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1.  Life with too much polyprenol–polyprenol reductase deficiency 
Molecular genetics and metabolism  2011;105(4):642-651.
Congenital disorders of glycosylation (CDG) are caused by a dysfunction of glycosylation, an essential step in the manufacturing process of glycoproteins. This paper focuses on a 6-year-old patient with a new type of CDG-I caused by a defect of the steroid 5α reductase type 3 gene (SRD5A3). The clinical features were psychomotor retardation, pathological nystagmus, slight muscular hypotonia and microcephaly. SRD5A3 was recently identified encoding the polyprenol reductase, an enzyme catalyzing the final step of the biosynthesis of dolichol, which is required for the assembly of the glycans needed for N-glycosylation.
Although an early homozygous stop-codon (c.57G> A [W19X]) with no functional protein was found in the patient, about 70% of transferrin (Tf) was correctly glycosylated. Quantification of dolichol and unreduced polyprenol in the patient's fibroblasts demonstrated a high polyprenol/dolichol ratio with normal amounts of dolichol, indicating that high polyprenol levels might compete with dolichol for the initiation of N-glycan assembly but without supporting normal glycosylation and that there must be an alternative pathway for dolichol biosynthesis.
PMCID: PMC3428379  PMID: 22304929
Congenital disorders of glycosylation; SRD5A3-CDG; Polyprenol reductase; Dolichol; Psychomotor retardation; Consanguinity
2.  Next generation sequencing in a family with autosomal recessive Kahrizi syndrome (OMIM 612713) reveals a homozygous frameshift mutation in SRD5A3 
As part of a large-scale, systematic effort to unravel the molecular causes of autosomal recessive mental retardation, we have previously described a novel syndrome consisting of mental retardation, coloboma, cataract and kyphosis (Kahrizi syndrome, OMIM 612713) and mapped the underlying gene to a 10.4-Mb interval near the centromere on chromosome 4. By combining array-based exon enrichment and next generation sequencing, we have now identified a homozygous frameshift mutation (c.203dupC; p.Phe69LeufsX2) in the gene for steroid 5α-reductase type 3 (SRD5A3) as the disease-causing change in this interval. Recent evidence indicates that this enzyme is required for the conversion of polyprenol to dolichol, a step that is essential for N-linked protein glycosylation. Independently, another group has recently observed SRD5A3 mutations in several families with a type 1 congenital disorder of glycosylation (CDG type Ix, OMIM 212067), mental retardation, cerebellar ataxia and eye disorders. Our results show that Kahrizi syndrome and this CDG Ix subtype are allelic disorders, and they illustrate the potential of next-generation sequencing strategies for the elucidation of single gene defects.
PMCID: PMC3039499  PMID: 20700148
SRD5A3; next generation sequencing; congenital disorder of glycosylation; mental retardation; autosomal recessive; consanguinity
3.  From glycosylation disorders to dolichol biosynthesis defects: a new class of metabolic diseases 
Polyisoprenoid alcohols are membrane lipids that are present in every cell, conserved from archaea to higher eukaryotes. The most common form, alpha-saturated polyprenol or dolichol is present in all tissues and most organelle membranes of eukaryotic cells. Dolichol has a well defined role as a lipid carrier for the glycan precursor in the early stages of N-linked protein glycosylation, which is assembled in the endoplasmic reticulum of all eukaryotic cells. Other glycosylation processes including C- and O-mannosylation, GPI-anchor biosynthesis and O-glucosylation also depend on dolichol biosynthesis via the availability of dolichol-P-mannose and dolichol-P-glucose in the ER. The ubiquity of dolichol in cellular compartments that are not involved in glycosylation raises the possibility of additional functions independent of these protein post-translational modifications. The molecular basis of several steps involved in the synthesis and the recycling of dolichol and its derivatives is still unknown, which hampers further research into this direction. In this review, we summarize the current knowledge on structural and functional aspects of dolichol metabolites. We will describe the metabolic disorders with a defect in known steps of dolichol biosynthesis and recycling in human and discuss their pathogenic mechanisms. Exploration of the developmental, cellular and biochemical defects associated with these disorders will provide a better understanding of the functions of this lipid class in human.
PMCID: PMC3137772  PMID: 21384228
4.  A novel carbohydrate-deficient glycoprotein syndrome characterized by a deficiency in glucosylation of the dolichol-linked oligosaccharide. 
Journal of Clinical Investigation  1998;102(4):647-652.
Carbohydrate-deficient glycoprotein syndromes (CDGS) type I are a group of genetic diseases characterized by a deficiency of N-linked protein glycosylation in the endoplasmic reticulum. The majority of these CDGS patients have phosphomannomutase (PMM) deficiency (type A). This enzyme is required for the synthesis of GDP-mannose, one of the substrates in the biosynthesis of the dolichol-linked oligosaccharide Glc3Man9GlcNAc2. This oligosaccharide serves as the donor substrate in the N-linked glycosylation process. We report on the biochemical characterization of a novel CDGS type I in fibroblasts of four related patients with normal PMM activity but a strongly reduced ability to synthesize glucosylated dolichol-linked oligosaccharide leading to accumulation of dolichol-linked Man9GlcNAc2. This deficiency in the synthesis of dolichol-linked Glc3Man9GlcNAc2 oligosaccharide explains the hypoglycosylation of serum proteins in these patients, because nonglucosylated oligosaccharides are suboptimal substrates in the protein glycosylation process, catalyzed by the oligosaccharyltransferase complex. Accordingly, the efficiency of N-linked protein glycosylation was found to be reduced in fibroblasts from these patients.
