Deficiency of the first mannosylation step in the N-glycosylation pathway causes congenital disorder of glycosylation type Ik

Defects of N-linked glycosylation represent diseases with multiple organ involvements that are classified as congenital disorders of glycosylation (CDG). In recent years, several CDG types have been attributed to defects of dolichol-linked oligosaccharide assembly in the endoplasmic reticulum. The profiling of [3H]mannose-labeled lipid-linked oligosaccharides was instrumental in identifying most of these glycosylation disorders. However, this method is poorly suited for the identification of short lipid-linked oligosaccharide biosynthesis defects. To adequately resolve deficiencies affecting the first steps of lipid-linked oligosaccharide formation, we have used a non-radioactive procedure employing the fluorescence detection of 2-aminobenzamide-coupled oligosaccharides after HPLC separation. By applying this method, we have detected the accumulation of dolichylpyrophosphate-GlcNAc2 in a previously untyped CDG patient. The accumulation pattern suggested a deficiency of the ALG1 beta1,4 mannosyltransferase, which adds the first mannose residue to lipid-linked oligosaccharides. This was supported by the finding that this CDG patient was compound heterozygous for three mutations in the ALG1 gene, leading to the amino acid substitutions S150R and D429E on one allele and S258L on the other. The detrimental effect of these mutations on ALG1 protein function was demonstrated in a complementation assay using alg1 Saccharomyces cerevisiae yeast mutants. The ALG1 mannosyltransferase defect described here represents a novel type of CDG, which should be referred to as CDG-Ik.     

Severe ALG8-CDG (CDG-Ih) associated with homozygosity for two novel missense mutations detected by exome sequencing of candidate genes

Posttranslationally glycosylated proteins are important in many biological processes in humans and Congenital disorders of glycosylation (CDGs) are associated with a broad range of phenotypes. Type I CDGs are a group of rare autosomal recessive conditions. To date 17 subtypes have been enzymatically and molecularly characterized. Impaired function of the enzyme dolichyl pyrophosphate Glc₁Man₉GlcNAc₂ alpha-1,3-glucosyltransferase encoded by the ALG8 gene, causes ALG8-CDG (CDG-Ih, OMIM #608104). This enzyme facilitates the transfer of a second glucose molecule to a growing lipid-linked oligosaccharide chain, a process that transpires in the endoplasmic reticulum (ER). We present a female patient of consanguineous parents, with pre- and postnatal growth retardation, dysmorphic features, significant developmental delay, visual impairment and an electrophoretic serum transferrin pattern indicative of a type I CDG. Type I CDG subgroup was determined by exome sequencing facilitated by homozygosity analysis. The patient was homozygous for two variants, nine nucleotides apart, in exon 8 of ALG8; c.799T > C [p.Ser267Pro] and c.808T > C [p.Phe270Leu]. Both missense mutations are predicted to affect a conserved region of an intraluminal ER loop of dolichyl pyrophosphate Glc₁Man₉GlcNAc₂ alpha-1,3-glucosyltransferase. To our knowledge, the current report describes the ninth published case of ALG8-CDG, contributing to the further delineation of this rare and variable disorder.     

Improved diagnostics lead to identification of three new patients with congenital disorder of glycosylation-Ip

Congenital disorders of glycosylation (CDG) comprise a clinically and biochemically heterogeneous group of monogenetic-inherited, multisystemic diseases that affect the biosynthesis of N- and/or O-glycans linked to glycoconjugates. Recently, we identified the first patient with a defect in the cytosolic-orientated GDP-mannose:Man(3-4) GlcNAc(2)-PP-dolichol alpha-1,2-mannosyltransferase (ALG11), who presented an accumulation of shortened dolichol-linked oligosaccharides leading to CDG-Ip (ALG11-CDG). Here we describe an improved metabolic labeling method that allowed the identification of three new CDG-Ip cases that were missed so far in routine diagnostics. Although all CDG-Ip patients carry different mutations in the ALG11 gene, they share a variety of clinical syndromes like an unremarkable prenatal period followed by developmental delay, psychomotor, and mental retardation, strabismus convergens and seizures occurring in the first year of life.     

