Deficiency of Dol-P-Man Synthase Subunit DPM3 Bridges the Congenital Disorders of Glycosylation with the Dystroglycanopathies

Alpha-dystroglycanopathies such as Walker Warburg syndrome represent an important subgroup of the muscular dystrophies that have been related to defective O-mannosylation of alpha-dystroglycan. In many patients, the underlying genetic etiology remains unsolved. Isolated muscular dystrophy has not been described in the congenital disorders of glycosylation (CDG) caused by N-linked protein glycosylation defects. Here, we present a genetic N-glycosylation disorder with muscular dystrophy in the group of CDG type I. Extensive biochemical investigations revealed a strongly reduced dolichol-phosphate-mannose (Dol-P-Man) synthase activity. Sequencing of the three DPM subunits and complementation of DPM3-deficient CHO2.38 cells showed a pathogenic p.L85S missense mutation in the strongly conserved coiled-coil domain of DPM3 that tethers catalytic DPM1 to the ER membrane. Cotransfection experiments in CHO cells showed a reduced binding capacity of DPM3(L85S) for DPM1. Investigation of the four Dol-P-Man-dependent glycosylation pathways in the ER revealed strongly reduced O-mannosylation of alpha-dystroglycan in a muscle biopsy, thereby explaining the clinical phenotype of muscular dystrophy. This mild Dol-P-Man biosynthesis defect due to DPM3 mutations is a cause for alpha-dystroglycanopathy, thereby bridging the congenital disorders of glycosylation with the dystroglycanopathies.     

Enzymes that recognize dolichols participate in three glycosylation pathways and are required for protein secretion.

We have explored the structure, function, and membrane topography of enzymes that recognize dolichols and participate in glycosylation pathways in the endoplasmic reticulum. Enzymes that interact with dolichols, including dolichyl phosphate mannose (Dol-P-Man) synthase and UDP-GlcNAc:Dol-P-transferase, revealed a conserved amino acid sequence in membrane-spanning regions. The consensus is Phe-Ile/Val-Xaa-Phe/Try-Xaa-Xaa-Ile-Pro-Phe-Xaa-Phe/Tyr, and we propose it is involved in dolichol recognition. We have used yeast mutants to demonstrate the role of dolichols in three glycosylation pathways. At its nonpermissive temperature, a Dol-P-Man synthase mutant (dpm1) was blocked in N-glycosylation, O-mannosylation, and glycosyl phosphoinositol membrane anchoring of protein, most likely because Dol-P-Man serves as mannosyl donor in all three pathways. The secretion mutant sec59 has a similar phenotype to dpm1, and the presence of a dolichol recognition sequence in the SEC59 protein gave a clue to its defect, which is in dolichol kinase. Comparison of yeast glycosylation mutant suggests that the ability to carry out N-glycosylation alone is sufficient to allow yeast to secrete glycoproteins and that an N-linked saccharide of a minimum size must be attached to proteins for cells to be able to secrete them and maintain a functional secretory pathway.     

Congenital disorders of glycosylation type Ig is defined by a deficiency in dolichyl-P-mannose:Man7GlcNAc2-PP-dolichyl mannosyltransferase

Type I congenital disorders of glycosylation (CDG I) are diseases presenting multisystemic lesions including central and peripheral nervous system deficits. The disease is characterized by under-glycosylated serum glycoproteins and is caused by mutations in genes encoding proteins involved in the stepwise assembly of dolichol-oligosaccharide used for protein N-glycosylation. We report that fibroblasts from a type I CDG patient, born of consanguineous parents, are deficient in their capacity to add the eighth mannose residue onto the lipid-linked oligosaccharide precursor. We have characterized cDNA corresponding to the human ortholog of the yeast gene ALG12 that encodes the dolichyl-P-Man:Man(7)GlcNAc(2)-PP-dolichyl alpha6-mannosyltransferase that is thought to accomplish this reaction, and we show that the patient is homozygous for a point mutation (T571G) that causes an amino acid substitution (F142V) in a conserved region of the protein. As the pathological phenotype of the fibroblasts of the patient was largely normalized upon transduction with the wild type gene, we demonstrate that the F142V substitution is the underlying cause of this new CDG, which we suggest be called CDG Ig. Finally, we show that the fibroblasts of the patient are capable of the direct transfer of Man(7)GlcNAc(2) from dolichol onto protein and that this N-linked structure can be glucosylated by UDP-glucose:glycoprotein glucosyltransferase in the endoplasmic reticulum.     