PMCID: PMC508925  PMID: 9710431
5.  The Compartmentalisation of Phosphorylated Free Oligosaccharides in Cells from a CDG Ig Patient Reveals a Novel ER-to-Cytosol Translocation Process 
PLoS ONE  2010;5(7):e11675.
Biosynthesis of the dolichol linked oligosaccharide (DLO) required for protein N-glycosylation starts on the cytoplasmic face of the ER to give Man5GlcNAc2-PP-dolichol, which then flips into the ER for further glycosylation yielding mature DLO (Glc3Man9GlcNAc2-PP-dolichol). After transfer of Glc3Man9GlcNAc2 onto protein, dolichol-PP is recycled to dolichol-P and reused for DLO biosynthesis. Because de novo dolichol synthesis is slow, dolichol recycling is rate limiting for protein glycosylation. Immature DLO intermediates may also be recycled by pyrophosphatase-mediated cleavage to yield dolichol-P and phosphorylated oligosaccharides (fOSGN2-P). Here, we examine fOSGN2-P generation in cells from patients with type I Congenital Disorders of Glycosylation (CDG I) in which defects in the dolichol cycle cause accumulation of immature DLO intermediates and protein hypoglycosylation.
Methods and Principal Findings
In EBV-transformed lymphoblastoid cells from CDG I patients and normal subjects a correlation exists between the quantities of metabolically radiolabeled fOSGN2-P and truncated DLO intermediates only when these two classes of compounds possess 7 or less hexose residues. Larger fOSGN2-P were difficult to detect despite an abundance of more fully mannosylated and glucosylated DLO. When CDG Ig cells, which accumulate Man7GlcNAc2-PP-dolichol, are permeabilised so that vesicular transport and protein synthesis are abolished, the DLO pool required for Man7GlcNAc2-P generation could be depleted by adding exogenous glycosylation acceptor peptide. Under conditions where a glycotripeptide and neutral free oligosaccharides remain predominantly in the lumen of the ER, Man7GlcNAc2-P appears in the cytosol without detectable generation of ER luminal Man7GlcNAc2-P.
Conclusions and Significance
The DLO pools required for N-glycosylation and fOSGN2-P generation are functionally linked and this substantiates the hypothesis that pyrophosphatase-mediated cleavage of DLO intermediates yields recyclable dolichol-P. The kinetics of cytosolic fOSGN2-P generation from a luminally-generated DLO intermediate demonstrate the presence of a previously undetected ER-to-cytosol translocation process for either fOSGN2-P or DLO.
PMCID: PMC2907391  PMID: 20652024
6.  Glycoprotein Biosynthesis in a Eukaryote Lacking the Membrane Protein Rft1* 
The Journal of Biological Chemistry  2013;288(28):20616-20623.
Background: The membrane protein Rft1 was proposed to flip Man5GlcNAc2-PP-dolichol (M5-DLO) to the ER lumen for N-glycoprotein biosynthesis.
Results: Rft1-null Trypanosoma brucei has a normal steady-state level of mature dolichol-linked oligosaccharide (mDLO) and significant N-glycosylation.
Conclusion: Rft1 is not required for M5-DLO flipping in vivo but aids conversion of M5-DLO to mDLO by another mechanism.
Significance: The M5-DLO flippase remains to be identified.
Mature dolichol-linked oligosaccharides (mDLOs) needed for eukaryotic protein N-glycosylation are synthesized by a multistep pathway in which the biosynthetic lipid intermediate Man5GlcNAc2-PP-dolichol (M5-DLO) flips from the cytoplasmic to the luminal face of the endoplasmic reticulum. The endoplasmic reticulum membrane protein Rft1 is intimately involved in mDLO biosynthesis. Yeast genetic analyses implicated Rft1 as the M5-DLO flippase, but because biochemical tests challenged this assignment, the function of Rft1 remains obscure. To understand the role of Rft1, we sought to analyze mDLO biosynthesis in vivo in the complete absence of the protein. Rft1 is essential for yeast viability, and no Rft1-null organisms are currently available. Here, we exploited Trypanosoma brucei (Tb), an early diverging eukaryote whose Rft1 homologue functions in yeast. We report that TbRft1-null procyclic trypanosomes grow nearly normally. They have normal steady-state levels of mDLO and significant N-glycosylation, indicating robust M5-DLO flippase activity. Remarkably, the mutant cells have 30–100-fold greater steady-state levels of M5-DLO than wild-type cells. All N-glycans in the TbRft1-null cells originate from mDLO indicating that the M5-DLO excess is not available for glycosylation. These results suggest that rather than facilitating M5-DLO flipping, Rft1 facilitates conversion of M5-DLO to mDLO by another mechanism, possibly by acting as an M5-DLO chaperone.
