Patricie Burda

The dolichol pathway of N-linked glycosylation.

The oligosaccharide substrate for the N-linked protein glycosylation is assembled at the membrane of the endoplasmic reticulum. Dolichyl pyrophosphate serves as a carrier in this biosynthetic pathway. In this review, we discuss the function of the lipid carrier dolichol in oligosaccharide assembly and give an overview of the biosynthesis of the different sugar donors required for the building of the oligosaccharide. Yeast genetic techniques have made it possible to identify many different loci encoding specific glycosyltransferases required for the precise and ordered assembly of the dolichyl pyrophosphate-linked oligosaccharide. Based on the knowledge obtained from studying this pathway in yeast, we compare it to the process of N-linked protein glycosylation in archaea. We suggest that N-linked glycosylation in eukaryotes and in archaea share a common evolutionary origin.

STT3, a highly conserved protein required for yeast oligosaccharyl transferase activity in vivo

N‐linked glycosylation is a ubiquitous protein modification, and is essential for viability in eukaryotic cells. A lipid‐linked core‐oligosaccharide is assembled at the membrane of the endoplasmic reticulum and transferred to selected asparagine residues of nascent polypeptide chains by the oligosaccharyl transferase (OTase) complex. Based on the synthetic lethal phenotype of double mutations affecting the assembly of the lipid‐linked core‐oligosaccharide and the OTase activity, we have performed a novel screen for mutants in Saccharomyces cerevisiae with altered N‐linked glycosylation. Besides novel mutants deficient in the assembly of the lipid‐linked oligosaccharide (alg mutants), we identified the STT3 locus as being required for OTase activity in vivo. The essential STT3 protein is approximately 60% identical in amino acid sequence to its human homologue. A mutation in the STT3 locus affects substrate specificity of the OTase complex in vivo and in vitro. In stt3–3 cells very little glycosyl transfer occurs from incomplete lipid‐linked oligosaccharide, whereas the transfer of full‐length Glc3Man9GlcNAc2 is hardly affected as compared with wild‐type cells. Depletion of the STT3 protein results in loss of transferase activity in vivo and a deficiency in the assembly of OTase complex.

Deficiency of dolichol-phosphate-mannose synthase-1 causes congenital disorder of glycosylation type Ie

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 beta-trace protein. Lipid-linked oligosaccharides in the endoplasmic reticulum of patient fibroblasts showed an accumulation of the dolichyl pyrophosphate Man(5)GlcNAc(2) 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.

Multiple phenotypes in phosphoglucomutase 1 deficiency

BACKGROUND Congenital disorders of glycosylation are genetic syndromes that result in impaired glycoprotein production. We evaluated patients who had a novel recessive disorder of glycosylation, with a range of clinical manifestations that included hepatopathy, bifid uvula, malignant hyperthermia, hypogonadotropic hypogonadism, growth retardation, hypoglycemia, myopathy, dilated cardiomyopathy, and cardiac arrest. METHODS Homozygosity mapping followed by whole-exome sequencing was used to identify a mutation in the gene for phosphoglucomutase 1 (PGM1) in two siblings. Sequencing identified additional mutations in 15 other families. Phosphoglucomutase 1 enzyme activity was assayed on cell extracts. Analyses of glycosylation efficiency and quantitative studies of sugar metabolites were performed. Galactose supplementation in fibroblast cultures and dietary supplementation in the patients were studied to determine the effect on glycosylation. RESULTS Phosphoglucomutase 1 enzyme activity was markedly diminished in all patients. Mass spectrometry of transferrin showed a loss of complete N-glycans and the presence of truncated glycans lacking galactose. Fibroblasts supplemented with galactose showed restoration of protein glycosylation and no evidence of glycogen accumulation. Dietary supplementation with galactose in six patients resulted in changes suggestive of clinical improvement. A new screening test showed good discrimination between patients and controls. CONCLUSIONS Phosphoglucomutase 1 deficiency, previously identified as a glycogenosis, is also a congenital disorder of glycosylation. Supplementation with galactose leads to biochemical improvement in indexes of glycosylation in cells and patients, and supplementation with complex carbohydrates stabilizes blood glucose. A new screening test has been developed but has not yet been validated. (Funded by the Netherlands Organization for Scientific Research and others.).