HPLC and mass spectrometry analysis of dolichol-phosphates at the cell culture scale

Dolichols (Dol) are polyprenol lipids that are essential structural components of eukaryotic membranes. In addition, the phosphorylated derivatives of Dol function as lipid anchors of mono- and oligosaccharide precursors involved in protein glycosylation. The biological importance of Dol phosphates (Dol-P) is illustrated by the severe outcome of human disorders linked to Dol biosynthetic defects, such as Dol-kinase deficiency. For characterization of inherited human diseases and evaluation of therapeutic trials, cultured cells often serve as a sole possible source for experimentation. Limited amounts of cell culture material render the quantitative analysis of Dol a challenging task. Here, we present HPLC- and mass spectrometry-based approaches to analyze and quantitate Dol-P from cultured human cells. The composition of naturally occurring Dol-P and the saturation state of the α-isoprene units was identified by negative-ion electrospray ionization mass spectrometry. Furthermore, fluorescently labeled Dol-P were separated by HPLC and quantified by comparison to known amounts of the internal standard polyprenol-P. The effect of pravastatin, a 3-hydroxy-3-methyl-glutaryl coenzyme-A reductase inhibitor, on the formation of Dol-P in HeLa cells was investigated. As expected, this treatment led to a decrease of Dol-P down to 35% of normal levels.     

Mannose supplementation corrects GDP-mannose deficiency in cultured fibroblasts from some patients with Congenital Disorders of Glycosylation (CDG)

Congenital Disorders of Glycosylation (CDG) are human deficiencies in glycoprotein biosynthesis. Previous studies showed that 1 mM mannose corrects defective protein N-glycosylation in cultured fibroblasts from some CDG patients. We hypothesized that these CDG cells have limited GDP-mannose (GDP-Man) and that exogenous mannose increases the GDP-Man levels. Using a well established method to measure GDP-Man, we found that normal fibroblasts had an average of 23.5 pmol GDP-Man/10(6) cells, whereas phosphomannomutase (PMM)-deficient fibroblasts had only 2.3-2.7 pmol/10(6) cells. Adding 1 mM mannose to the culture medium increased the GDP-Man level in PMM-deficient cells to approximately 15.5 pmol/10(6) cells, but had no significant effect on GDP-Man levels in normal fibroblasts. Similarly, mannose supplementation increased GDP-Man from 4.6 pmol/10(6) cells to 24.6 pmol/10(6) cells in phosphomannose isomerase (PMI)-deficient fibroblasts. Based on the specific activity of the GDP-[(3)H]Man pool present in [2-(3)H]mannose labeled cells, mannose supplementation also partially corrected the impaired synthesis of mannosylphosphoryldolichol (Man-P-Dol) and Glc(0)(-)(3)Man(9)GlcNAc(2)-P-P-Dol. These results confirm directly that deficiencies in PMM and PMI result in lowered cellular GDP-Man levels that are corrected by the addition of mannose. In contrast to these results, GDP-Man levels in fibroblasts from a CDG-Ie patient, who is deficient in Man-P-Dol synthase, were normal and unaffected by mannose supplementation even though mannose addition was found to correct abnormal lipid intermediate synthesis in another study (Kim et al. [2000] J. Clin. Invest., 105, 191-198). The mechanism by which mannose supplementation corrects abnormal protein N-glycosylation in Man-P-Dol synthase deficient cells is unknown, but this observation suggests that the regulation of Man-P-Dol synthesis and utilization may be more complex than is currently understood.     