PMCID: PMC3711325  PMID: 23720757
Endoplasmic Reticulum (ER); Glycobiology; Glycoprotein Biosynthesis; Lipid Synthesis; Membrane Proteins; Trypanosoma brucei; Flippase
7.  Dolichol phosphate mannose synthase (DPM1) mutations define congenital disorder of glycosylation Ie (CDG-Ie) 
Journal of Clinical Investigation  2000;105(2):191-198.
Congenital disorders of glycosylation (CDGs) are metabolic deficiencies in glycoprotein biosynthesis that usually cause severe mental and psychomotor retardation. Different forms of CDGs can be recognized by altered isoelectric focusing (IEF) patterns of serum transferrin (Tf). Two patients with these symptoms and similar abnormal Tf IEF patterns were analyzed by metabolic labeling of fibroblasts with [2-3H]mannose. The patients produced a truncated dolichol-linked precursor oligosaccharide with 5 mannose residues, instead of the normal precursor with 9 mannose residues. Addition of 250 μΜ mannose to the culture medium corrected the size of the truncated oligosaccharide. Microsomes from fibroblasts of these patients were approximately 95% deficient in dolichol-phosphate-mannose (Dol-P-Man) synthase activity, with an apparent Km for GDP-Man ∼6-fold higher than normal. DPM1, the gene coding for the catalytic subunit of Dol-P-Man synthase, was altered in both patients. One patient had a point mutation, C274G, causing an R92G change in the coding sequence. The other patient also had the C274G mutation and a 13-bp deletion that presumably resulted in an unstable transcript. Defects in DPM1 define a new glycosylation disorder, CDG-Ie.
PMCID: PMC377427  PMID: 10642597
8.  Two Argentinean Siblings with CDG-Ix: A Novel Type of Congenital Disorder of Glycosylation? 
JIMD Reports  2011;1:65-72.
Congenital disorders of glycosylation (CDG) are genetic diseases caused by abnormal protein and lipid glycosylation. In this chapter, we report the clinical, biochemical, and molecular findings in two siblings with an unidentified CDG (CDG-Ix). They are the first and the third child of healthy consanguineous Argentinean parents. Patient 1 is now a 11-year-old girl, and patient 2 died at the age of 4 months. Their clinical picture involved liver dysfunction in the neonatal period, psychomotor retardation, microcephaly, seizures, axial hypotonia, feeding difficulties, and hepatomegaly. Patient 1 also developed strabismus and cataract. They showed a type 1 pattern of serum sialotransferrin. Enzymatic analysis for phosphomannomutase and phosphomannose isomerase in leukocytes and fibroblasts excluded PMM2-CDG and MPI-CDG. Lipid-linked oligosaccharide (LLO) analysis showed a normal profile. Therefore, this result could point to a deficiency in the dolichol metabolism. In this context, ALG8-CDG, DPAGT1-CDG, and SRD5A3-CDG were analyzed and no defects were identified. In conclusion, we could not identify the genetic deficiency in these patients yet. Further studies are underway to identify the basic defect in them, taking into account the new CDG types that have been recently described.
PMCID: PMC3509812  PMID: 23430830
Congenital disorders of glycosylation; Isoelectrofocusing; N- and O-glycosylation
9.  A predicted geranylgeranyl reductase reduces the ω-position isoprene of dolichol phosphate in the halophilic archaeon, Haloferax volcanii 
Biochimica et Biophysica Acta  2012;1821(6):923-933.
In N-glycosylation in both Eukarya and Archaea, N-linked oligosaccharides are assembled on dolichol phosphate prior to transfer of the glycan to the protein target. However, whereas only the α-position isoprene subunit is saturated in eukaryal dolichol phosphate, both the α- and ω-position isoprene subunits are reduced in the archaeal lipid. The agents responsible for dolichol phosphate saturation remain largely unknown. The present study sought to identify dolichol phosphate reductases in the halophilic archaeon, Haloferax volcanii. Homology-based searches recognize HVO_1799 as a geranylgeranyl reductase. Mass spectrometry revealed that cells deleted of HVO_1799 fail to fully reduce the isoprene chains of Hfx. volcanii membrane phospholipids and glycolipids. Likewise, the absence of HVO_1799 led to a loss of saturation of the ω-position isoprene subunit of C55 and C60 dolichol phosphate, with the effect of HVO_1799 deletion being more pronounced with C60 dolichol phosphate than with C55 dolichol phosphate. Glycosylation of dolichol phosphate in the deletion strain occurred preferentially on that version of the lipid saturated at both the α- and ω-position isoprene subunits.
PMCID: PMC3340491  PMID: 22469971
Archaea; dolichol phosphate; geranylgeranyl reductase; Haloferax volcanii; isoprene; reductase
10.  Defects in the N-Linked Oligosaccharide Biosynthetic Pathway in a Trypanosoma brucei Glycosylation Mutant†  
Eukaryotic Cell  2004;3(2):255-263.