Reduced heparan sulfate accumulation in enterocytes contributes to protein-losing enteropathy in a congenital disorder of glycosylation

Intestinal biopsy in a boy with gastroenteritis-induced protein-losing enteropathy (PLE) showed loss of heparan sulfate (HS) and syndecan-1 core protein from the basolateral surface of the enterocytes, which improved after PLE subsided. Isoelectric focusing analysis of serum transferrin indicated a congenital disorder of glycosylation (CDG) and subsequent analysis showed three point mutations in the ALG6 gene encoding an alpha1,3-glucosyltransferase needed for the addition of the first glucose to the dolichol-linked oligosaccharide. The maternal mutation, C998T, causing an A333V substitution, has been shown to cause CDG-Ic, whereas the two paternal mutations, T391C (Y131H) and C924A (S308R) have not previously been reported. The mutations were tested for their ability to rescue faulty N:-linked glycosylation of carboxypeptidase Y in an ALG6-deficient Saccharomyces cerevisiae strain. Normal human ALG6 rescues glycosylation and A333V partially rescues, whereas the combined paternal mutations (Y131H and S308R) are ineffective. Underglycosylation resulting from each of these mutations is much more severe in rapidly dividing yeast. Similarly, incomplete protein glycosylation in the patient is most severe in rapidly dividing enterocytes during gastroenteritis-induced stress. Incomplete N:-linked glycosylation of an HS core protein and/or other biosynthetic enzymes may explain the selective localized loss of HS and PLE.     

Congenital disorders of glycosylation: an update on defects affecting the biosynthesis of dolichol-linked oligosaccharides

Defects in the biosynthesis of the oligosaccharide precursor for N-glycosylation lead to decreased occupancy of glycosylation sites and thereby to diseases known as congenital disorders of glycosylation (CDG). In the last 20 years, approximately 1,000 CDG patients have been identified presenting with multiple organ dysfunctions. This review sets the state of the art by listing all mutations identified in the 15 genes (PMM2, MPI, DPAGT1, ALG1, ALG2, ALG3, ALG9, ALG12, ALG6, ALG8, DOLK, DPM1, DPM3, MPDU1, and RFT1) that yield a deficiency of dolichol-linked oligosaccharide biosynthesis. The present analysis shows that most mutations lead to substitutions of strongly conserved amino acid residues across eukaryotes. Furthermore, the comparison between the different forms of CDG affecting dolichol-linked oligosaccharide biosynthesis shows that the severity of the disease does not relate to the position of the mutated gene along this biosynthetic pathway. Hum Mutat 30:1–14, 2009. © 2009 Wiley-Liss, Inc.     

Novel variants and clinical symptoms in four new ALG3‐CDG patients,review of the literature,and identification of AAGRP‐ALG3 as a novel ALG3 variant with alanine and glycine‐rich N‐terminus

ALG3‐CDG is one of the very rare types of congenital disorder of glycosylation (CDG) caused by variants in the ER‐mannosyltransferase ALG3. Here, we summarize the clinical, biochemical, and genetic data of four new ALG3‐CDG patients, who were identified by a type I pattern of serum transferrin and the accumulation of Man₅GlcNAc₂‐PP‐dolichol in LLO analysis. Additional clinical symptoms observed in our patients comprise sensorineural hearing loss, right‐descending aorta, obstructive cardiomyopathy, macroglossia, and muscular hypertonia. We add four new biochemically confirmed variants to the list of ALG3‐CDG inducing variants: c.350G>C (p.R117P), c.1263G>A (p.W421*), c.1037A>G (p.N346S), and the intron variant c.296+4A>G. Furthermore, in Patient 1 an additional open‐reading frame of 141 bp (AAGRP) in the coding region of ALG3 was identified. Additionally, we show that control cells synthesize, to a minor degree, a hybrid protein composed of the polypeptide AAGRP and ALG3 (AAGRP‐ALG3), while in Patient 1 expression of this hybrid protein is significantly increased due to the homozygous variant c.160_196del (g.165C>T). By reviewing the literature and combining our findings with previously published data, we further expand the knowledge of this rare glycosylation defect.     

Stored dolichyl pyrophosphoryl oligosaccharides in Batten disease.

