RFT1-CDG: deafness as a novel feature of congenital disorders of glycosylation.

Congenital disorders of glycosylation (CDG) are genetic diseases due to defects in the synthesis of glycans and in the attachment of glycans to lipids and proteins. Actually, some 42 CDG are known including defects in protein N-glycosylation, in protein O-glycosylation, in lipid glycosylation, and in multiple and other glycosylation pathways. Most CDG are multisystem diseases and a large number of signs and symptoms have already been reported in CDG. An exception to this is deafness. This symptom has not been observed as a consistent feature in CDG. In 2008, a novel defect was identified in protein N-glycosylation, namely in RFT1. This is a defect in the assembly of N-glycans. RFT1 is involved in the transfer of Man(5)GlcNAc(2)-PP-Dol from the cytoplasmic to the luminal side of the endoplasmic reticulum. According to the novel nomenclature (non-italicized gene symbol followed by -CDG) this defect is named RFT1-CDG. Recently, three other patients with RFT1-CDG have been reported and here we report two novel patients. Remarkably, all six patients with RFT1-CDG show sensorineural deafness as part of a severe neurological syndrome. We conclude that RFT1-CDG is the first 'deafness-CDG'. CDG should be included in the work-up of congenital, particularly syndromic, hearing loss.

A new case of ALG8 deficiency (CDG Ih).

UNLABELLED: Congenital disorders of glycosylation (CDG) represent an expanding group of inherited diseases. One of them, ALG8 deficiency (CDG Ih), leads to protein N-glycosylation defects caused by malfunction of glucosyltransferase 2 (Dol-P-Glc:Glc1-Man(9)-GlcNAc(2)-P-P-Dol glucosyltransferase) resulting in inefficient addition of the second glucose residue onto lipid-linked oligosaccharides. So far, only five patients have been described with ALG8 deficiency. We present a new patient with neonatal onset. The girl was born at the 29th week of gestation complicated by oligohydramnios. Although the early postnatal adaptation was uneventful (Apgar score 8 and 9 at 5 and 10 min), generalized oedema, multifocal myoclonic seizures, and bleeding due to combined coagulopathy were present from the first day. Diarrhoea progressing to protein-losing enteropathy with ascites and pericardial effusion developed in the third week of life. Pharmacoresistant seizures and cortical, cerebellar and optic nerve atrophy indicated neurological involvement. No symptoms of liver disease except coagulopathy were observed; however, steatofibrosis with cholestasis was found at autopsy. The girl died at the age of 2 months owing to the progressive general oedema, bleeding and cardio-respiratory insufficiency. Molecular analysis revealed two heterozygous mutations in the ALG8 gene: c.139A>C (p.T47P) and the novel mutation c.1090C>T (p.R364X). CONCLUSION: The prognosis of patients with ALG8 deficiency is unfavourable. The majority of affected children have early onset of the disease with heterogeneous symptoms including multiple organ dysfunction, coagulopathy and protein-losing enteropathy. Neurological impairment is not a general clinical symptom, but it has to be taken into consideration when thinking about ALG8 deficiency.

On the nomenclature of congenital disorders of glycosylation (CDG).

A new nomenclature of CDG is proposed because the current one is too complex for clinicians and provides no added value.

Congenital disorder of glycosylation (CDG) Ig: report on a patient and review of the literature.

We report a new patient with CDG Ig and review the five other known patients. From the data on this small number of patients, it seems that the association of psychomotor retardation, male hypogenitalism and decreased serum IgG in a patient with a type 1 pattern of serum sialotransferrins might be a clue to the diagnosis of CDG Ig.

Congenital disorder of glycosylation (CDG) type Ie. A new patient.