Concanavalin A (ConA) kills the procyclic (insect) form of Trypanosoma brucei by binding to its major surface glycoprotein, procyclin. We previously isolated a mutant cell line, ConA 1-1, that is less agglutinated and more resistant to ConA killing than are wild-type (WT) cells. Subsequently we found that the ConA resistance phenotype in this mutant is due to the fact that the procyclin either has no N-glycan or has an N-glycan with an altered structure. Here we demonstrate that the alteration in procyclin N-glycosylation correlates with two defects in the N-linked oligosaccharide biosynthetic pathway. First, ConA 1-1 has a defect in activity of polyprenol reductase, an enzyme involved in synthesis of dolichol. Metabolic incorporation of [3H]mevalonate showed that ConA 1-1 synthesizes equal amounts of dolichol and polyprenol, whereas WT cells make predominantly dolichol. Second, we found that ConA 1-1 synthesizes and accumulates an oligosaccharide lipid (OSL) precursor that is smaller in size than that from WT cells. The glycan of OSL in WT cells is apparently Man9GlcNAc2, whereas that from ConA 1-1 is Man7GlcNAc2. The smaller OSL glycan in the ConA 1-1 explains how some procyclin polypeptides bear a Man4GlcNAc2 modified with a terminal N-acetyllactosamine group, which is poorly recognized by ConA.
PMCID: PMC387663  PMID: 15075256
11.  Carbohydrate-deficient glycoprotein syndrome: not an N-linked oligosaccharide processing defect, but an abnormality in lipid-linked oligosaccharide biosynthesis? 
Journal of Clinical Investigation  1994;94(5):1901-1909.
The carbohydrate-deficient glycoprotein syndrome (CDGS) is a developmental disease associated with an abnormally high isoelectric point of serum transferrin. Carbohydrate analyses of this glycoprotein initially suggested a defect in N-linked oligosaccharide processing, although more recent studies indicate a defect in the attachment of these sugar chains to the protein. We studied both serum glycoproteins and fibroblast-derived [2-3H]mannose-labeled oligosaccharides from CDGS patients and normal controls. While there was a decrease in the glycosylation of serum glycoproteins of affected individuals, differences were not seen in either monosaccharide composition or oligosaccharide structures. The lectin-binding profiles of glycopeptides from [2-3H]-mannose-labeled fibroblasts were likewise indistinguishable. However, the incorporation of [2-3H]mannose into both glycoproteins and the dolichol-linked oligosaccharide precursor was significantly reduced. Thus, at least in some patients, CDGS is not due to a defect in processing of N-linked oligosaccharides, but rather to defective synthesis and transfer of nascent dolichol-linked oligosaccharide precursors. This abnormality could result in both a failure to glycosylate some sites on some proteins, as well as secondary abnormalities in overall glycoprotein processing and/or function.
PMCID: PMC294598  PMID: 7962535
12.  SRD5A3: A Surprising Role in Glycosylation 
Cell  2010;142(2):196-198.
The steroid 5α-reductase (SRD5A) family of enzymes produces steroid hormones that regulate male sexual development. Now, Cantagrel et al. (2010) identify a member of this family, SRD5A3, as a polyprenol reductase with a crucial role in N-linked protein glycosylation and pinpoint SRD5A3 mutations as the cause of a rare Mendelian disease.
PMCID: PMC3104503  PMID: 20655462
13.  Mannose corrects altered N-glycosylation in carbohydrate-deficient glycoprotein syndrome fibroblasts. 
Journal of Clinical Investigation  1996;97(6):1478-1487.
Type I carbohydrate-deficient glycoprotein syndrome (CDGS) patients fail to add entire N-linked oligosaccharide chains to some serum glycoproteins. Here we show that four CDGS fibroblast cell lines have two related glycosylation abnormalities. First, they incorporate 3-10-fold less [3H] mannose into proteins, and, second, the size of the lipid-linked oligosaccharide precursor (LLO) is much smaller than in controls. Addition of exogenous mannose, but not glucose, to these CDGS cells corrects both the lowered [3H] mannose incorporation and the size of LLO. These corrections are not permanent, and the defects immediately reappear when mannose is removed. To explore further the basis of mannose correction, we analyzed the amount of 3H-labeled LLO intermediates. Except for dolichol-P-mannose, other precursors, including mannose, mannose-6-phosphate, mannose-1-phosphate, and GDP-mannose, all showed a 3-10-fold decrease in CDGS cells. Thus, there are no obvious lesions in the intracellular conversion of mannose into LLO, and, once inside the cell, [3H]mannose appeared to be metabolized normally. Initial velocities of [3H]mannose uptake were two- to threefold less in CDGS cells compared with controls, and this slower transport may partially explain the reduced [3H]mannose incorporation in CDGS cells. Since we previously showed that the enzymes converting glucose to mannose-6-phosphate appear to be normal, our results suggest that cells may acquire or generate mannose in other ways. Although we have not identified the primary defect in CDGS, these studies show that intracellular mannose is limited and that some patients might benefit from including mannose in their regular diets.