N-glycosylation site occupancy in serum glycoproteins using multiple reaction monitoring liquid chromatography-mass spectrometry

Congenital disorders of glycosylation (CDGs) are a family of N-linked glycosylation defects associated with severe clinical manifestations. In CDG type-I, deficiency of lipid-linked oligosaccharide assembly leads to the underoccupancy of N-glycosylation sites on glycoproteins. Although the level of residual glycosylation activity is known to correlate with the clinical phenotype linked to individual CDG mutations, it is not known whether the degree of N-glycosylation site occupancy by itself correlates with the severity of the disease. To quantify the extent of underglycosylation in healthy control and in CDG samples, we developed a quantitative method of N-glycosylation site occupancy based on multiple reaction monitoring LC-MS/MS. Using isotopically labeled standard peptides, we directly quantified the level of N-glycosylation site occupancy on selected serum proteins. In healthy control samples, we determined 98-100% occupancy for all N-glycosylation sites of transferrin and alpha(1)-antitrypsin. In CDG type-I samples, we observed a reduction in N-glycosylation site occupancy that correlated with the severity of the disease. In addition, we noticed a selective underglycosylation of N-glycosylation sites, indicating preferential glycosylation of acceptor sequons of a given glycoprotein. In transferrin, a preferred occupancy for the first N-glycosylation site was observed, and a decreasing preference for the first, third, and second N-glycosylation sites was observed in alpha(1)-antitrypsin. This multiple reaction monitoring LC-MS/MS method can be extended to multiple glycoproteins, thereby enabling a glycoproteomics survey of N-glycosylation site occupancies in biological samples.     

The Saccharomyces cerevisiae DPM1 gene encoding dolichol-phosphate-mannose synthase is able to complement a glycosylation-defective mammalian cell line.

The Saccharomyces cerevisiae DPM1 gene product, dolichol-phosphate-mannose (Dol-P-Man) synthase, is involved in the coupled processes of synthesis and membrane translocation of Dol-P-Man. Dol-P-Man is the lipid-linked sugar donor of the last four mannose residues that are added to the core oligosaccharide transferred to protein during N-linked glycosylation in the endoplasmic reticulum. We present evidence that the S. cerevisiae gene DPM1, when stably transfected into a mutant Chinese hamster ovary cell line, B4-2-1, is able to correct the glycosylation defect of the cells. Evidence for complementation includes (i) fluorescence-activated cell sorter analysis of differential lectin binding to cell surface glycoproteins, (ii) restoration of Dol-P-Man synthase enzymatic activity in crude cell lysates, (iii) isolation and high-performance liquid chromatography fractionation of the lipid-linked oligosaccharides synthesized in the transfected and control cell lines, and (iv) the restoration of endoglycosidase H sensitivity to the oligosaccharides transferred to a specific glycoprotein synthesized in the DPM1 CHO transfectants. Indirect immunofluorescence with a primary antibody directed against the DPM1 protein shows a reticular staining pattern of protein localization in transfected hamster and monkey cell lines.     

Laboratory diagnosis of congenital disorders of glycosylation type I by analysis of transferrin glycoforms

Congenital disorders of glycosylation (CDG) are being recognized as a rapidly growing and complex group of disorders. The pathophysiology results from depressed synthesis or remodeling of oligosaccharide moieties of glycoproteins. The ultimate result is the formation of abnormal glycoproteins affecting their structure and metabolic functions. The most thoroughly studied subset of CDG are the type I defects affecting N-glycosylation. Causal mutations occur in at least 12 different genes which encode primarily monosaccharide transferases necessary for N-glycosylation in the endoplasmic reticulum. The broad clinical presentation of these glycosylation defects challenge clinicians to test for these defects in a variety of clinical settings. The first described CDG was a phosphomannomutase deficiency (CDG-Ia). The original method used to define the glycosylation defect was isoelectric focusing (IEF) of transferrin. More recently, the use of other charge separation methods and electrospray-mass spectrometry (ESI-MS) has proven valuable in detecting type I CDG defects. By mass resolution, the under-glycosylation of transferrin is characterized as the total absence of one or both N-linked oligosaccharide. Beyond providing a new understanding of the structure of transferrin in type I CDG patients, it is adaptable to high throughput serum analysis. The use of transferrin under-glycosylation to detect the type I CDG provides limited insight into the specific site of the defect in oligosaccharide assembly since its value is constrained to observation of the final product of glycoprotein synthesis. New analytical targets and tools are converging with the clinical need for diagnosis of CDG. Defining the biosynthetic sites responsible for specific CDG phenotypes is in progress, and ten more type I defects have been putatively identified. This review discusses current methods, such as IEF and targeted proteomics using mass spectrometry, that are used routinely to test for type I CDG disorders, along with some newer approaches to define the defective synthetic sites responsible for the type I CDG defects. All diagnostic endeavors are followed by the quest for a reliable treatment. The isolated success of CDG-Ib treatment will be described with the hope that this may expand to other type I CDG disorders.     

