the results obtained by direct inlet mass spectrometry of permethylated glycosphingolipids and by 360-MHz ¹H nuclear magnetic resonance spectroscopy (Egge, H., and Hanfland, P., 1981, Arch. Biochem. Biophys., 210, 396–404; Dabrowski, J., Hanfland, P., Egge, H., and Dabrowski, U., 1981, Arch. Biochem. Biophys., 210, 405–411) the complete structures of the oligosaccharide chains of the Le^a-, Le^b-, and H-type 1-active glycolipids were established: Galβ1 → 3GlcNAc(4 ← 1αFuc)β1 → 3Galβ1 → 4Glcβ1 → 1 Cer for the Le^a antigens; Fucα1 → 2Galβ1 → 3GlcNAc(4 ← 1αFuc)β1 → 3Galβ1 → 4Glcβ1 → 1 Cer for the Le^b antigens; and Fucα1 → 2Galβ1 → 3GlcNAcβ1 → 3Galβ1 → 4Glcβ1 → 1 Cer for the H-type 1 (Le^dH) glycolipids. The diverse antigens of the same blood-group specificity obviously differ from one another in their lipid residue. In addition, plasmatic neolactotetraosylceramide could be identified, differing from that of human erythrocytes by a slower migration behavior in thin-layer chromatography.