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Congenital disorders of glycosylation (CDG) diseases are characterized by defects of N-linked glycosylation. In CDG-Ic, several genetic defects cause a shortage of dolichol-linked oligosaccharides, which leads to underglycosylation of nascent glycoproteins (Freeze et al., 2006). N-linked glycosylation is critical for proper folding and trafficking of glycoproteins. Inhibition of glycosylation leads to buildup of misfolded proteins in the endoplasmic reticulum, which induces a protective reaction known as the unfolded protein response (UPR) (Chapman et al., 1998). This dissertation determined for the first time the scope and extent of the UPR in cells with CDG-Ic disorder. Results reported indicate that endoplasmic stress in CDG-Ic cells was similar, though more moderate, to the response induced by tunicamycin-mediated inhibition of N-glycosylation. The moderate UPR detected in CDG-Ic cells indicates that these cells do undergo ER-stress probably by accumulation of some unfolded proteins, but does not lead to the collapse of the secretory pathway.
10.2 Summary of experiments and their findings
In the present study, we show that the altered glucosylation of the oligosaccharide core caused by a mutation in the ALG6 a1,3-glucosyltransferase gene leads to hypoglycosylation of proteins that is only partial filling of the available glycosylation sites of the model secretory protein employed in this study. The CDG caused by ALG6 mutation as type-Ic represents a defect in glycosylation localized in the endoplasmic reticulum, proximally from the transfer of the oligomannose core to nascent proteins. The effect of the A333V substitution on the properties of the Alg6p is yet unclear. The replacement of an alanine by a more bulky amino acid like valine has been shown in various cases to be linked to profound alterations of protein functions. The proximity of the A333V mutation to a large domain of the Alg6p that is conserved between yeast and man suggests a possible alteration of the catalytic properties of Alg6p.
ALG6 deficient S. cerevisiae strains inadequately glycosylate their glycoproteins. The vacuolar carboxypeptidase (CPY) has four N-linked chains, but the alg6 knockout yeast strain lacks one or two of these chains. Full glycosylation can be restored by transformation of the knockout strain with the yeast or human ALG6 cDNA. The human ALG6 gene restores CPY glycosylation to an extent when transformed into the alg6 knockout strain. In contrast, the cDNA encoding the A333V Alg6 protein leads to misglycosylation because CPY glycoforms lacking both 1 and 2 oligosaccharide chains are seen. Endoglycosidase H and Peptide: N-Glycosidase F digests CPY glycoprotein to release all of the sugar chains, produces only one band indicating that the size differences in immunoprecipitated CPY are based on the number of oligosaccharide chains. These results suggest that CDG-Ic patients have a more substantial loss of ALG6 activity which could explain the severe clinical presentation.
The deficiencies in the patient could occur because most secretory and membrane proteins require glycosylation for correct folding and thereby for the further transport through the secretory pathway. In mammalian cells, calnexin and calreticulin seem to be important for quality control. They are lectins that bind specifically with partially trimmed, monoglucosylated N-linked oligosaccharides. Thus, the absence of terminal glucose on the N-linked chains can modify their association with the quality control apparatus, which can delay their movement through the secretory pathway or lead to their degradation in the endoplasmic reticulum. Furthermore, the OST complex, in charge for the transfer of the sugar chains to the polypeptide, has a decreased affinity for the incomplete substrate. Thus, glucosylation is crucial for efficient transfer of the LLO to the protein and thereafter for proper quality control. However, a mutant form of the yeast vacuolar protease, CPY, lacking all glycosylation sites is still transported to the vacuole, but at a slower rate than fully glycosylated CPY. Sensitizing CDG-Ic yeast strains with small quantity of tunicamycin presented altered CPY misglycosylation pattern lacking 1 to 4 oligosaccharide chains. This could be a result of chemicalââ‚¬"genetic epistatic interactions, in which CDG-Ic disorder was hypersensitive to a normally sub lethal concentration of the small molecule inhibitor tunicamycin. It is possible that enteric protein loss seen in some individuals results from environmental stresses that exceed their glycosylation capacity.