Identification of intercellular cell adhesion molecule 1 (ICAM-1) as a hypoglycosylation marker in congenital disorders of glycosylation cells

Many human inherited disorders cause protein N-glycosylation defects, but there are few cellular markers to test gene complementation for such defects. Plasma membrane glycoproteins are potential biomarkers because they may be reduced or even absent in plasma membranes of glycosylation-deficient cells. We combined stable isotope labeling by amino acids in cell culture (SILAC) with linear ion trap mass spectrometry (LTQ Orbitrap(TM)) to identify and quantify membrane proteins from wild-type CHO and glycosylation-deficient CHO (Lec9) cells. We identified 165 underrepresented proteins from 1447 unique quantified proteins, including 18 N-glycosylated plasma membrane proteins. Using various methods, we found that intercellular cell adhesion molecule 1 (ICAM-1) was reduced in Lec9 cells and in fibroblasts from 31 congenital disorder of glycosylation (CDG) patients compared with normal controls. Mannose supplementation of phosphomannose isomerase-deficient CDG-Ib (MPI-CDG) cells and complementation with PMM2 in PMM2-deficient CDG-Ia (PMM2-CDG) cells partially corrected hypoglycosylation based on increased ICAM-1 presence on the plasma membrane. These data indicate that ICAM-1 could be a useful hypoglycosylation biomarker to assess gene complementation of CDG-I patient cells and to monitor improved glycosylation in response to therapeutic drugs.     

How Golgi glycosylation meets and needs trafficking: the case of the COG complex

Protein glycosylation is one of the major biosynthetic functions occurring in the endoplasmic reticulum and Golgi compartments. It requires an amazing number of enzymes, chaperones, lectins and transporters whose actions delicately secure the fidelity of glycan structures. Over the past 30 years, glycobiologists hammered that glycan structures are not mere decorative elements but serve crucial cellular functions. This becomes dramatically illustrated by a group of mostly severe, inherited human disorders named congenital disorders of glycosylation (CDG). To date, many types of CDG have been defined genetically and most of the time the defects impair the biosynthesis, transfer and remodeling of N-glycans. Recently, the identification of the several types of CDG caused by deficiencies in the conserved oligomeric Golgi (COG) complex, a complex involved in vesicular Golgi trafficking, expanded the field of CDG but also brought novel insights in glycosylation. The molecular mechanisms underlying the complex pathway of N-glycosylation in the Golgi are far from understood. The availability of COG-deficient CDG patients and patients' cells offered a new way to study how COG, and its different subunits, could influence the Golgi N-glycosylation machinery and localization. This review summarizes the recent findings on the implication of COG in Golgi glycosylation. It highlights the need for a dynamic, finely tuned balance between anterograde and retrograde trafficking for the correct localization of Golgi enzymes to assure the stepwise maturation of N-glycan chains.     

Functional analysis of T-cell mutants defective in the biosynthesis of glycosylphosphatidylinositol anchor. Relative importance of glycosylphosphatidylinositol anchor versus N-linked glycosylation in T-cell activation.

The glycosylphosphatidylinositol (GPI) anchor, potentially capable of generating a number of second messengers, such as diacylglycerol, phosphatidic acid, and inositol phosphate glycan, has been postulated to be involved in signal transduction in various cell types, including T-cells. We have identified a panel of T-cell hybridoma mutants that are defective at various steps of GPI anchor biosynthesis. Since they were derived from a functional T-T hybridoma, we were able to determine the precise role of the GPI anchor in T-cell activation. Two mutants were chosen for this analysis. The first mutant is defective at the first step of GPI anchor biosynthesis, i.e. in the transfer of N-acetylglucosamine to a phosphatidylinositol acceptor. Thus, it cannot form any GPI precursors or GPI-like compounds. Interestingly, this mutant can be activated by antigen, superantigen, and concanavalin A in a manner comparable to the wild-type hybridoma. These data strongly suggest that the GPI anchor, its precursor, or its potential cleavage product, inositol phosphate glycan, is not required for the early phase of T-cell activation. The second mutant is able to synthesize the first two GPI precursors, but is not able to add mannose residues to them due to a deficiency in dolichol-phosphate-mannose (Dol-P-Man) biosynthesis. Unexpectedly, all of the Dol-P-Man mutants are defective in activation by antigen, suprantigen, and concanavalin A despite normal T-cell receptor expression. Here, we show that the activation defect was due to a pleiotropic glycosylation abnormality because Dol-P-Man is required for both GPI anchor and N-linked oligosaccharide biosynthesis. When the yeast Dol-P-Man synthase gene was stably transfected into the mutants, full expression of surface GPI-anchored proteins was restored. However, N-linked glycosylation was either partially or completely corrected in different transfectants. Reconstitution of activation defects correlates well with the status of N-linked glycosylation, but not with the expression of GPI-anchored proteins. These results thus reveal an unexpected role of N-linked glycosylation in T-cell activation.     