CDG Ie is caused by a deficiency of dolichol-phosphate-mannose synthase 1 (DPM1), an enzyme involved in N-glycan assembly in the endoplasmic reticulum. Three proteins are known to be part of the synthase complex: DPM 1, DPM2 and DPM3. Only mutations in DPM1, the catalytic subunit, have been described in three families. One was homozygous for the c274C>G (R92G) mutation in DPM1 and two others were compound heterozygous for R92G and a c628delC deletion or a c331-343del13, respectively. Clinical features were a severe infantile encephalopathy, early intractable seizures, acquired microcephaly, and some dysmorphic features. We report a patient with milder symptoms: microcephaly, dysmorphic features, developmental delay, optic atrophy, and cerebellar dysfunction without cerebellar atrophy. The patient is homozygous for a new mutation in exon 9 of the DPM1 gene (c742T>C (S248P)). Our findings extend the spectrum of CDG Ie.

The galactosyltransferase family.

Galactose is transferred via several linkages to acceptor structures by galactosyltransferase enzymes. In prokaryotes, galactose is mainly found on lipopolysaccharides and capsular polysaccharides. In eukaryotes, galactosyltransferases, which are localized in the Golgi apparatus, are involved in the formation of several classes of glycoconjugates and in lactose biosynthesis. Although they sometimes catalyze identical reactions, prokaryotic and eukaryotic galactosyltransferases share only little structural similarities. In mammals, 19 distinct galactosyltransferase enzymes have been characterized to date. These enzymes catalyze the transfer of galactose via beta1-4, beta1-3, alpha1-3 and alpha1-4 linkages. The present review focuses on the description of these mammalian galactosyltransferases.

MPDU1 mutations underlie a novel human congenital disorder of glycosylation, designated type If.

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.

Biosynthesis of the linkage region of glycosaminoglycans: cloning and activity of galactosyltransferase II, the sixth member of the beta 1,3-galactosyltransferase family (beta 3GalT6).

A family of five beta1,3-galactosyltransferases has been characterized that catalyze the formation of Galbeta1,3GlcNAcbeta and Galbeta1,3GalNAcbeta linkages present in glycoproteins and glycolipids (beta3GalT1, -2, -3, -4, and -5). We now report a new member of the family (beta3GalT6), involved in glycosaminoglycan biosynthesis. The human and mouse genes were located on chromosomes 1p36.3 and 4E2, respectively, and homologs are found in Drosophila melanogaster and Caenorhabditis elegans. Unlike other members of the family, beta3GalT6 showed a broad mRNA expression pattern by Northern blot analysis. Although a high degree of homology across several subdomains exists among other members of the beta3-galactosyltransferase family, recombinant enzyme did not utilize glucosamine- or galactosamine-containing acceptors. Instead, the enzyme transferred galactose from UDP-galactose to acceptors containing a terminal beta-linked galactose residue. This product, Galbeta1,3Galbeta is found in the linkage region of heparan sulfate and chondroitin sulfate (GlcAbeta1,3Galbeta1,3Galbeta1,4Xylbeta-O-Ser), indicating that beta3GalT6 is the so-called galactosyltransferase II involved in glycosaminoglycan biosynthesis. Its identity was confirmed in vivo by siRNA-mediated inhibition of glycosaminoglycan synthesis in HeLa S3 cells. Its localization in the medial Golgi indicates that this is the major site for assembly of the linkage region.

Cloning of a mouse beta 1,3 N-acetylglucosaminyltransferase GlcNAc(beta 1,3)Gal(beta 1,4)Glc-ceramide synthase gene encoding the key regulator of lacto-series glycolipid biosynthesis.

