Precursor cells in mouse islets generate new beta-cells in vivo during aging and after islet injury.

Whereas it is believed that the pancreatic duct contains endocrine precursors, the presence of insulin progenitor cells residing in islets remain controversial. We tested whether pancreatic islets of adult mice contain precursor beta-cells that initiate insulin synthesis during aging and after islet injury. We used bigenic mice in which the activation of an inducible form of Cre recombinase by a one-time pulse of tamoxifen results in the permanent expression of a floxed human placental alkaline phosphatase (PLAP) gene in 30% of pancreatic beta-cells. If islets contain PLAP(-) precursor cells that differentiate into beta-cells (PLAP(-)IN(+)), a decrease in the percentage of PLAP(+)IN(+) cells per total number of IN(+) cells would occur. Conversely, if islets contain PLAP(+)IN(-) precursors that initiate synthesis of insulin, the percentage of PLAP(+)IN(+) cells would increase. Confocal microscope analysis revealed that the percentage of PLAP(+)IN(+) cells in islets increased from 30 to 45% at 6 months and to 60% at 12 months. The augmentation in the level of PLAP in islets with time was confirmed by real-time PCR. Our studies also demonstrate that the percentage of PLAP(+)IN(+) cells in islets increased after islet injury and identified putative precursors in islets. We postulate that PLAP(+)IN(-) precursors differentiate into insulin-positive cells that participate in a slow renewal of the beta-cell mass during aging and replenish beta-cells eliminated by injury.

Regulation of Paramecium primaurelia glycosylphosphatidyl-inositol biosynthesis via dolichol phosphate mannose synthesis.

A set of glycosylinositol-phosphoceramides, belonging to a family of glycosylphosphatidyl-inositols (GPIs) synthesized in a cell-free system prepared from the free-living protozoan Paramecium primaurelia has been described. The final GPI precursor was identified and structurally characterized as: ethanolamine-phosphate-6Man alpha 1-2Man alpha 1-6(mannosylphosphate) Man alpha 1-4glucosamine-inositol-phospho-ceramide. During our investigations on the biosynthesis of the acid-labile modification, the additional mannosyl phosphate substitution, we observed that the use of the nucleotide triphosphate analogue GTP gamma S (guanosine 5-O-(thiotriphosphate)) blocks the biosynthesis of the mannosylated GPI glycolipids. We show that GTP gamma S inhibits the synthesis of dolichol-phosphate-mannose, which is the donor of the mannose residues for GPI biosynthesis. Therefore, we investigated the role of GTP binding regulatory 'G' proteins using cholera and pertussis toxins and an intracellular second messenger cAMP analogue, 8-bromo-cAMP. All the data obtained suggest the involvement of classical heterotrimeric G proteins in the regulation of GPI-anchor biosynthesis through dolichol-phosphate-mannose synthesis via the activation of adenylyl cyclase and protein phosphorylation. Furthermore, our data suggest that GTP gamma S interferes with synthesis of dolichol monophosphate, indicating that the dolichol kinase is regulated by the heterotrimeric G proteins.

Signposts in the assembly of chylomicrons.

Intestinal cells synthesize and secrete chylomicrons in the postprandial state. Synthesis of these particles is defective in abetalipoproteinemia and chylomicron retention disease. Chylomicrons are very large, heterogeneous, lipid-rich particles ranging in diameters from 75 to 450 nm and function to transport dietary fat and fat-soluble vitamins to blood. The size heterogeneity of the secreted particles depends on the rate of fat absorption, type and amount of fat absorbed. The fatty acid composition of triglycerides present in chylomicrons reflects the composition of dietary fat, whereas the fatty acid composition of chylomicron phospholipids does not. The differences in the fatty acid compositions are also observed when lipids are labeled with glycerol. Thus, the differences are not due to differential incorporation of dietary fatty acids into different lipids but are mainly due to different pools of lipids used for chylomicron assembly. It has been suggested that preformed phospholipids and nascent triglycerides are preferentially used for intestinal lipoprotein assembly. Biosynthesis of chylomicrons requires apoB48. ApoB48 is translated from apoB mRNA that is post-transcriptionally edited in the intestinal cells to incorporate a stop codon. Nascent apoB48 may be cotranslationally lipidated and this process is critically dependent on the presence of microsomal triglyceride transfer protein. Two different models have been proposed for the assembly of chylomicrons. In the independent model, intestinal cells are hypothesized to synthesize VLDL and chylomicron by two independent pathways. The chylomicron assembly pathway is hypothesized to be sensitive to a surfactant, Pluronic L81, but that of VLDL assembly is not. In the sequential assembly model, synthesis of all lipoproteins is hypothesized to begin with the assembly of apoB-containing primordial lipoprotein particles. The primordial particles are suggested to fuse with triglyceride-rich lipid droplets that are synthesized independently of apoB. This process results in the core expansion of primordial particles and the synthesis of nascent lipoproteins. Differences in the size of secreted lipoproteins may be due to differences in the size of triglyceride-rich lipid droplets. Pluronic L81 is hypothesized to inhibit the formation of large triglyceride-rich droplets that serve as precursors for chylomicron assembly. In this review, we have discussed some signposts that might be unique to different steps in the assembly of chylomicrons. First, it is proposed that the association of preformed phospholipids with nascent apoB in the endoplasmic reticulum may serve as a signpost for the very early steps in the assembly of chylomicrons. Second, association of large amounts of newly synthesized triglycerides compared to preformed triglycerides may serve as a signpost for the assembly of larger lipoproteins. Third, the incorporation of retinyl esters may serve as markers for the final stages of chylomicron assembly. These signposts may be helpful in the identification and characterization of various intermediates in the assembly of chylomicrons. The knowledge about the molecular assembly of chylomicrons may lead to better therapeutic agents for controlling various hyperlipidemias, obesity, and atherosclerosis.