One critical protein property in the cell was its subcellular localization, which provides critical information about how a protein works inside a cell. This property was frequently determined by visual interpretation of fluorescence microscope images. A major advance therefore came from the creation of the yeast GFP fusion localization database at the University of California, San Francisco, USA (UCSF) in which around 4500 yeast proteins are available for visualisation owing to the GFP-gene fused to individual genes. Deletion or mutations in the ALG6 genes involved in the biosynthesis of glycosylation were expected to show a paucity of mature glycoproteins. Yeast alg6 mutants accumulate lipid-linked Man9GlcNAc2, suggesting that this locus encodes an endoplasmic glucosyltransferase. We generated a complete set of Wildtype GFP, Î"alg6 GFP and CDG-Ic GFP collection via modified mass mating technique. Individual protein localization in alg6 variants were compared to wildtype yeast cells under high throughput confocal microscopy and automated images were acquired. Our observation revealed that the majority of the proteins including glycoproteins passed through the protein secretory pathway without accumulating in the endoplasmic reticulum. Few glycoproteins were observed to mislocalise as a result of the CDG-Ic disorder. Interestingly PDR5 glycoprotein which is a plasma membrane ATP-binding cassette transporter was observed in internal punctuate structures in yeast with CDG-Ic disorder, providing a useful morphological phenotype of the lesion the disease mutation causes. In the wildtype yeast cells, PDR5 when fused to GFP shows protein localization in the cell periphery and deletion of ALG6 gene or C998T point mutation on the human ALG6 gene in yeast leads to accumulation of small percentage of glycoproteins in the protein secretory pathway.
Within the lumen of the endoplasmic reticulum (ER), a variety of resident ER proteins assist newly translocated nascent polypeptides to fold into their correct tertiary and quaternary structures. These resident proteins include molecular chaperones that recognize and stabilize partially folded intermediates during polypeptide folding and assembly, as well as enzymes that catalyze rate-determining steps in folding, such as protein disulfide isomerase and peptidyl prolyl isomerases. Under normal growth conditions these chaperones and folding catalysts are synthesized constitutively and abundantly. However, their rates of synthesis can be increased significantly by the accumulation of mutant proteins in the endoplasmic Reticulum or by a variety of stress conditions whose common denominator is thought to be the accumulation in the ER of unfolded polypeptides. This unfolded protein response operates in yeast and higher eukaryotes to regulate the levels of ER chaperones and protein folding catalysts. The unfolded protein response acts to reduce level of misfolded proteins by enhancing proteins to fold to their native state, promoting transit to the distal secretory pathway and enhancing the rate of ERAD while simultaneously reducing the formation of misfolded proteins. Loss of ERAD function results in accumulation of unfolded proteins in the ER and activates the UPR. We tested for constructive activation of the UPR in yeast cells with either alg6 knockout or yeast cells with CDG-Ic disorder. Our observation revealed 3 fold induction of UPR in yeast alg6 knockout cells and more than 2 fold induction of UPR in yeast cells with CDG-Ic disorder. The misfolding of proteins is likely to reflect the inherent difficulty of folding secretory and membrane proteins, which unlike cytosolic proteins often require covalent modifications, such as disulfide bonds and glycosylation, as well as the precise ordering of transmembrane domains across and within the lipid bilayer. Conversely, when the UPR is available in a cell with diminished ERAD capacity, misfolded proteins can still be handled by mechanisms like refolding by chaperones in the endoplasmic reticulum.
In S cerevisiae, more than 80% of the around 6200 predicted genes are nonessential, implying that the genome is buffered from the phenotypic consequences of genetic perturbation. To evaluate function, a method for systematic construction of double mutants, termed synthetic genetic array (SGA) analysis was used, in which alg6 knockout strain or yeast with CDG-Ic disorder as a query strain was crossed to an array of about 4700 non-essential deletion mutants. To enable high-throughput synthetic lethal analysis, an ordered array of about 4700 viable yeast gene-deletion mutants was assembled and performed. A series of selection steps in which mating and meiotic recombinations were used to generate haploid double mutants. To evaluate the colony sizes of double mutants generated from a query screen, we compare them to a reference set of wildtype control screens. In addition to visual inspection of the double mutants, we have a computer-based scoring system, which generates an estimate of relative growth rates from the area of individual colonies, as measured from digital images of the double-mutant plates. Following normalization of the images derived from control and double mutant plates, statistical significance can be determined for each strain by comparing the measurements between the mutants and wild-type controls. The inviable double-mutant meiotic progeny potentially identify functional relationships between genes. When the results of the double mutants were analysed, a difference of only one negative genetic interaction was observed between alg6 knockout SGA and CDG-Ic SGA. Out of 26 negative interactions, 10 genes were involved in transferase activity when analysed with SGD gene ontology slim mapper. The genes involved in the transferase activity play crucial role in glycosylation process and interact with the ALG6 gene in the glycosylation pathway. Other negative interactions observed were related to protein binding, lipid binding, DNA binding and RNA binding process.