The STT3 protein is a component of the yeast oligosaccharyltransferase complex

Abstract N-linked protein glycosylation is an essential process in eukaryotic cells. In the central reaction, the oligosaccharyltransferase (OTase) catalyzes the transfer of the oligosaccharide Glc3Man9GlcNAc2 from dolicholpyrophosphate onto asparagine residues of nascent polypeptide chains in the lumen of the endoplasmic reticulum. The product of the essential gene STT3 is required for OTase activity in vivo, but is not present in highly purified OTase preparations. Using affinity purification of a tagged Stt3 protein, we now demonstrate that other components of the OTase complex, namely Ost1p, Wbp1p and Swp1p, specifically co-purify with the Stt3 protein. In addition, different conditional stt3 alleles can be suppressed by overexpression of either OST3 and OST4, which encode small components of the OTase complex. These genetic and biochemical data show that the highly conserved Stt3p is a component of the oligosaccharyltransferase complex.

LC-MS/MS based assay and reference intervals in children and adolescents for oxysterols elevated in Niemann-Pick diseases.

BACKGROUND Niemann-Pick type C (NP-C) is a rare progressive neurodegenerative lipid storage disorder with heterogeneous clinical presentation and challenging diagnostic procedures. Recently oxysterols have been reported to be specific biomarkers for NP-C but knowledge on the intra-individual variation and on reference intervals in children and adolescents are lacking. METHODS We established a LC-MS/MS assay to measure Cholestane-3β, 5α, 6β-triol (C-triol) and 7-Ketocholesterol (7-KC) following Steglich esterification. To assess reference intervals and intra-individual variation we determined oxysterols in 148 children and adolescents from 0 to 18 years and repeat measurements in 19 of them. RESULTS The reported method is linear (r>0.99), sensitive (detection limit of 0.03 ng/mL [0.07 nM] for C-triol, and 0.54 ng/mL [1.35 nM] for 7-KC) and precise, with an intra-day imprecision of 4.8% and 4.1%, and an inter-day imprecision of 7.0% and 11.0% for C-triol (28 ng/ml, 67 nM) and 7-KC (32 ng/ml, 80 nM), respectively. Recoveries for 7-KC and C-triol range between 93% and 107%. The upper reference limit obtained for C-triol is 40.4 ng/mL (95% CI: 26.4-61.7 ng/mL, 96.0 nM, 95% CI: 62.8-146.7 nM) and 75.0 ng/mL for 7-KC (95% CI: 55.5-102.5 ng/mL, 187.2 nM, 95% CI: 138.53-255.8 nM), with no age or gender dependency. Both oxysterols have a broad intra-individual variation of 46%±23% for C-triol and 52%±29% for 7-KC. Nevertheless, all Niemann-Pick patients showed increased C-triol levels including Niemann-Pick type A and B patients. CONCLUSIONS The LC-MS/MS assay is a robust assay to quantify C-triol and 7-KC in plasma with well documented reference intervals in children and adolescents to screen for NP-C in the pediatric population. In addition our results suggest that especially the C-triol is a biomarker for all three Niemann-Pick diseases.

Molecular mechanisms leading to three different phenotypes in the cblD defect of intracellular cobalamin metabolism

The cblD defect of intracellular vitamin B(12) metabolism can lead to isolated methylmalonic aciduria (cblD-MMA) or homocystinuria (cblD-HC), or combined methylmalonic aciduria and homocystinuria (cblD-MMA/HC). We studied the mechanism whereby MMADHC mutations can lead to three phenotypes. The effect of various expression vectors containing MMADHC modified to contain an enhanced mitochondrial leader sequence or mutations changing possible downstream sites of reinitiation of translation or mutations introducing stop codons on rescue of adenosyl- and methylcobalamin (MeCbl) formation was studied. The constructs were transfected into cell lines derived from various cblD patients fibroblasts. Expression of 10 mutant alleles from 15 cblD patients confirmed that the nature and location of the mutations correlate with the biochemical phenotype. In cblD-MMA/HC cells, improving mitochondrial targeting of MMADHC clearly increased the formation of adenosylcobalamin (AdoCbl) with a concomitant decrease in MeCbl formation. In cblD-MMA cells, this effect was dependent on the mutation and showed a negative correlation with endogenous MMADHC mRNA levels. These findings support the hypothesis that a single protein exists with two different functional domains that interact with either cytosolic or mitochondrial targets. Also a delicate balance exists between cytosolic MeCbl and mitochondrial AdoCbl synthesis, supporting the role of cblD protein as a branch point in intracellular cobalamin trafficking. Furthermore, our data indicate that the sequence after Met116 is sufficient for MeCbl synthesis, whereas the additional sequence between Met62 and Met116 is required for AdoCbl synthesis. Accordingly, western blot studies reveal proteins of the size expected from the stop codon position with subsequent reinitiation of translation.