The Schizosaccharomyces pombe GPI8 gene complements a Saccharomyces cerevisiae GPI8 anchoring mutant.

The final step in glycosylphosphatidylinositol (GPI) anchoring of cell surface proteins consists of a transamidation reaction, in which preassembled GPI donors are substituted for C-terminal signal sequences in nascent polypeptides. The Saccharomyces cerevisiae GPI8 gene (ScGPI8) encodes a protein which is involved in the GPI transamidation reaction. We have cloned and isolated the Schizosaccharomyces pombe GPI8 homologous gene (SpGPI8). The SpGPI8 gene encodes a protein of 411 amino acids with a calculated molecular weight of about 47 kDa. It shows 53.5% identity with the ScGPI8 and complements a S. cerevisiae GPI8 anchoring mutant.

Processing and localisation of a GPI-anchored Plasmodium falciparum surface protein expressed by the baculovirus system.

We describe the expression, in insect cells using the baculovirus system, of two protein fragments derived from the C-terminus of merozoite surface protein 1(MSP-1) of the human malaria parasite Plasmodium falciparum, and their glycosylation and intracellular location. The transport and intracellular localisation of the intact C-terminal MSP-1 fragment, modified by addition of a signal sequence for secretion, was compared with that of a similar control protein in which translation of the GPI-cleavage/attachment site was abolished by insertion of a stop codon into the DNA sequence. Both proteins could only be detected intracellularly, most likely in the endoplasmic reticulum. This lack of transport to the cell surface or beyond, was confirmed for both proteins by immunofluorescence with a specific antibody and characterisation of their N-glycans. The N-glycans had not been processed by enzymes localised in post-endoplasmic reticulum compartments. In contrast to MSP-1, the surface antigen SAG-1 of Toxoplasma gondii was efficiently transported out of the endoplasmic reticulum of insect cells and was located, at least in part, on the cell surface. No GPI-anchor could be detected for either of the MSP-1 constructs or SAG-1, showing that the difference in transport is a property of the individual proteins and cannot be attributed to the lack of a GPI-anchor. The different intracellular location and post-translational modification of recombinant proteins expressed in insect cells, as compared to the native proteins expressed in parasites, and the possible implications for vaccine development are discussed.

An early step of glycosylphosphatidyl-inositol anchor biosynthesis is abolished in lepidopteran insect cells following baculovirus infection.

The expression of recombinant proteins in their native state has become a prerequisite for a variety of functional and structural studies, as well as vaccine development. Many biochemical properties and functions of proteins are dependent on or reside in posttranslational modifications, such as glycosylation. The baculovirus system has increasingly become the system of choice due to it capabilities of performing posttranslational modifications and usually high yields of recombinant proteins. The Toxoplasma gondii surface antigen SAG1 was used as a model for a glycosylphosphatidyl-inositol (GPI)-anchored protein and expressed in insect cells using the baculovirus system. We show that the T. gondii SAG1 surface antigen expressed in this system was not modified by a GPI-anchor. In vitro and in vivo studies demonstrate that uninfected insect cells are able to produce GPI-precursors and to transfer a mature GPI-anchor to nascent proteins. These cells however are not capable to produce GPI-precursors following infection. We also show that the biosynthesis of the early GPI intermediate GlcNH(2)-PI is blocked in baculovirus-infected H5 cells, thus preventing the subsequent mannosylation steps for the synthesis of the conserved GPI-core-glycan. We therefore conclude that the baculovirus system is not appropriate for the expression of GPI-anchored proteins.