Expression of UDP-N-acetylgalactosamine: beta-galactose beta 1,4-N-acetylgalactosaminyltransferase in functionally defined T-cell clones.

To measure UDP-N-acetylgalactosamine: beta-galactose beta 1,4-N-acetylgalactosaminyltransferase (beta 1,4-GalNActransferase) in crude cell and tissue extracts we designed an assay containing UDP-[3H]N-acetylgalactosamine as donor and biotinylated human glycophorin A as an acceptor. After incubation the labelled acceptor was separated by the use of avidin-agarose from extract-derived endogenous acceptors. This assay permitted one to measure specifically the beta 1,4-GalNActransferase in crude extracts. This glycosyltransferase has previously been shown to be involved in the biosynthesis of Vicia villosa (hairy winter vetch)-lectin (VV)-binding sites of the murine cytotoxic T-cell line B6.1. Since VV-binding sites are a distinct marker for the cytotoxic subclass of murine T-lymphocytes, we used this assay to determine enzyme levels in a panel of functionally defined murine T-cell clones. Non-cytolytic T-cell lines generally have low activity, whereas most cytotoxic lines have high levels of activity. However, one cytotoxic T-cell line does not express the enzyme, although it has large numbers of VV-binding sites. This suggests the existence of another type of VV-binding sites which is independent of the beta 1,4-GalNActransferase in some cytotoxic-T-lymphocyte lines. The enzyme was also assayed in a variety of other tissues and found to have a very high activity in the intestine but a low activity in most other tissues. This was in considerable contrast with the ubiquitously high expression of UDP-GalNAc:peptide alpha 1-GalNActransferase. Therefore, the beta 1,4-GalNActransferase seems to be regulated during differentiation.     

The N-glycosylation defect of cwh8Delta yeast cells causes a distinct defect in sphingolipid biosynthesis

CWH8/YGR036c of Saccharomyces cerevisiae has been identifiedas a dolichylpyrophosphate (Dol-PP) phosphatase that removesa phosphate from the Dol-PP generated by the oligosaccharyltransferase(OST), while it adds N-glycans to nascent glycoproteins in theendoplasmic reticulum (ER). Lack of CWH8 was proposed to interruptthe so called dolichol (Dol) cycle by trapping Dol in the formof Dol-PP in the ER lumen. Indeed, cwh8D mutants display a severedeficiency in N-glycosylation. We find that cwh8D mutants havestrongly reduced levels of inositolphosphorylceramide (IPC),whereas its derivative, mannosyl-(inositol-P)₂-ceramide (M(IP)₂C)is not affected. Microsomes of cwh8D contain normal ceramidesynthase and IPC synthesis activities. Within a large panelof mutants affecting Dol dependent pathways such as N- or O-glycosylation,or glycosylphosphatidyl inositol (GPI)-anchoring, only the mutantshaving a deficiency of N-glycan addition show the defect inIPC biosynthesis. By mutating genes required for the additionof N-glycans or by treating cells with tunicamycin (Tm) onecan similarly reduce the steady state level of IPC and exactlyreproduce the phenotype of cwh8D cells. Some potential mechanismsby which the lack of N-glycans could lead to the sphingolipidabnormality were further explored.     

Glycine reduces platelet aggregation

It has been demonstrated that a wide variety of white blood cells and macrophages (i.e. Kupffer cells, alveolar and peritoneal macrophages and neutrophils) contain glycine-gated chloride channels. Binding of glycine on the receptor stimulates Cl^? influx causing membrane hyperpolarization that prevents agonist-induced influx of calcium. Since platelet-aggregation is calcium-dependent, this study was designed to test the hypothesis that glycine would inhibit platelet aggregation. Rats were fed diets rich of glycine for 5 days, while controls received isonitrogenous valine. The bleeding time and ADP- and collagen-induced platelet aggregation were measured. Dietary glycine significantly increased bleeding time about twofold compared to valine-treated controls. Furthermore, the amplitude of platelet aggregation stimulated with ADP or collagen was significantly decreased in whole blood drawn from rats fed 2.5 or 5 % dietary glycine by over 50 %. Addition of glycine in vitro (1–10 mM) also blunted rat platelet aggregation in a dose-dependent manner. Strychnine, a glycine receptor antagonist, abrogated the inhibitory effect of glycine on platelet-aggregation in vitro suggesting the glycine works via a glycine receptor. Glycine also blunted aggregation of human platelets. Further, the glycine receptor was detected in both rat and human platelets by western blotting. Based on these data, it is concluded that glycine prevents aggregation of platelets in a dose-dependent manner via mechanisms involving a glycine receptor.     

Mokola virus glycoprotein and chimeric proteins can replace rabies virus glycoprotein in the rescue of infectious defective rabies virus particles.