Asparagine 81, an invariant glycosylation site near apyrase conserved region 1, is essential for full enzymatic activity of ecto-nucleoside triphosphate diphosphohydrolase 3

N-linked glycosylation is important for the function, cellular localization, and oligomerization of membrane-bound ecto-nucleoside triphosphate diphosphohydrolases (eNTPDases). NTPDase3 is a prototypical cell membrane-associated eNTPDase, which is equally related and enzymatically intermediate to the other two cell surface membrane NTPDases (NTPDase1 and 2). The protein sequence of NTPDase3 contains seven putative N-glycosylation sites located in the ecto-domain. Only one of these putative glycosylation sites, asparagine 81 in NTPDase3, which is located near apyrase conserved region 1 (ACR1), is invariant in all the cell surface membrane eNTPDases. Using site-directed mutagenesis, mutants were constructed to eliminate this highly conserved N-glycosylation site in NTPDase3. The results indicate that glycosylation at this position is essential for full enzymatic activity, with mutant ATPase activity decreased more than ADPase activity. Enzymatic deglycosylation of this site is shown to be responsible for the inactivation of the wild-type enzyme by treatment with peptide N-glycosidase-F. In addition, glycosylation of this conserved site is necessary for the stabilization/stimulation of nucleotidase activity upon treatment with the lectin concanavalin A. However, lack of glycosylation at this site did not result in large changes in tertiary or quaternary structure, as measured by Cibacron blue binding, chemical cross-linking, and native gel electrophoretic analysis. Since this N-glycosylation site is invariant in cell membrane eNTPDases, it is postulated that glycosylation of this residue near ACR1 is crucial for full enzymatic activity of the cell membrane NTPDases.     

DDOST mutations identified by whole-exome sequencing are implicated in congenital disorders of glycosylation

Congenital disorders of glycosylation (CDG) are inherited autosomal-recessive diseases that impair N-glycosylation. Approximately 20% of patients do not survive beyond the age of 5 years old as a result of widespread organ dysfunction. Although most patients receive a CDG diagnosis based on abnormal glycosylation of transferrin, this test cannot provide a genetic diagnosis; indeed, many patients with abnormal transferrin do not have mutations in any known CDG genes. Here, we combined biochemical analysis with whole-exome sequencing (WES) to identify the genetic defect in an untyped CDG patient, and we found a 22 bp deletion and a missense mutation in DDOST, whose product is a component of the oligosaccharyltransferase complex that transfers the glycan chain from a lipid carrier to nascent proteins in the endoplasmic reticulum lumen. Biochemical analysis with three biomarkers revealed that N-glycosylation was decreased in the patient's fibroblasts. Complementation with wild-type-DDOST cDNA in patient fibroblasts restored glycosylation, indicating that the mutations were pathological. Our results highlight the power of combining WES and biochemical studies, including a glyco-complementation system, for identifying and confirming the defective gene in an untyped CDG patient. This approach will be very useful for uncovering other types of CDG as well.     

Defect in N-glycosylation of proteins is tissue-dependent in congenital disorders of glycosylation Ia

The biochemical hallmark of Congenital Disorders of Glycosylation (CDG) including type Ia is a defective N-glycosylation of serum glycoproteins. Hypoglycosylated forms of alpha1-antitrypsin have been detected by Western blot in serum from CDG Ia patients. In contrast we were not able to detect hypoglycosylation in alpha1-antitrypsin synthesized by fibroblasts, keratinocytes, enterocytes, and leukocytes. Similarly no hypoglycosylation was detectable in a membrane-associated N-linked glycoprotein, the facilitative glucose transporter GLUT-1 and also in serum immunoglobulin G isolated from sera of CDG Ia patients. We conclude that the phenotypic expression of CDG Ia is tissue-dependent.     

