Golgi Glycosylation Process And Its Biological Function In Saccharomyces Cerevisiae

Since Saccharomyces cerevisiae has several advantages, such as safe, easy to cultivate, having glycosylation and a clear biochemical and genetic background, it has been one of the commonly used organisms for heterogenous protein expression. S. cerevisiae is the first sequenced genome eukaryotes, and the gene annotations in yeast genome database provide powerful information for structure-function studies, for identification of essential domains, and for elucidation of topography of membrane-bound glycosylation enzymes. This single-celled organism is also important in understanding cellular and molecular processes in eukaryotes. As some stages of glycosylation are highly conserved among eukaryotes, yeast glycosylation mutants can be used to isolate cDNAs encoding enzymes of these pathways from other species. The availability of genes encoding glycosylation enzymes in the ER and Golgi will be useful to identify their targeting mechanisms to these subcellular compartments. Depending on the proteins, glycans may contribute to their conformation, stability, and appropriate targeting. Furthermore, in multicellular eukaryotes, specific carbohydrate structures are known to participate in biological recognition processes. In eukaryotic cells, secreted and membrane proteins are frequently modified with complex glycan structures. The syntheses of these N-glycans, initiating in the endopiasmic reticulum (ER), are catalyzed by the enzyme oligosaccharyltransferase complex (OST), transfering the glycans from the lipid carrier (dolichol) to asparagine residues in the polypeptide chains. After the export of predominantly Man₈GlcNAc₂-containing glycoproteins to the Golgi, the core oligosaccharide may be hypermannosylated with up to 200 mannose residues. This work focused on the Golgi glycosylation pathway and the biofunction of the outer chains.The OCH1 gene encodes al, 6-mannosyltransferase functional in the initiating stage of mannose outer chain addition to the to the ER-form core oligosaccharide. Golgiα1, 3-mannosyltransferase (Mnn1p) is known to be responsible for the addition of the fourth mannose residue on N-linked chains, and it has been postulated to terminally mannosylate the core and outer chains on N-linked glycans as well. To get a mutant deficient in Golgi glycosylation, we deleted the two mannosyltransferase, Mnn1p and Och1p. The null disruptions of MNN1. 0CH1 were carried out basing on an overlap extension PCR strategy. MNNI, OCHI was replaced by the S. cerevisiae URA3, HIS3, respectively. Transformants were confirmed by amplifying and sequencing the recombinant genomic region. Therefore, we generated two glycosylation mutants, mnn1 mutant and mnn1 och1 mutant. To characterize the N-glycosylation in the mnn1 och1 mutant, mannoproteins were obtained by hot citrate buffer extraction after the mnn1 och1 cells were crumbled. The extracted mannoprotein was precipitated by ethanol, and further purified by concanavalin A-sepharose 4B. The N-oligomannose saccharides were released from mannoprotein by PNGase F digestion, and then peptides and detergents were removed by passage through ion exchange columns. For desalting, glycans were applied to porous graphitic-carbon cartridge. 2-aminopyridine pyridylaminated sugars were profiled and purified by size fractionation HPLC with Shim-pack clc-NH2 column, and result showed dominantly a single peak. MALDI TOF/MS analysis of this peak revealed that its molecular weight was 1796.5 Da, which corresponds to the calculated mass of Man₈GlcNAc₂-PA. These results indicated that disruptions of MNN1 and OCH1 eliminated the hypermannosylation of the N-linked glycans, and glycoproteins were glycosylated with a single core type glycan, Man₈GlcNAc₂, in the mnn1 och1 mutant.N-glycosylation pathway involves the synthesis of lipid-linked oligosaccharide precursor and the subsequent processing events in the ER and the Golgi. It functions by modifying proteins with appropriate oligosaccharide structures, thus influencing their properties and bioactivities. N-glycan matures in Golgi apparatus and perturbations in Golgi N-glycosylation correlate with, and may result from, other malfunctions of the Golgi pathway. The mnn1 mutant yeast cells exhibit no observable change compared to the wild type strain at all temperature. The mnn1 och1 double mutant showed a slower growth rate and a thinner cell density. The Golgi glycosylation mutation also affected the cell viability; the mnn1 och1 mutant became temperature-sensitive and trypan blue dye staining showed more than 35% of cells died at nonpermissive temperature for 20h. However, these defects could be rescued in the presence of osmotic stabilizers. Additionally, the mnn1 och1 mutations impaired cell cytokinesis—most of the mother cells sporulated with two or three daughter cells. The double null mutant cells grew extremely clumped together. Even sonication could not disrupt cell clumps efficiently, indicating strong cell-cell interactions. DAPI staining revealed a nuclear migration defect in mnn1 och1 mutant cells; some of the buds were anucleate. The loss of mannosyl-phosphate accepting sites in mnn1 och1 also resulted in a loss of charge repulsion between cell surfaces and impairment of the surface hydration layer, causing cells to aggregate.Apoptosis functions to clear unused or potentially harmful cells remained in the unicellular organism. Here, for the first time, we showed that the accidental cell death and programmed cell death induced with defect in the outer sugar chain by blocking the Golgi N-glycosylation elongation in S. cerevisiae. Microscopic visualization of trypan blue stained mnn1 och1 cells at 37℃showed that the surface of some dead cells displayed a loose and wrinkled appearance, one of the characteristics of ageing cells. To test whether the mnn1 och1 mutation induces apoptosis at nonpermissive temperature, we examined the morphological and biochemical features of apoptosis. Results showed that the mnn1 och1 cells displayed chromatin condensation and nuclear fragmentation. PS was also exposed on the outer surface of the plasma membrane when cells were still metabolically active and able to exclude the vital dye PI. These evidences indicated that the mnn1 och1 mutant underwent a program cell death. The production of reactive oxygen species in the mnn1 och1 mutant was also detected by Dihydrorhodamine 123. As the ROS have been shown to be a regulator of inducing apoptosis in yeast, the program cell death of mnn1 och1 mutant was probably due to the accumulation of ROS. The mnn1 och1 mutant also underwent a necrotic cell death caused by the cell wall defects. The phenomenon was verified by the increase of cell survival in the presence of an osmotic stabilizer, increase of susceptibility to glucuronidase digestion and sensitivity to the Congo red. Therefore, our study provides a new insight into the correlation between glycosylation with the cell death in yeast.Beta Human interferon (HuIFN-β) is a glycoprotein, secreted by fibroblasts in response to viral infection or exposure to double-stranded RNA. It has an antiviral activity, and has also been used in chemotherapy of certain types of tumors and therapy of multiple sclerosis. To study the HuIFN-βexpression in different strains, the human interferon-βgene was inserted in the Hind III clone site of the secretion-expression plasmid YFD18, and the recombinant plasmid, pYFD18-HuIFN was constructed. The recombinant plasmid was then transformed into strain W303-1A, mnn1 mutant and mnn1 och1 respectively by electroporation. Western blot was applied to analyse the HuIFN-βexpression. However, results showed the HuIFN-βremained intracellular, and the alpha-factor secretion signal had not led the HuIFN-βto secrete into fermentation liquid. Probably, the expression of the HuIFN-βwas very toxic for S. cerevisiae, which affected the HuIFN-βexport to Golgi modification and secretion pathway. Therefore, the HuIFN-βaccumulated intracellularly as a fusion protein with alpha-factor signal peptide.