PMCID: PMC507208  PMID: 8617881
14.  Molecular characterization of the cis-prenyltransferase of Giardia lamblia 
Glycobiology  2010;20(7):824-832.
Giardia lamblia, the protist that causes diarrhea, makes an Asn-linked-glycan (N-glycan) precursor that contains just two sugars (GlcNAc2) attached by a pyrophosphate linkage to a polyprenol lipid. Because the candidate cis-prenyltransferase of Giardia appears to be more similar to bacterial enzymes than to those of most eukaryotes and because Giardia is missing a candidate dolichol kinase (ortholog to Saccharomyces cerevisiae SEC59 gene product), we wondered how Giardia synthesizes dolichol phosphate (Dol-P), which is used to make N-glycans and glycosylphosphatidylinositol (GPI) anchors. Here we show that cultured Giardia makes an unsaturated polyprenyl pyrophosphate (dehydrodolichol), which contains 11 and 12 isoprene units and is reduced to dolichol. The Giardia cis-prenyltransferase that we have named Gl-UPPS because the enzyme primarily synthesizes undecaprenol pyrophosphate is phylogenetically related to those of bacteria and Trypanosoma rather than to those of other protists, metazoans and fungi. In transformed Saccharomyces, the Giardia cis-prenyltransferase also makes a polyprenol containing 11 and 12 isoprene units and supports normal growth, N-glycosylation and GPI anchor synthesis of a rer2Δ, srt1Δ double-deletion mutant. Finally, despite the absence of an ortholog to SEC59, Giardia has cytidine triphosphate-dependent dolichol kinase activity. These results suggest that the synthetic pathway for Dol-P is conserved in Giardia, even if some of the important enzymes are different from those of higher eukaryotes or remain unidentified.
PMCID: PMC2900897  PMID: 20308470
cis-prenyltransferase; dolichol; Giardia; recombinant expression; Saccharomyces
15.  Deficiency of dolichol-phosphate-mannose synthase-1 causes congenital disorder of glycosylation type Ie 
Journal of Clinical Investigation  2000;105(2):233-239.
Congenital disorders of glycosylation (CDG), formerly known as carbohydrate-deficient glycoprotein syndromes, lead to diseases with variable clinical pictures. We report the delineation of a novel type of CDG identified in 2 children presenting with severe developmental delay, seizures, and dysmorphic features. We detected hypoglycosylation on serum transferrin and cerebrospinal fluid β-trace protein. Lipid-linked oligosaccharides in the endoplasmic reticulum of patient fibroblasts showed an accumulation of the dolichyl pyrophosphate Man5GlcNAc2 structure, compatible with the reduced dolichol-phosphate-mannose synthase (DolP-Man synthase) activity detected in these patients. Accordingly, 2 mutant alleles of the DolP-Man synthase DPM1 gene, 1 with a 274C>G transversion, the other with a 628delC deletion, were detected in both siblings. Complementation analysis using DPM1-null murine Thy1-deficient cells confirmed the detrimental effect of both mutations on the enzymatic activity. Furthermore, mannose supplementation failed to improve the glycosylation status of DPM1-deficient fibroblast cells, thus precluding a possible therapeutic application of mannose in the patients. Because DPM1 deficiency, like other subtypes of CDG-I, impairs the assembly of N-glycans, this novel glycosylation defect was named CDG-Ie.
PMCID: PMC377434  PMID: 10642602
16.  RFT1 Deficiency in Three Novel CDG Patients 
Human mutation  2009;30(10):10.1002/humu.21085.
The medical significance of N-glycosylation is underlined by a group of inherited human disorders called Congenital Disorders of Glycosylation (CDG). One key step in the biosynthesis of the Glc3Man9Glc-NAc2-PP-dolichol precursor, essential for N-glycosylation, is the translocation of Man5GlcNAc2-PP-dolichol across the endoplasmic reticulum membrane. This step is facilitated by the RFT1 protein. Recently, the first RFT1-deficient CDG (RFT1-CDG) patient was identified and presented a severe N-glycosylation disorder. In the present study, we describe three novel CDG patients with an RFT1 deficiency. The first patient was homozygous for the earlier reported RFT1 missense mutation (c.199C4T; p.R67C), whereas the two other patients were homozygous for the missense mutation c.454A4G (p.K152E) and c.892G4A (p.E298 K), respectively. The pathogenic character of the novel mutations was illustrated by the accumulation of Man5GlcNAc2-PP-dolichol and by reduced recombinant DNase 1 secretion. Both the glycosylation pattern and recombinant DNase 1 secretion could be normalized by expression of normal RFT1 cDNA in the patients’ fibroblasts. The clinical phenotype of these patients comprised typical CDG symptoms in addition to sensorineural deafness, rarely reported in CDG patients. The identification of additional RFT1-deficient patients allowed to delineate the main clinical picture of RFT1-CDG and confirmed the crucial role of RFT1 in Man5GlcNAc2-PPdolichol translocation.
PMCID: PMC3869400  PMID: 19701946
glycosylation; CDG; RFT1; dolichol
17.  Analysis of Glycosylation in CDG-Ia Fibroblasts by Fluorophore-assisted Carbohydrate Electrophoresis 
The Journal of biological chemistry  2005;280(18):17901-17909.