The distinction between the different classes of glycolipids is conditioned by the action of specific core transferases. The entry point for lacto-series glycolipids is catalyzed by the beta1,3 N-acetylglucosaminyltransferase GlcNAc(beta1,3)Gal(beta1,4)Glc-ceramide (Lc3) synthase enzyme. The Lc3 synthase activity has been shown to be regulated during development, especially during brain morphogenesis. Here, we report the molecular cloning of a mouse gene encoding an Lc3 synthase enzyme. The mouse cDNA included an open reading frame of 1131 base pairs encoding a protein of 376 amino acids. The Lc3 synthase protein shared several structural motifs previously identified in the members of the beta1,3 glycosyltransferase superfamily. The Lc3 synthase enzyme efficiently utilized the lactosyl ceramide glycolipid acceptor. The identity of the reaction products of Lc3 synthase-transfected CHOP2/1 cells was confirmed by thin-layer chromatography immunostaining using antibodies TE-8 and 1B2 that recognize Lc3 and Gal(beta1,4)GlcNAc(beta1,3)Gal(beta1,4)Glc-ceramide (nLc4) structures, respectively. In addition to the initiating activity for lacto-chain synthesis, the Lc3 synthase could extend the terminal N-acetyllactosamine unit of nLc4 and also had a broad specificity for gangliosides GA1, GM1, and GD1b to generate neolacto-ganglio hybrid structures. The mouse Lc3 synthase gene was mainly expressed during embryonic development. In situ hybridization analysis revealed that that the Lc3 synthase was expressed in most tissues at embryonic day 11 with elevated expression in the developing central nervous system. Postnatally, the expression was restricted to splenic B-cells, the placenta, and cerebellar Purkinje cells where it colocalized with HNK-1 reactivity. These data support a key role for the Lc3 synthase in regulating neolacto-series glycolipid synthesis during embryonic development.

Congenital disorders of glycosylation: genetic model systems lead the way.

N-linked glycosylation is the most frequent modification of secretory proteins in eukaryotic cells. The highly conserved glycosylation process is initiated in the endoplasmic reticulum (ER), where the Glc(3)Man(9)GlcNAc(2) oligosaccharide is assembled on the lipid carrier dolichylpyrophosphate and then transferred to selected asparagine residues of polypeptide chains. In recent years, several inherited human diseases, congenital disorders of glycosylation (CDG), have been associated with deficiencies in this pathway. The ER-associated glycosylation pathway has been studied in the budding yeast Saccharomyces cerevisiae, and this model system has been invaluable in elucidating the molecular basis of novel types of CDG.

Multi-allelic origin of congenital disorder of glycosylation (CDG)-Ic.

Congenital disorders of glycosylation (CDG), formerly known as carbohydrate-deficient glycoprotein syndrome, represent a family of genetic diseases with variable clinical presentations. Common to all types of CDG characterized to date is a defective Asn-linked glycosylation caused by enzymatic defects of N-glycan synthesis. Previously, we have identified a mutation in the ALG6 alpha1,3 glucosyltransferase gene as the cause of CDG-Ic in four related patients. Here, we present the identification of seven additional cases of CDG-Ic among a group of 35 untyped CDG patients. Analysis of lipid-linked oligosaccharides in fibroblasts confirmed the accumulation of dolichyl pyrophosphate-Man9GlcNAc2 in the CDG-Ic patients. The genomic organization of the human ALG6 gene was determined, revealing 14 exons spread over 55 kb. By polymerase chain reaction amplification and sequencing of ALG6 exons, three mutations, in addition to the previously described A333 V substitution, were detected in CDG-Ic patients. The detrimental effect of these mutations on ALG6 activity was confirmed by complementation of alg6 yeast mutants. Haplotype analysis of CDG-Ic patients revealed a founder effect for the ALG6 allele bearing the A333 V mutation. Although more than 80% of CDG are type Ia, CDG-Ic may be the second most common form of the disease.

The beta 1,3-galactosyltransferase beta 3GalT-V is a stage-specific embryonic antigen-3 (SSEA-3) synthase.

We have previously reported the molecular cloning of beta1, 3-galactosyltransferase-V (beta3GalT-V), which catalyzes the transfer of Gal to GlcNAc-based acceptors with a preference for the core3 O-linked glycan GlcNAc(beta1,3)GalNAc structure. Further characterization indicated that the recombinant beta3GalT-V enzyme expressed in Sf9 insect cells also utilized the glycolipid Lc3Cer as an efficient acceptor. Surprisingly, we also found that beta3GalT-V catalyzes the transfer of Gal to the terminal GalNAc unit of the globoside Gb4, thereby synthesizing the glycolipid Gb5, also known as the stage-specific embryonic antigen-3 (SSEA-3). The SSEA-3 synthase activity of beta3GalT-V was confirmed in vivo by stable expression of the human beta3GalT-V gene in F9 mouse teratocarcinoma cells, as detected with the monoclonal antibody MC-631 by flow cytometry analysis and immunostaining of extracted glycolipids. The biological relation between SSEA-3 formation and beta3GalT-V was further documented by showing that F9 cells treated with the differentiation-inducing agent retinoic acid induced the expression of both the SSEA-3 epitope and the endogenous mouse beta3GalT-V gene. This study represents the first example of a glycosyltransferase, which utilizes two kinds of sugar acceptor substrates without requiring any additional modifier molecule.