Each of the 3 childhood forms of Batten disease, juvenile (JB), late-infantile (LIB), and infantile (IB), have abnormally high brain concentrations of dolichyl pyrophosphoryl oligosaccharides (Dol-PP-OS). In this study, the carbohydrate portions of Dol-PP-OS were analysed: in JB and LIB, they range in size from Man2GlcNAc2 to Glc3Man9GlcNAc2, predominant components being Man5-7GlcNAc2 and Glc3Man7GlcNAc2. In IB, they range from Man6-9GlcNAc2, no glucose containing oligosaccharides being identified. In Batten disease, the main subcellular location of Dol-PP-OS is within storage material, where it represents up to 7% of the dry weight. [3H]-Mannose incorporation experiments with cultured fibroblasts show that synthesis of Dol-PP-OS in JB is normal. We infer that the glycosylation intermediate Glc3Man9GlcNAc2-PP-dolichol is synthesised normally within the endoplasmic reticulum in Batten disease, but that catabolic derivatives accumulate within the lysosomes. It is unclear whether this process is central to the pathogenesis of the disease, though in IB a defect in the release of mannose residues from Dol-PP-OS is a distinct possibility.     

Evidence for processing of dolichol-linked oligosaccharides in patients with neuronal ceroid-lipofuscinosis.

In agreement with reports from other laboratories, we have shown that patients with the juvenile or late infantile forms of neuronal ceroid-lipofuscinosis (NCL) have greatly increased levels (5-fold to 20-fold) of dolichyl pyrophosphoryl oligosaccharides in their cerebral gray matter. Oligosaccharides containing 2 GlcNAc residues and 3 to 9 mannose residues were liberated by mild acid hydrolysis. The oligosaccharide profile given by brain tissue from 2 patients with infantile NCL was markedly different from that of late infantile and juvenile NCL brain, with Man9GlcNAc2 as the most abundant component and decreasing amounts of Man8- Man7- and Man6GlcNAc2. By contrast, Man5GlcNAc2 was the most abundant oligosaccharide present in all juvenile NCL brain samples analyzed. Both the susceptibility of the isolated Man5GlcNAc2 to endoglucosaminidase H digestion and permethylation analysis clearly indicated that it is not an intermediate in the biosynthesis of Glc3Man9GlcNAc2-PP-dolichol but has undergone catabolism, probably either in the endoplasmic reticulum or in the Golgi apparatus. Treatment of cultured skin fibroblasts for 7 days with N-methyldeoxynojirimycin, a potent inhibitor of the endoplasmic reticulum processing enzymes glucosidase I and II, resulted in an accumulation of the same Man5GlcNAc2-PP-dolichol species that was elevated in juvenile NCL brain. The level in untreated fibroblasts was undetectable, suggesting that inhibition of processing glucosidases has interfered with the regulation and compartmentalization of lipid-linked oligosaccharides.     

Characterization of protein-linked oligosaccharides in trypanosomatid flagellates

Protein-linked, endo-beta-N-acetylglucosaminidase H-sensitive oligosaccharides were isolated from several trypanosomatids incubated with [U-14C]glucose. Structural analysis of the compounds revealed that Man9GlcNAc2 was the oligosaccharide transferred from dolichol-P-P derivatives to proteins in Trypanosoma dionisii, Trypanosoma conorhini, Leptomonas samueli and Herpetomonas samuelpessoai and Man6GlcNAc2 in Blastocrithidia culicis and Leishmania adleri. In all cases, transiently glucosylated compounds were detected: Glc1Man7-9GlcNAc2 in T. dionisii, T. conorhini, L. samueli; Glc1Man9GlcNAc2 in H. samuelpessoai, Glc1Man6GlcNAc2 in B. culicis and Glc1Man6GlcNAc2 and Glc1Man5GlcNAc2 in L. adleri. The mechanism of protein glycosylation in T. dionisii and T. conorhini appeared to be similar to that described before for Trypanosoma cruzi epimastigotes, although some differences were found between the structures of the main isomers of Man7GlcNAc2 and Man8GlcNAc2 present in T. conorhini and T. cruzi. Differences between the mechanisms of glycosylation occurring in Leishmania mexicana and L. adleri were also found: Man6GlcNAc2 in the latter microorganism was demannosylated to Man5GlcNAc2, a step not detected in the former parasite. A novel substituent in N-linked high mannose-type oligosaccharides was found in L. samueli and H. samuelpessoai: galactose in the furanose configuration. In the latter trypanosomatid, Man9GlcNAc2 was demannosylated only to Man8GlcNAc2, whereas in all other parasites in which the same oligosaccharide was transferred to proteins, Man5-7GlcNAc2 were also detected.     