Phosphomannomutase (PMM) deficiency causes congenital disorder of glycosylation (CDG)-Ia, a broad spectrum disorder with developmental and neurological abnormalities. PMM converts mannose 6-phosphate (M6P) to mannose-1-phosphate, a precursor of GDP-mannose used to make Glc3Man9GlcNAc2-P-P-dolichol (lipid-linked oligosaccharide; LLO). LLO, in turn, is the donor substrate of oligosaccharyltransferase for protein N-linked glycosylation. Hepatically produced N-linked glycoproteins in CDG-Ia blood are hypoglycosylated. Upon labeling with [3H]mannose, CDG-Ia fibroblasts have been widely reported to accumulate [3H]LLO intermediates. Since these are thought to be poor oligosaccharyltransferase substrates, LLO intermediate accumulation has been the prevailing explanation for hypoglycosylation in patients. However, this is discordant with sporadic reports of specific glycoproteins (detected with antibodies) from CDG-Ia fibroblasts being fully glycosylated. Here, fluorophore-assisted carbohydrate electrophoresis (FACE, a nonradioactive technique) was used to analyze steady-state LLO compositions in CDG-Ia fibroblasts. FACE revealed that low glucose conditions accounted for previous observations of accumulated [3H]LLO intermediates. Additional FACE experiments demonstrated abundant Glc3Man9GlcNAc2-P-P-dolichol, without hypoglycosylation, in CDG-Ia fibroblasts grown with physiological glucose. This suggested a “missing link” to explain hypoglycosylation in CDG-Ia patients. Because of the possibility of its accumulation, the effects of M6P on glycosylation were explored in vitro. Surprisingly, M6P was a specific activator for cleavage of Glc3Man9GlcNAc2-P-P-dolichol. This led to futile cycling of the LLO pathway, exacerbated by GDP-mannose/PMM deficiency. The possibilities that M6P may accumulate in hepatocytes and that M6P-stimulated LLO cleavage may account for both hypoglycosylation and the clinical failure of dietary mannose therapy with CDG-Ia patients are discussed.
PMCID: PMC1282451  PMID: 15708848
18.  Dolichol-linked oligosaccharide selection by the oligosaccharyltransferase in protist and fungal organisms 
The Journal of Cell Biology  2007;177(1):29-37.
The dolichol-linked oligosaccharide Glc3Man9GlcNAc2-PP-Dol is the in vivo donor substrate synthesized by most eukaryotes for asparagine-linked glycosylation. However, many protist organisms assemble dolichol-linked oligosaccharides that lack glucose residues. We have compared donor substrate utilization by the oligosaccharyltransferase (OST) from Trypanosoma cruzi, Entamoeba histolytica, Trichomonas vaginalis, Cryptococcus neoformans, and Saccharomyces cerevisiae using structurally homogeneous dolichol-linked oligosaccharides as well as a heterogeneous dolichol-linked oligosaccharide library. Our results demonstrate that the OST from diverse organisms utilizes the in vivo oligo saccharide donor in preference to certain larger and/or smaller oligosaccharide donors. Steady-state enzyme kinetic experiments reveal that the binding affinity of the tripeptide acceptor for the protist OST complex is influenced by the structure of the oligosaccharide donor. This rudimentary donor substrate selection mechanism has been refined in fungi and vertebrate organisms by the addition of a second, regulatory dolichol-linked oligosaccharide binding site, the presence of which correlates with acquisition of the SWP1/ribophorin II subunit of the OST complex.
PMCID: PMC2064103  PMID: 17403929
19.  The thermoacidophilic archaeon Sulfolobus acidocaldarius contains an unsually short, highly reduced dolichol phosphate 
Biochimica et biophysica acta  2011;1811(10):607-616.
Polyprenoids, polymers containing varied numbers of isoprene subunits, serve numerous roles in biology. In Eukarya, dolichyl phosphate, a phosphorylated polyprenol bearing a saturated α-end isoprene subunit, serves as the glycan carrier during N-glycosylation, namely that post-translational modification whereby glycans are covalently linked to select asparagine residues of a target protein. As in Eukarya, N-glycosylation in Archaea also relies on phosphorylated dolichol. In this report, LC-ESI/MS/MS was employed to identify a novel dolichyl phosphate (DolP) in the thermoacidophilic archaeon, Sulfolobus acidocaldarius. The unusually short S. acidocaldarius DolP presents a degree of saturation not previously reported. S. acidocaldarius DolP contains not only the saturated α- and ω-end isoprene subunits observed in other archaeal DolPs, but also up to five saturated intra-chain isoprene subunits. The corresponding dolichol and hexose-charged DolP species were also detected. The results of the present study offer valuable information on the biogenesis and potential properties of this unique DolP. Furthermore, elucidation of the mechanism of the α-isoprene unit reduction in S. acidocaldarius dolichol may facilitate the identification of the alternative, as yet unknown polyprenol reductase in Eukarya.