A reverse genetics approach which allows the generation of infectious defective rabies virus (RV) particles entirely from plasmid-encoded genomes and proteins (K.-K. Conzelmann and M. Schnell, J. Virol. 68:713-719, 1994) was used to investigate the ability of a heterologous lyssavirus glycoprotein (G) and chimeric G constructs to function in the formation of infectious RV-like particles. Virions containing a chloramphenicol acetyltransferase (CAT) reporter gene (SDI-CAT) were generated in cells simultaneously expressing the genomic RNA analog, the RV N, P, M, and L proteins, and engineered G constructs from transfected plasmids. The infectivity of particles was determined by a CAT assay after passage to helper virus-infected cells. The heterologous G protein from Eth-16 virus (Mokola virus, lyssavirus serotype 3) as well as a construct in which the ectodomain of RV G was fused to the cytoplasmic and transmembrane domains of the Eth-16 virus G rescued infectious SDI-CAT particles. In contrast, a chimeric protein composed of the amino-terminal half of the Eth-16 virus G and the carboxy-terminal half of RV G failed to produce infectious particles. Site-directed mutagenesis was used to convert the antigenic site III of RV G to the corresponding sequence of Eth-16 G. This chimeric protein rescued infectious SDI-CAT particles as efficiently as RV G. Virions containing the chimeric protein were specifically neutralized by an anti-Eth-16 virus serum and escaped neutralization by a monoclonal antibody directed against RV antigenic site III. The results show that entire structural domains as well as short surface epitopes of lyssavirus G proteins may be exchanged without affecting the structure required to mediate infection of cells.     

A domain-oriented approach to the reduction of combinatorial complexity in signal transduction networks

Background:  

Receptors and scaffold proteins possess a number of distinct domains and bind multiple partners. A common problem in modeling signaling systems arises from a combinatorial explosion of different states generated by feasible molecular species. The number of possible species grows exponentially with the number of different docking sites and can easily reach several millions. Models accounting for this combinatorial variety become impractical for many applications.     

Incorporation and metabolism of ganglioside GM2 in skin fibroblasts from normal and GM2 gangliosidosis subjects

Ganglioside GM2, 3H-labeled in the sphingoid base, was added to the culture medium of normal and GM2 gangliosidosis fibroblasts. Ganglioside was found to adsorb rapidly to the cell surface, most of it could however be removed by trypsination. The trypsin-resistant incorporation was about 10 nmol/mg cell protein, after 48 h. The rates of adsorption and incorporation depended strongly on the concentration of fetal calf serum in the medium, higher serum concentrations being inhibitory. After various incubation times, the lipids were extracted, separated by thin-layer chromatography and visualized by fluorography. In normal cells a variety of degradation products as well as sphingomyelin was found whereas in GM2 gangliosidosis cells, only trace amounts of such products (mainly GA2) were found. In contrast, the higher gangliosides GM1 and GD1a were formed in comparable amounts (2.2-3.6% of total radioactivity after 92 h) in normal and pathologic cell lines. Supplementation of cells from GM2 gangliosidosis, variant AB, with purified GM2-activator protein restored ganglioside GM2 degradation to almost normal rates but had no effect on its glycosylation to gangliosides GM1 and GD1a. From these results we conclude that the synthesis of higher gangliosides from incorporated GM2 can occur by direct glycosylation and not only via lysosomal degradation and resynthesis from [3H]sphinganine-containing degradation products. Preliminary studies with subcellular fractionation after various times of [3H]ganglioside incorporation indicated biphasic kinetics for the net transport of membrane-inserted ganglioside to lysosomes, compatible with the notion that a portion of the glycolipids can also escape from secondary lysosomes and migrate to Golgi compartment or cell surface.     

Occurrence of lysoganglioside lyso-GM2 (II3-Neu5Ac-gangliotriaosylsphingosine) in GM2 gangliosidosis brain

Lysoganglioside lyso-GM2 (sialylgangliotriaosylsphingosine) was detected in a brain sample of a patient with variant B of infantile GM2 gangliosidosis (Tay-Sachs disease) at a concentration of approx. 15 nmol/g wet tissue. Neither lysoganglioside lyso-GM2 nor the corresponding GM1 derivative could be detected in normal brain.     

Anchoring of membrane proteins via phosphatidylinositol is deficient in two classes of Thy-1 negative mutant lymphoma cells.

Recent evidence shows that mature Thy-1 glycoprotein lacks amino acids 113-143 predicted from the cDNA sequence and is anchored to the plasma membrane by a phosphatidylinositol-containing glycolipid attached to amino acid 112. Previously characterized Thy-1-deficient mutant lymphoma lines of complementation classes A and E were analysed. They make detergent binding Thy-1 precursors but, in contrast to wild-type, the detergent binding moiety cannot be removed by phospholipase C. Moreover, tryptophan which only occurs at position 124 is incorporated into mutant but not parental Thy-1. This suggests that the mutants make a Thy-1 precursor of 143 amino acids but fail to replace its C-terminal end by a glycolipid anchor.     

Human homologues of LAG1 reconstitute Acyl-CoA-dependent ceramide synthesis in yeast

Lag1p and Lac1p are two highly homologous membrane proteins of the endoplasmic reticulum. lag1delta lac1delta double mutants in Saccharomyces cerevisiae lack an acyl-CoA-dependent ceramide synthase and are either very sick or nonviable, depending on the genetic background. LAG1 and LAC1 are members of a large eukaryotic gene family that shares the Lag1 motif, and some members of this family additionally contain a DNA-binding HOX homeodomain. Here we show that several human LAG1 homologues can rescue the viability of lag1delta lac1delta yeast cells and restore acyl-CoA-dependent ceramide and sphingolipid biosynthesis. When tested in a microsomal assay, Lac1p and Lag1p had a strong preference for C26:0-CoA over C24:0-CoA, C20-CoA, and C16-CoA, whereas some human homologues preferred C24:0-CoA and CoA derivatives with shorter fatty acids. This suggests that LAG1 proteins are related to substrate recognition and to the catalytic activity of ceramide synthase enzymes. CLN8, another human LAG1 homologue implicated in ceroid lipofuscinosis, could not restore viability to lag1delta lac1delta yeast mutants.