Dolichol phosphate mannose synthase is required in vivo for glycosyl phosphatidylinositol membrane anchoring, O mannosylation, and N glycosylation of protein in Saccharomyces cerevisiae.

Glycosyl phosphatidylinositol (GPI) anchoring, N glycosylation, and O mannosylation of protein occur in the rough endoplasmic reticulum and involve transfer of precursor structures that contain mannose. Direct genetic evidence is presented that dolichol phosphate mannose (Dol-P-Man) synthase, which transfers mannose from GDPMan to the polyisoprenoid dolichol phosphate, is required in vivo for all three biosynthetic pathways leading to these covalent modifications of protein in yeast cells. Temperature-sensitive yeast mutants were isolated after in vitro mutagenesis of the yeast DPM1 gene. At the nonpermissive temperature of 37 degrees C, the dpm1 mutants were blocked in [2-3H]myo-inositol incorporation into protein and accumulated a lipid that could be radiolabeled with both [2-3H]myo-inositol and [2-3H]glucosamine and met existing criteria for an intermediate in GPI anchor biosynthesis. The likeliest explanation for these results is that Dol-P-Man donates the mannose residues needed for completion of the GPI anchor precursor lipid before it can be transferred to protein. Dol-P-Man synthase is also required in vivo for N glycosylation of protein, because (i) dpm1 cells were unable to make the full-length precursor Dol-PP-GlcNAc2Man9Glc3 and instead accumulated the intermediate Dol-PP-GlcNAc2Man5 in their pool of lipid-linked precursor oligosaccharides and (ii) truncated, endoglycosidase H-resistant oligosaccharides were transferred to the N-glycosylated protein invertase after a shift to 37 degrees C. Dol-P-Man synthase is also required in vivo for O mannosylation of protein, because chitinase, normally a 150-kDa O-mannosylated protein, showed a molecular size of 60 kDa, the size predicted for the unglycosylated protein, after shift of the dpm1 mutant to the nonpermissive temperature.     

Inhibition of squalene synthase upregulates PCSK9 expression in rat liver

Proprotein convertase subtilisin/kexin type 9 (PCSK9) plays a critical role in cholesterol metabolism by enhancing the degradation of the LDL receptor protein in the liver. It has previously been shown that administration of zaragozic acid A (ZA), a potent inhibitor of squalene synthase, also significantly increases the rate of degradation of hepatic LDL receptor protein. Thus, we decided to determine whether ZA administration might act to up regulate hepatic expression of the rat PCSK9 gene. Administration of ZA resulted in increased PCSK9 mRNA and protein levels in rat liver surprisingly in concert with an increase in hepatic LDL receptor mRNA levels, LDL receptor protein turnover, and decreased serum cholesterol levels. These observations suggest an involvement of PCSK9 in hepatic LDL receptor protein degradation and perhaps, in increasing the rate of LDL receptor cycling resulting in lower serum cholesterol levels in response to cholesterol biosynthesis inhibitors.     

Cloning and sequencing of the yeast gene for dolichol phosphate mannose synthase, an essential protein