Clinical and biochemical characteristics of congenital disorder of glycosylation type Ic, the first recognized endoplasmic reticulum defect in N-glycan synthesis.

We report on 8 patients with a recently described novel subtype of congenital disorder of glycosylation type Ic (CDG-Ic). Their clinical presentation was mainly neurological with developmental retardation, muscular hypotonia, and epilepsy. Several symptoms commonly seen in CDG-Ia such as inverted nipples, abnormal fat distribution, and cerebellar hypoplasia were not observed. The clinical course is milder overall, with a better neurological outcome, than in CDG-Ia. The isoelectric focusing pattern of serum transferrin in CDG-Ia and CDG-Ic is indistinguishable. Interestingly, beta-trace protein in cerebrospinal fluid derived from immunoblot analysis of the brain showed a less pronounced hypoglycosylation pattern in CDG-Ic patients than in CDG-Ia patients. Analysis of lipid-linked oligosaccharides revealed an accumulation of Man9GlcNAc2 intermediates due to dolichol pyrophosphate-Man9GlcNAc2 alpha-1,3 glucosyltransferase deficiency. All patients were homozygous for an A333V mutation.

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.

Secretion and purification of recombinant beta1-4 galactosyltransferase from insect cells using pFmel-protA, a novel transposition-based baculovirus transfer vector.

The palette of transfer vectors available for generation of recombinant baculoviruses based on transposition-mediated recombination has been enlarged by constructing the pFmel-protA vector. The pFmel-protA plasmid includes the honeybee melittin secretion signal and a Staphylococcus aureus protein A fusion protein tag, which allows the secretion and purification of recombinant proteins. Using this system, the human beta1-4 galactosyltransferase-I protein was expressed in Sf9 insect cells at a level ranging from 22 to 28 U (4.8 to 6.0 mg)/L. The protein A tag enabled a simple monitoring of recombinant protein expression by enzyme-linked immunosorbent assay and Western blotting. Single step purification was achieved by immunoglobulin G affinity chromatography achieving a recovery yield of 28% and a specific activity of 1.9 U per mg of recombinant protein.

The remodeling of glycoconjugates in mice.

A role for glycoconjugates in mediating cellular interactions is well established. To further understand the formation, function and regulation of various glycoconjugates in vivo, gene targeting approaches have been applied to glycosyltransferase and glycosidase enzymes involved in different biosynthetic pathways. The growing number of gene targeted mice generated have brought confirmations of the importance of both core and terminal glycosylation enzymes in normal development and physiology. Of particular interest has been the degree of cell and tissue specificity of phenotypes generated by systemic null mutations as well as the number of enzymes belonging to multigene families having overlapping activities.

Molecular cloning of a human UDP-galactose:GlcNAcbeta1,3GalNAc beta1, 3 galactosyltransferase gene encoding an O-linked core3-elongation enzyme.

Using the full-length amino-acid sequences of the human beta1,3 galactosyltransferase (beta3GalT)-I, -II and III enzymes as query, we have identified an additional member of the beta3GalT gene family within a sequenced region of the human chromosome 21 as found in GenBank. The novel human beta3GalT-V gene included an open reading frame of 933 bp encoding a protein of 310 amino acids with a short N-terminal cytoplasmic tail, a single predicted transmembrane domain and a large lumenal catalytic domain. The human beta3GalT-V protein showed 34%, 27%, 31% and 23% sequence identity with the human beta3GalT-I, -II, -III and -IV enzymes, respectively. The expression of beta3GalT-V as a recombinant protein in Sf9 insect cells confirmed the galactosyltransferase activity catalyzed by this enzyme. Similarly to beta3GalT-I, -II and -III, the beta3GalT-V enzyme used beta-linked GlcNAc as an acceptor, but unlike the former enzymes beta3GalT-V exhibited a marked preference for the O-linked core3 GlcNAcbeta1,3GalNAc substrate. The beta3GalT-V gene was mainly expressed in human small intestine and to a lesser extent in pancreas and testis. Although beta3GalT-V transcripts were not detected in normal colon tissue, based on Northern analysis, beta3GalT-V mRNA was found in the adenocarcinoma cell line Colo 205.

