Biochimica Et Biophysica Acta: Molecular Basis Of Disease, Sep 1, 2009
Dolichol phosphate is a lipid carrier embedded in the endoplasmic reticulum (ER) membrane essenti... more Dolichol phosphate is a lipid carrier embedded in the endoplasmic reticulum (ER) membrane essential for the synthesis of N-glycans, GPI-anchors and protein C-and O-mannosylation. The availability of dolichol phosphate on the cytosolic site of the ER is rate-limiting for Nglycosylation. The abundance of dolichol phosphate is influenced by its de novo synthesis and the recycling of dolichol phosphate from the luminal leaflet to the cytosolic leaflet of the ER. Enzymatic defects affecting the de novo synthesis and the recycling of dolichol phosphate result in glycosylation defects in yeast or cell culture models, and are expected to cause glycosylation disorders in humans termed congenital disorders of glycosylation (CDG). Currently only one disorder affecting the dolichol phosphate metabolism has been described. In CDG-Im, the final step of the de novo synthesis of dolichol phosphate catalyzed by the enzyme dolichol kinase is affected. The defect causes a severe phenotype with death in early infancy. The present review summarizes the biosynthesis of dolichol-phosphate and the recycling pathway with respect to possible defects of the dolichol phosphate metabolism causing glycosylation defects in humans.
Congenital dyserythropoietic anemia type II (CDA II) is the most frequent type of congenital dyse... more Congenital dyserythropoietic anemia type II (CDA II) is the most frequent type of congenital dyserythropoietic anemia. More than 200 cases have been described, but with the exception of a report by the International CDA II Registry, these reports include only small numbers of cases and no data on the lifetime evolution of the disease. Since 1967, we were able to follow 48 cases of CDA II from 43 families for up to 35 years. All patients exhibit chronic anemia of variable severity requiring regular red cell transfusions only in a minority of children; 60% developed gallstones before the age of 30 years, and 16 patients had cholecystectomy between 8 and 34 years of age. Iron overload was a frequent complication. In 16 cases, iron depletion started between 7 and 36 years. Three patients died from secondary hemochromatosis. Splenectomy, performed in 22 cases, led to mod-erate increases in hemoglobin values and eliminated the need for transfusions but did not prevent further iron loading. The current recommendation is to consider splenectomy if the anemia compromises patients' performance, and to manage iron overload according to the guidelines derived from patients with thalassemia.
Congenital disorders of glycosylation – a second patient of CDG type Id deficiency: different clinical phenotype, molecular analysis and prenatal diagnosis
Congenital disorder of glycosylation type Id is an inherited glycosylation disorder based on a de... more Congenital disorder of glycosylation type Id is an inherited glycosylation disorder based on a defect of the first mannosyltransferase involved in N-glycan biosynthesis inside the endoplasmic reticulum. Only one patient with this disease has been described until now. In this article, a second patient and an affected fetus are described. The patient showed abnormal glycosylation of several plasma proteins as demonstrated by isoelectric focusing and Western blot. Lipid-linked oligosaccharides in the endoplasmic reticulum, reflecting early N-glycan assembly, revealed an accumulation of immature Man 5 GlcNAc 2glycans in fibroblasts of the patient. Chorion cells of the affected fetus showed the same characteristic lipid-linked oligosaccharides pattern. However, the fetus had a normal glycosylation of several plasma proteins. Some fetal glycoproteins are known to be derived from the mother, but even glycoproteins that do not cross the placenta were normally glycosylated in the affected fetus. Maternal or placental factors that partially compensate for the glycosylation defect in the fetal stage must be proposed and may be relevant for the therapy of these disorders in the future. (Pediatr Res 58: 248-253, 2005) Abbreviations CDG, congenital disorder of glycosylation IEF, isoelectric focusing LLO, lipid-linked oligosaccharides
Characterization of the N-glycosylation phenotype of erythrocyte membrane proteins in congenital dyserythropoietic anemia type II (CDA II/HEMPAS)
Glycoconjugate Journal, Dec 29, 2007
Congenital dyserythropoetic anemia type II (CDA II) is characterized by bi- and multinucleated er... more Congenital dyserythropoetic anemia type II (CDA II) is characterized by bi- and multinucleated erythroblasts and an impaired N-glycosylation of erythrocyte membrane proteins. Several enzyme defects have been proposed to cause CDA II based on the investigation of erythrocyte membrane glycans pinpointing to defects of early Golgi processing steps. Hitherto no molecular defect could be elucidated. In the present study, N-glycosylation of erythrocyte membrane proteins of CDA II patients and controls was investigated by SDS-Page, lectin binding studies, and MALDI-TOF/MS mapping in order to allow an embracing view on the glycosylation defect in CDA II. Decreased binding of tomato lectin was a consistent finding in all typical CDA II patients. New insights into tomato lectin binding properties were found indicating that branched polylactosamines are the main target. The binding of Aleuria aurantia, a lectin preferentially binding to alpha1-6 core-fucose, was reduced in western blots of CDA II erythrocyte membranes. MALDI-TOF analysis of band 3 derived N-glycans revealed a broad spectrum of truncated structures showing the presence of high mannose and hybrid glycans and mainly a strong decrease of large N-glycans suggesting impairment of cis, medial and trans Golgi processing. Truncation of N-glycans is a consistent finding in CDA II erythrocytes indicating the diagnostic value of tomato-lectin studies. However, structural data of erythrocyte N-glycans implicate that CDA II is not a distinct glycosylation disorder but caused by a defect disturbing Golgi processing in erythroblasts.