3-methylcrotonyl-CoA carboxylase deficiency: Clinical, biochemical, enzymatic and molecular studies in 88 individuals

BackgroundIsolated 3-methylcrotonyl-CoA carboxylase (MCC) deficiency is an autosomal recessive disorder of leucine metabolism caused by mutations in MCCC1 or MCCC2 encoding the α and β subunit of MCC, respectively. The phenotype is highly variable ranging from acute neonatal onset with fatal outcome to asymptomatic adults.MethodsWe report clinical, biochemical, enzymatic and mutation data of 88 MCC deficient individuals, 53 identified by newborn screening, 26 diagnosed due to clinical symptoms or positive family history and 9 mothers, identified following the positive newborn screening result of their baby.ResultsFifty-seven percent of patients were asymptomatic while 43% showed clinical symptoms, many of which were probably not related to MCC deficiency but due to ascertainment bias. However, 12 patients (5 of 53 identified by newborn screening) presented with acute metabolic decompensations. We identified 15 novel MCCC1 and 16 novel MCCC2 mutant alleles. Additionally, we report expression studies on 3 MCCC1 and 8 MCCC2 mutations and show an overview of all 132 MCCC1 and MCCC2 variants known to date.ConclusionsOur data confirm that MCC deficiency, despite low penetrance, may lead to a severe clinical phenotype resembling classical organic acidurias. However, neither the genotype nor the biochemical phenotype is helpful in predicting the clinical course.

Insights into severe 5,10-methylenetetrahydrofolate reductase deficiency: molecular genetic and enzymatic characterization of 76 patients.

5,10‐Methylenetetrahydrofolate reductase (MTHFR) deficiency is the most common inherited disorder of folate metabolism and causes severe hyperhomocysteinaemia. To better understand the relationship between mutation and function, we performed molecular genetic analysis of 76 MTHFR deficient patients, followed by extensive enzymatic characterization of fibroblasts from 72 of these. A deleterious mutation was detected on each of the 152 patient alleles, with one allele harboring two mutations. Sixty five different mutations (42 novel) were detected, including a common splicing mutation (c.1542G>A) found in 21 alleles. Using an enzyme assay in the physiological direction, we found residual activity (1.7%–42% of control) in 42 cell lines, of which 28 showed reduced affinity for nicotinamide adenine dinucleotide phosphate (NADPH), one reduced affinity for methylenetetrahydrofolate, five flavin adenine dinucleotide‐responsiveness, and 24 abnormal kinetics of S‐adenosylmethionine inhibition. Missense mutations causing virtually absent activity were found exclusively in the N‐terminal catalytic domain, whereas missense mutations in the C‐terminal regulatory domain caused decreased NADPH binding and disturbed inhibition by S‐adenosylmethionine. Characterization of patients in this way provides a basis for improved diagnosis using expanded enzymatic criteria, increases understanding of the molecular basis of MTHFR dysfunction, and points to the possible role of cofactor or substrate in the treatment of patients with specific mutations.

A single mutation in MCCC1 or MCCC2 as a potential cause of positive screening for 3-methylcrotonyl-CoA carboxylase deficiency

Isolated 3-Methylcrotonyl-CoA carboxylase deficiency (MCC deficiency) is an organic aciduria presenting with a highly variable phenotype and has been part of newborn screening programs in various countries, in particular in the US. Here we present enzymatic and genetic characterisation of 22 individuals with increased 3-hydroxyisovalerylcarnitine and/or 3-methylcrotonylglycine suggesting MCC deficiency, but only partially reduced 3-methylcrotonyl-CoA carboxylase activity. Among these, 21 carried a single mutant allele in either MCCC1 (n=20) or MCCC2 (n=1). Our results suggest that heterozygosity for such a single deleterious mutation may lead to misdiagnosis of MCC deficiency.