Dolichol phosphate mannose (Dol-P-Man) synthase (EC 2.4.1.83) catalyzes the formation of Dol-P-Man from Dol-P and GDP-Man. The structural gene for yeast Dol-P-Man synthase (DPM1) was isolated by screening a yeast genomic DNA library for colonies that overexpressed Dol-P-Man synthase activity. This approach relied on a method to screen for Dol-P-Man synthase activity in lysed yeast colonies and used a yeast mutant with very low Dol-P-Man synthase activity in colony lysates. Transformants isolated using this technique expressed Dol-P-Man synthase activity 9-14-fold higher than that of a wild type strain, and all seven plasmids conferring this overproduction had a common region in their yeast genomic DNA insert. DPM1 is the structural gene for yeast Dol-P-Man synthase since Escherichia coli transformants harboring this gene express Dol-P-Man synthase activity in vitro. DNA sequencing of the DPM1 gene revealed an open reading frame of 801 bases. The 30-kDa size of the predicted protein is in excellent agreement with the size of the purified yeast enzyme (Haselbeck, A., and Tanner, W. (1982) Proc. Natl. Acad. Sci. U. S. A. 79, 1520-1524). Analysis of the predicted amino acid sequence reveals the protein has a potential membrane spanning domain of 25 amino acids at its COOH terminus. The protein's NH2 terminus, though not hydrophobic, meets existing criteria for yeast signal sequences, but there is no site for cleavage by signal peptidase. If the NH2 terminus is a functional signal sequence, the protein is predicted to be oriented toward the lumen of the endoplasmic reticulum with both NH2 and COOH termini serving as membrane anchors. If there is no signal sequence, the enzyme is predicted to face the cytoplasm and be anchored only by its COOH terminus. The DPM1 gene is essential for viability in yeast since disruption of the gene is lethal. We suspect Dol-P-Man synthase is not an essential protein due to its role in N-glycosylation since mutations in other genes that affect the late steps in lipid-linked oligosaccharide synthesis do not affect cell growth. Instead, DPM1 may be an essential gene because its product is required for O-glycosylation in yeast or because Dol-P-Man synthase is needed in some unidentified pathway.     

Overexpression of GDP-mannose pyrophosphorylase in Saccharomyces cerevisiae corrects defects in dolichol-linked saccharide formation and protein glycosylation

Thermosensitive mutants of Saccharomyces cerevisiae, affected in the endoplasmic reticulum (ER) located glycosylation, i.e. in Dol-P-Man synthase (dpm1), in beta-1,4 mannosyl transferase (alg1) and in alpha-1,3 mannosyltransferase (alg2), were used to assess the role of GDP-Man availability for the synthesis of dolichol-linked saccharides. The mutants were transformed with the yeast gene MPG1 (PSA1/VIG9) encoding GDP-Man pyrophosphorylase catalyzing the final step of GDP-Man formation. We found that overexpression of MPG1 allows growth at non-permissive temperature and leads to an increase in the cellular content of GDP-Man. In the alg1 and alg2 mutants, complemented with MPG1 gene, N-glycosylation of invertase was in part restored, to a degree comparable to that of the wild-type control. In the dpm1 mutant, the glycosylation reactions that depend on the formation of Dol-P-Man, i.e. elongation of Man(5)GlcNAc(2)-PP-Dol, O-mannosylation of chitinase and synthesis of GPI anchor were normal when MPG1 was overexpressed.Our data indicate that an increased level of GDP-Man is able to correct defects in mannosylation reactions ascribed to the ER and to the Golgi.     

Proteome-wide analysis of single-nucleotide variations in the N-glycosylation sequon of human genes

N-linked glycosylation is one of the most frequent post-translational modifications of proteins with a profound impact on their biological function. Besides other functions, N-linked glycosylation assists in protein folding, determines protein orientation at the cell surface, or protects proteins from proteases. The N-linked glycans attach to asparagines in the sequence context Asn-X-Ser/Thr, where X is any amino acid except proline. Any variation (e.g. non-synonymous single nucleotide polymorphism or mutation) that abolishes the N-glycosylation sequence motif will lead to the loss of a glycosylation site. On the other hand, variations causing a substitution that creates a new N-glycosylation sequence motif can result in the gain of glycosylation. Although the general importance of glycosylation is well known and acknowledged, the effect of variation on the actual glycoproteome of an organism is still mostly unknown. In this study, we focus on a comprehensive analysis of non-synonymous single nucleotide variations (nsSNV) that lead to either loss or gain of the N-glycosylation motif. We find that 1091 proteins have modified N-glycosylation sequons due to nsSNVs in the genome. Based on analysis of proteins that have a solved 3D structure at the site of variation, we find that 48% of the variations that lead to changes in glycosylation sites occur at the loop and bend regions of the proteins. Pathway and function enrichment analysis show that a significant number of proteins that gained or lost the glycosylation motif are involved in kinase activity, immune response, and blood coagulation. A structure-function analysis of a blood coagulation protein, antithrombin III and a protease, cathepsin D, showcases how a comprehensive study followed by structural analysis can help better understand the functional impact of the nsSNVs.