Biomarkers and diagnosis of congenital disorders of glycosylation
Expert opinion on medical diagnostics, 2009
Congenital disorders of glycosylation (CDG) are clinically and biochemically versatile disorders ... more Congenital disorders of glycosylation (CDG) are clinically and biochemically versatile disorders affecting the N- or O-glycosylation of proteins or lipids. The rapidly growing number of distinct CDG increasingly makes clear that a comprehensive screening for the vast majority of CDG is not possible on a simple basis. The prevailing methods for the screening and the diagnosis of CDG are presented, showing the potential and the limits of the diagnostic and screening tools used, orientated at suitability for daily use. Although valid and comprehensive diagnostic tests for CDG type I exist, there are limitations in the diagnosis of CDG type II, and many undiagnosed CDG type IIs must be expected. The apparent limitations of the methods used at present imply a necessity for new and broad diagnostic tools that comprise modern methodology to meet the requirements for screening tools.
Glycoproteomics of N‐glycosylation by in‐gel deglycosylation and matrix‐assisted laser desorption/ionisation‐time of flight mass spectrometry mapping: Application to congenital disorders of glycosylation
PROTEOMICS, 2005
A general strategy for the structural evaluation of N‐glycosylation, a common post‐translational ... more A general strategy for the structural evaluation of N‐glycosylation, a common post‐translational protein modification, is presented. The methods for the release of N‐linked glycans from the gel‐separated proteins, their isolation, purification and matrix‐assisted laser desorption/ionisation‐mass spectrometry (MALDI‐MS) analysis of their mixtures were optimised. Since many glycoproteins are available only at low quantities from sodium dodecyl sulphate‐polyacrylamide gel electrophoresis or two‐dimensional gels, high attention was paid to obtain N‐glycan mixtures representing their actual composition in human plasma by in‐gel deglycosylation. The relative sensitivity of solid MALDI matrices for MS analysis of acidic N‐glycans was compared. The most favourable results for native acidic N‐glycans were obtained with 2,4,6‐trihydroxyacetophenone monohydrate/diammoniumcitrate as a matrix. This matrix provided good results for both neutral and acidic mixtures as well as for methylated N‐glyc...
Genetics of α-dystroglycanopathies – More questions than answers
Novel ALG8 mutations expand the clinical spectrum of congenital disorder of glycosylation type Ih
Molecular Genetics and Metabolism, 2009
Congenital disorders of glycosylation (CDG) are an expanding group of inherited disorders caused ... more Congenital disorders of glycosylation (CDG) are an expanding group of inherited disorders caused by defects in the N- or O-Glycosylation of proteins and lipids. Several CDG subtypes have been described so far, including CDG type Ih which is caused by a deficiency of the dolichyl-P-Glc:Glc(1)Man(9)GlcNAc(2)-PP-dolichyl alpha1,3-glucosyltransferase (hALG8). The defect leads to an accumulation of Dol-PP-GlcNAc(2)Man(9) and Dol-PP-GlcNAc(2)Man(9)Glc(1) in the endoplasmic reticulum of patients' fibroblasts that can be detected by analyzing the lipid-linked oligosaccharyl intermediates. Five patients with CDG-Ih have been described so far. The clinical presentation of four of these patients was severe with death in early infancy. In this report, we describe two mildly affected siblings with CDG-Ih caused by two novel mutations. While one mutation (c.1434delC) causes a frame shift resulting in a premature termination codon (p.485X), the point mutation of the other allele (c.845C>T, p.A282V) causes an amino acid replacement in a highly conserved region of the hALG8 gene. The two siblings show similar symptoms, including pseudo-gynecomastia, epicanthus, muscular hypotonia, mental retardation and ataxia, expanding the genetic and clinical spectrum of CDG-Ih.