PMCID: PMC3156894  PMID: 21745590
Archaea; dolichol; electrospray ionization mass spectrometry; polyprenol; polyprenol reductase; Sulfolobus acidocaldarius
20.  Adult phenotype and further phenotypic variability in SRD5A3-CDG 
BMC Medical Genetics  2014;15:10.
SRD5A3 is responsible for SRD5A3-CDG, a type of congenital disorder of glycosylation, and mutations have been reported in 15 children. All the mutations are recessive and truncating.
Case presentation
We present 2 brothers at the age of 38 and 40 years with an initial diagnosis of cerebellar ataxia. We found the candidate disease loci via linkage analysis using data from single nucleotide polymorphism genome scans and homozygous truncating mutation SRD5A3 p.W19X, which was previously reported in 3 unrelated children, by exome sequencing.
Clinical investigations included physical and ocular examinations and blood tests. Severe ocular involvement with retinal bone spicule pigmentation and optic atrophy are the most prominent disabling clinical features of the disease. The serum transferrin isoelectric focusing (TIEF) pattern is abnormal in the patient investigated.
Our patients are older, with later onset and milder clinical phenotypes than all patients with SRD5A3-CDG reported so far. They also have atypical ocular findings and variable phenotypes. Our findings widen the spectrum of phenotypes resulting from SRD5A3 mutations and the clinical variability of SRD5A3-CDG, and suggest screening for SRD5A3 mutations in new patients with at least a few of the clinical symptoms of SRD5A3-CDG.
PMCID: PMC3898029  PMID: 24433453
SRD5A3; SRD5A3-CDG; CDG; Glycosylation defect
21.  Autosomal Recessive Dilated Cardiomyopathy due to DOLK Mutations Results from Abnormal Dystroglycan O-Mannosylation 
PLoS Genetics  2011;7(12):e1002427.
Genetic causes for autosomal recessive forms of dilated cardiomyopathy (DCM) are only rarely identified, although they are thought to contribute considerably to sudden cardiac death and heart failure, especially in young children. Here, we describe 11 young patients (5–13 years) with a predominant presentation of dilated cardiomyopathy (DCM). Metabolic investigations showed deficient protein N-glycosylation, leading to a diagnosis of Congenital Disorders of Glycosylation (CDG). Homozygosity mapping in the consanguineous families showed a locus with two known genes in the N-glycosylation pathway. In all individuals, pathogenic mutations were identified in DOLK, encoding the dolichol kinase responsible for formation of dolichol-phosphate. Enzyme analysis in patients' fibroblasts confirmed a dolichol kinase deficiency in all families. In comparison with the generally multisystem presentation in CDG, the nonsyndromic DCM in several individuals was remarkable. Investigation of other dolichol-phosphate dependent glycosylation pathways in biopsied heart tissue indicated reduced O-mannosylation of alpha-dystroglycan with concomitant functional loss of its laminin-binding capacity, which has been linked to DCM. We thus identified a combined deficiency of protein N-glycosylation and alpha-dystroglycan O-mannosylation in patients with nonsyndromic DCM due to autosomal recessive DOLK mutations.
Author Summary
Idiopathic dilated cardiomyopathy (DCM) is estimated to be of genetic origin in 20%–48% of the patients. Almost all currently known genetic defects show dominant inheritance, although especially in younger children recessive causes have been proposed to contribute considerably to DCM. Knowledge of the genetic causes and pathophysiological mechanisms is essential for prognosis and treatment. Here, we studied several individual young patients (5–13 years old) with idiopathic and sometimes asymptomatic dilated cardiomyopathy. The key to identification of the gene was the finding of abnormal protein N-glycosylation. Via homozygosity mapping and functional knowledge of the N-glycosylation pathway, the causative gene could be identified as dolichol kinase (DOLK). Since DCM is very rare in N-glycosylation disorders (Congenital Disorders of Glycosylation, CDG) and most patients with CDG present with a multisystem involvement, we studied the underlying pathophysiological cause of this life-threatening disease. Biochemical experiments in affected heart tissue showed deficient O-mannosylation of alpha-dystroglycan, which could be correlated with the dilated cardiomyopathy. Our results thus highlight nonsyndromic DCM as a novel presentation of DOLK-CDG, via deficient O-mannosylation of alpha-dystroglycan.