A mutation in the human ortholog of the Saccharomyces cerevisiae ALG6 gene causes carbohydrate-deficient glycoprotein syndrome type-Ic.

Carbohydrate-deficient glycoprotein syndrome (CDGS) represents a class of genetic diseases characterized by abnormal N-linked glycosylation. CDGS patients show a large number of glycoprotein abnormalities resulting in dysmorphy, encephalopathy, and other organ disorders. The majority of CDGSs described to date are related to an impaired biosynthesis of dolichyl pyrophosphate-linked Glc3Man9GlcNAc2 in the endoplasmic reticulum. Recently, we identified in four related patients a novel type of CDGS characterized by an accumulation of dolichyl pyrophosphate-linked Man9GlcNAc2. Elaborating on the analogy of this finding with the phenotype of alg5 and alg6 Saccharomyces cerevisiae strains, we have cloned and analyzed the human orthologs to the ALG5 dolichyl phosphate glucosyltransferase and ALG6 dolichyl pyrophosphate Man9GlcNAc2 alpha1,3-glucosyltransferase in four novel CDGS patients. Although ALG5 was not altered in the patients, a C-->T transition was detected in ALG6 cDNA of all four CDGS patients. The mutation cosegregated with the disease in a Mendelian recessive manner. Expression of the human ALG5 and ALG6 cDNA could partially complement the respective S. cerevisiae alg5 and alg6 deficiency. By contrast, the mutant ALG6 cDNA of CDGS patients failed to revert the hypoglycosylation observed in alg6 yeasts, thereby proving a functional relationship between the alanine to valine substitution introduced by the C-->T transition and the CDGS phenotype. The mutation in the ALG6 alpha1,3-glucosyltransferase gene defines an additional type of CDGS, which we propose to refer to as CDGS type-Ic.

A beta-1,3-N-acetylglucosaminyltransferase with poly-N-acetyllactosamine synthase activity is structurally related to beta-1,3-galactosyltransferases.

Human and mouse cDNAs encoding a new beta-1, 3-N-acetylglucosaminyltransferase (beta3GnT) have been isolated from fetal and newborn brain libraries. The human and mouse cDNAs included ORFs coding for predicted type II transmembrane polypeptides of 329 and 325 aa, respectively. The human and mouse beta3GnT homologues shared 90% similarity. The beta3GnT gene was widely expressed in human and mouse tissues, although differences in the transcript levels were visible, thus indicating possible tissue-specific regulation mechanisms. The beta3GnT enzyme showed a marked preference for Gal(beta1-4)Glc(NAc)-based acceptors, whereas no activity was detected on type 1 Gal(beta1-3)GlcNAc and O-glycan core 1 Gal(beta1-3)GalNAc acceptors. The new beta3GnT enzyme was capable of both initiating and elongating poly-N-acetyllactosamine chains, which demonstrated its identity with the poly-N-acetyllactosamine synthase enzyme (E.C. 2.4.1.149), showed no similarity with the i antigen beta3GnT enzyme described recently, and, strikingly, included several amino acid motifs in its protein that have been recently identified in beta-1,3-galactosyltransferase enzymes. The comparison between the new UDP-GlcNAc:betaGal beta3GnT and the three UDP-Gal:betaGlcNAc beta-1,3-galactosyltransferases-I, -II, and -III reveals glycosyltransferases that share conserved sequence motifs though exhibiting inverted donor and acceptor specificities. This suggests that the conserved amino acid motifs likely represent residues required for the catalysis of the glycosidic (beta1-3) linkage.