Pharmacological chaperones have been found to be effective in preventing misfolding of different disease-causing proteins, essentially reducing many protein-misfolding diseases (Bernier et al., 2004). In order to be functionally active, a protein has to acquire a unique 3D conformation via a complicated folding pathway. A small error in the folding process results in a misfolded structure, which can sometimes be lethal (Dobson, 1999, Forloni et al., 2002). Many proteins cannot fold properly by themselves and require the assistance of a special kind from the molecular chaperones present in the cell. One strategy was to prevent misfolding or correct a mutant proteinââ‚¬â„¢s lethal conformation was to influence the protein folding environment inside the cell. Pharmacological chaperones have proved very effective in rescuing a few receptor proteins from proteasomal degradation (Cohen and Kelly, 2003). Pharmacological compounds have shown to assist proteins to achieve a functionally active 3D structure and thus prevent the formation of a misfolded or aggregated structure, essentially enhancing folding efficiency by influencing the kinetics of the process and inhibiting events that lead to aggregation. We screened the CDG-Ic yeast mutant against 3000 pharmacologically active compounds with a wide range of biological activities and structural diversity using high-throughput library screens and identified ten compounds that rescued growth. One promising compound that rescued growth of the CDG-Ic yeast mutant was SMSC. From the CPY western blot assay SMSC emerged as the best pharmacological compound for improving the under-glycosylation phenotype of CPY glycoprotein in CDG-Ic yeast strain. Improvement in fully glycosylated CPY with incremental compound concentration suggested dose dependant rescue of CDG-Ic in yeast.
This thesis discovered a clear subcellular phenotype of the CDG-Ic mutation in yeast, namely partial relocation of the Pdr5p-GFP from cell periphery to significant amounts of punctate intracellular structures. In the presence of SMSC marked rescue in Pdr5p-GFP localization to the yeast cell periphery was observed (Figure 9.10). In another screen for UPR stress, a substantial decrease in the GFP/RFP intensity ratio was observed in CDG-Ic yeast strain with addition of 20 Ã¯ÂÂM/mL of SMSC revealing significant decrease in the UPR stress and indicated rescue effect caused by SMSC in the CDG-Ic yeast strain. In addition SMSC was observed to suppress the yeast mutant growth phenotype, causing cells that are exposed to the SMSC to grow normally.
10.3 Future Research Directions
10.3.1 Investigate the chemical kinetics of SMSC
Further studies are warranted on selenocysteine in S.cerevisiae and investigate its mode of action in rescuing CDG-Ic disorder, both from the point of view of its potential as a useful drug and as a probe for understanding cellular processes. Some immediate questions include, binding target of SMSC and its mode of action.
10.3.2 Localization refinement by co-localization
Yeast CDG-Ic mutant strains with specific subcellular localization labelled with GFP fluorescence should be mated with strains expressing reference proteins labelled with an alternate dye (e.g. RFP) and analysed by microscopy to identify co-localised GFP-RFP merged images. The GFP fluorescence and RFP-tagged reference proteins should allow resolution of what subcellular compartments within the yeast cell the punctate mutant phenotype relates to.
10.3.3 SGA Mapping
SGA mapping (SGAM) can be used to locate this SMSC suppressor to the locus in yeast, which contains a known mutation that appears to compromise ALG6 function. This laboratory had utilised the sophistication of yeast genetics to pursue the detailed targets of another small molecule inhibitor. The same strategies presumable could work in determining the molecular target of SMSC. Ordered arrays of marked yeast deletion strains provide an inherently powerful tool for high-resolution genetic mapping. When combined with SGA methodology, this mapping method can be automated and carried out in high throughput.
10.5 Research outcome
We have established phenotypes resulting from mis-glycosylation by generating assays for CDG-Ic. Yeast cells have been extremely helpful in identifying the biosynthetic machinery of protein glycosylation. The knowledge of the great majority of the mammalian genes involved in this pathway is built on information obtained in yeast. SMSC that caused reversion of the CDG-Ic phenotype to normal subcellular protein localization was seen to correct the glycosylation defects.
10.4 Concluding Remarks
SMSC apparently rescues the CDG-Ic mutation causing reversion of several mutation-related phenotypes to revert to normal wildtype and includes correction of the glycosylation defect. This is an important finding that should be followed up in cell cultures, mammalian systems and animal models. This compound could form the basis of future drug screens for human therapeutic intervention.