Characterization of the N-glycosylation phenotype of erythrocyte membrane proteins in congenital dyserythropoietic anemia type II (CDA II/HEMPAS)
Glycoconjugate Journal, 2007
Congenital dyserythropoetic anemia type II (CDA II) is characterized by bi- and multinucleated er... more Congenital dyserythropoetic anemia type II (CDA II) is characterized by bi- and multinucleated erythroblasts and an impaired N-glycosylation of erythrocyte membrane proteins. Several enzyme defects have been proposed to cause CDA II based on the investigation of erythrocyte membrane glycans pinpointing to defects of early Golgi processing steps. Hitherto no molecular defect could be elucidated. In the present study, N-glycosylation of erythrocyte membrane proteins of CDA II patients and controls was investigated by SDS-Page, lectin binding studies, and MALDI-TOF/MS mapping in order to allow an embracing view on the glycosylation defect in CDA II. Decreased binding of tomato lectin was a consistent finding in all typical CDA II patients. New insights into tomato lectin binding properties were found indicating that branched polylactosamines are the main target. The binding of Aleuria aurantia, a lectin preferentially binding to alpha1-6 core-fucose, was reduced in western blots of CDA II erythrocyte membranes. MALDI-TOF analysis of band 3 derived N-glycans revealed a broad spectrum of truncated structures showing the presence of high mannose and hybrid glycans and mainly a strong decrease of large N-glycans suggesting impairment of cis, medial and trans Golgi processing. Truncation of N-glycans is a consistent finding in CDA II erythrocytes indicating the diagnostic value of tomato-lectin studies. However, structural data of erythrocyte N-glycans implicate that CDA II is not a distinct glycosylation disorder but caused by a defect disturbing Golgi processing in erythroblasts.
Congenital dyserythropoietic anemia type II (CDA II) is the most frequent type of congenital dyse... more Congenital dyserythropoietic anemia type II (CDA II) is the most frequent type of congenital dyserythropoietic anemia. More than 200 cases have been described, but with the exception of a report by the International CDA II Registry, these reports include only small numbers of cases and no data on the lifetime evolution of the disease. Since 1967, we were able to follow 48 cases of CDA II from 43 families for up to 35 years. All patients exhibit chronic anemia of variable severity requiring regular red cell transfusions only in a minority of children; 60% developed gallstones before the age of 30 years, and 16 patients had cholecystectomy between 8 and 34 years of age. Iron overload was a frequent complication. In 16 cases, iron depletion started between 7 and 36 years. Three patients died from secondary hemochromatosis. Splenectomy, performed in 22 cases, led to moderate increases in hemoglobin values and eliminated the need for transfusions but did not prevent further iron loading....
Biochimica et Biophysica Acta (BBA) - Molecular Basis of Disease, 2009
Dolichol phosphate is a lipid carrier embedded in the endoplasmic reticulum (ER) membrane essenti... more Dolichol phosphate is a lipid carrier embedded in the endoplasmic reticulum (ER) membrane essential for the synthesis of N-glycans, GPI-anchors and protein C-and O-mannosylation. The availability of dolichol phosphate on the cytosolic site of the ER is rate-limiting for Nglycosylation. The abundance of dolichol phosphate is influenced by its de novo synthesis and the recycling of dolichol phosphate from the luminal leaflet to the cytosolic leaflet of the ER. Enzymatic defects affecting the de novo synthesis and the recycling of dolichol phosphate result in glycosylation defects in yeast or cell culture models, and are expected to cause glycosylation disorders in humans termed congenital disorders of glycosylation (CDG). Currently only one disorder affecting the dolichol phosphate metabolism has been described. In CDG-Im, the final step of the de novo synthesis of dolichol phosphate catalyzed by the enzyme dolichol kinase is affected. The defect causes a severe phenotype with death in early infancy. The present review summarizes the biosynthesis of dolichol-phosphate and the recycling pathway with respect to possible defects of the dolichol phosphate metabolism causing glycosylation defects in humans.
This study describes the discovery of a new inherited disorder of glycosylation named "CDG-Ik." C... more This study describes the discovery of a new inherited disorder of glycosylation named "CDG-Ik." CDG-Ik (congenital disorder of glycoslyation type Ik) is based on a defect of human mannosyltransferase I (MT-I [MIM 605907]), an enzyme necessary for the elongation of dolichol-linked chitobiose during N-glycan biosynthesis. Mutations in semiconserved regions in the corresponding gene, HMT-1 (yeast homologue, Alg1), in two patients caused drastically reduced enzyme activity, leading to a severe disease with death in early infancy. One patient had a homozygous point mutation (c.773CrT, S258L), whereas the other patient was compound heterozygous for the mutations c.773CrT and c.1025ArC (E342P). Glycosylation and growth of Alg1-deficient PRY56 yeast cells, showing a temperature-sensitive phenotype, could be restored by the human wild-type allele, whereas only slight restoration was observed after transformation with the patients' alleles.
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