PMCID: PMC3248466  PMID: 22242004
22.  Analysis and metabolic engineering of lipid-linked oligosaccharides in glycosylation-deficient CHO cells 
Glycosylation-deficient Chinese Hamster Ovary (CHO) cell lines can be used to expand our understanding of N-glycosylation pathways and to study Congenital Disorders of Glycosylation, diseases caused by defects in the synthesis of N-glycans. The mammalian N-glycosylation pathway involves the step-wise assembly of sugars onto a dolichol phosphate (P-Dol) carrier, forming a lipid-linked oligosaccharide (LLO), followed by the transfer of the completed oligosaccharide onto the protein of interest. In order to better understand how deficiencies in this pathway affect the availability of the completed LLO donor for use in N-glycosylation, we used a non-radioactive, HPLC-based assay to examine the intermediates in the LLO synthesis pathway for CHO-K1 cells and for three different glycosylation-deficient CHO cell lines. B4-2-1 cells, which have a mutation in the dolichol phosphate-mannose synthase (DPM2) gene, accumulated LLO with the structure Man5GlcNAc2-P-P-Dol, while MI8-5 cells, which lack glucosyltransferase I (ALG6) activity, accumulated Man9GlcNAc2-P-P-Dol. CHO-K1 and MI5-4 cells both produced primarily the complete LLO, Glc3Man9GlcNAc2-P-P-Dol, though the relative quantity was lower in MI5-4. MI5-4 cells have reduced hexokinase activity which could affect the availability of many of the substrates required for LLO synthesis and, consequently, impair production of the final LLO donor. Increasing hexokinase activity by overexpressing hexokinase II in MI5-4 caused a decrease in the relative quantities of the incomplete LLO intermediates from Man5GlcNAc2-PP-Dol through Glc1Man9GlcNAc2-PP-Dol, and an increase in the relative quantity of the final LLO donor, Glc3Man9GlcNAc2-P-P-Dol. This study suggests that metabolic engineering may be a useful strategy for improving LLO availability for use in N-glycosylation.
PMCID: PMC2887678  PMID: 20331963
Chinese Hamster Ovary; Congenital Disorders of Glycosylation; hexokinase; HPLC; N-linked glycan deficiency
23.  MPDU1 mutations underlie a novel human congenital disorder of glycosylation, designated type If 
Journal of Clinical Investigation  2001;108(11):1687-1695.
Deficiencies in the pathway of N-glycan biosynthesis lead to severe multisystem diseases, known as congenital disorders of glycosylation (CDG). The clinical appearance of CDG is variable, and different types can be distinguished according to the gene that is altered. In this report, we describe the molecular basis of a novel type of the disease in three unrelated patients diagnosed with CDG-I. Serum transferrin was hypoglycosylated and patients’ fibroblasts accumulated incomplete lipid-linked oligosaccharide precursors for N-linked protein glycosylation. Transfer of incomplete oligosaccharides to protein was detected. Sequence analysis of the Lec35/MPDU1 gene, known to be involved in the use of dolichylphosphomannose and dolichylphosphoglucose, revealed mutations in all three patients. Retroviral-based expression of the normal Lec35 cDNA in primary fibroblasts of patients restored normal lipid-linked oligosaccharide biosynthesis. We concluded that mutations in the Lec35/MPDU1 gene cause CDG. This novel type was termed CDG-If.
PMCID: PMC200989  PMID: 11733564
24.  Liquid chromatography/tandem mass spectrometry of dolichols and polyprenols, lipid sugar carriers across evolution* 
Biochimica et biophysica acta  2011;1811(11):800-806.
Across evolution, dolichols and polyprenols serve as sugar carriers in biosynthetic processes that include protein glycosylation and lipopolysaccharide biogenesis. Liquid chromatography coupled with electrospray ionization mass spectrometry offers a powerful tool for studying dolichols and polyprenols in their alcohol or glycan-modified forms in members of all three domains of life. In the following, recent examples of the how different versions of this analytical approach, namely reverse phase liquid chromatography-multiple reaction monitoring, normal phase liquid chromatography/tandem mass spectrometry and normal phase liquid chromatography-precursor ion scan detection have respectively served to address novel aspects of dolichol or polyprenol biology in Eukarya, Archaea and Bacteria. This article is part of a Special Issue entitled Lipodomics and Imaging Mass Spectrometry.
PMCID: PMC3419941  PMID: 21570481
Liquid chromatography/tandem mass; spectrometry; Multiple reaction monitoring; Precursor ion scan; Dolichol; Polyprenol
25.  Alg13p, the Catalytic Subunit of the Endoplasmic Reticulum UDP-GlcNAc Glycosyltransferase, Is a Target for Proteasomal Degradation 
Molecular Biology of the Cell  2008;19(5):2169-2178.
The second step of dolichol-linked oligosaccharide synthesis in the N-linked glycosylation pathway at the endoplasmic reticulum (ER) membrane is catalyzed by an unusual hetero-oligomeric UDP-N-acetylglucosamine transferase that in most eukaryotes is comprised of at least two subunits, Alg13p and Alg14p. Alg13p is the cytosolic and catalytic subunit that is recruited to the ER by the membrane protein Alg14p. We show that in Saccharomyces cerevisiae, cytosolic Alg13p is very short-lived, whereas membrane-associated Alg13 is relatively stable. Cytosolic Alg13p is a target for proteasomal degradation, and the failure to degrade excess Alg13p leads to glycosylation defects. Alg13p degradation does not require ubiquitin but instead, requires a C-terminal domain whose deletion results in Alg13p stability. Conversely, appending this sequence onto normally long-lived β-galactosidase causes it to undergo rapid degradation, demonstrating that this C-terminal domain represents a novel and autonomous degradation motif. These data lead to the model that proteasomal degradation of excess unassembled Alg13p is an important quality control mechanism that ensures proper protein complex assembly and correct N-linked glycosylation.
PMCID: PMC2366857  PMID: 18337470

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