Analysis of multiple mutations in the hALG6 gene in a patient with congenital disorder of glycosylation Ic

Congenital disorder of glycosylation Ic is caused by mutations in the hALG6 gene that encodes an alpha-1,3 glucosyltransferase. This enzyme is required for the addition of the first glucose residue to the lipid-linked oligosaccharide precursor for N-linked glycosylation. Here we describe the biochemical and molecular analysis of a patient with three mutations in the hALG6 gene. The maternal allele has an intronic G --> A mutation resulting in skipping of exon3 (IVS3 + 5G > A). This produces a nonfunctional enzyme as shown by its inability to restore normal glycosylation in a Saccharomyces cerevisiae strain lacking a functional ALG6. The paternal allele has two mutations. One is a deletion of three bases (895-897delATA) leading to an in-frame deletion of isoleucine 299 (delI299) located in a transmembrane domain. The second mutation on the same allele 911T > C causes a F304S change. When expressed in the ALG6 deficient yeast strain, this allele restores glycosylation but the mRNA is unstable or inefficiently transcribed, contributing to the impaired glycosylation in the patient.     

Expanding spectrum of congenital disorder of glycosylation Ig (CDG-Ig): sibs with a unique skeletal dysplasia, hypogammaglobulinemia, cardiomyopathy, genital malformations, and early lethality

In this report, we describe a brother and sister who presented at birth with short-limb skeletal dysplasia, polyhydramnios, prematurity, and generalized edema. Dysmorphic features included broad nose, thick ears, thin lips, micrognathia, inverted nipples, ulnar deviation at the wrists, spatulate fingers, fifth finger camptodactyly, nail hypoplasia, and talipes equinovarus. Other features included short stature, microcephaly, psychomotor retardation, B-cell lymphopenic hypogammaglobulinemia, sensorineural deafness, retinal detachment and blindness, intestinal malrotation with poor gastrointestinal motility, persistent hyponatremia, intermittent hypoglycemia, and thrombocytopenia. Cardiac anomalies included PDA, VSD, hypertrophic cardiomyopathy, and arrhythmias. The brother had a small penis with hypospadias, hypoplastic scrotum, and non-palpable testes. Skeletal findings included absent ossification of cervical vertebral bodies, pubic bones, knee epiphyses, and tali. Both sibs died before age 2 years, one of overwhelming sepsis and the other of cardiorespiratory failure associated with her cardiomyopathy. Metabolic studies showed a type 1 pattern of abnormal serum transferrin glycosylation. Fibroblasts synthesized truncated LLOs, primarily Man(7)GlcNAc(2), suggestive of CDG-Ig. Both sibs were compound heterozygotes for a novel 301 G > A (G101R) mutation and a previously described 437 G > A (R146Q) mutation in ALG12. Congenital disorders of glycosylation should be considered for children with undiagnosed multi-system disease including neurodevelopmental delay, skeletal dysplasia, immune deficiency, male genital hypoplasia, and cardiomyopathy.     

Identification of a frequent variant in ALG6, the cause of Congenital Disorder of Glycosylation-Ic

Congenital Disorder of Glycosylation (CDG) type Ic is caused by mutations in ALG6. This gene encodes an alpha1,3 glucosyltransferase used for synthesis of the lipid linked oligosaccharide (LLO) precursor of the protein N-glycosylation pathway. CDG-Ic patients have moderate to severe psychomotor retardation, seizures, hypotonia, strabismus, and feeding difficulties. We previously identified a typical patient with a heterozygous point mutation, c.391T>C (p.Tyr131His) in ALG6. Using complementation analysis of ALG6-deficient yeast, we show that this alteration is as severe as the most common disease-causing mutation, c998C>T (p. Ala333Val), which occurs in over half of all known CDG-Ic patients. The frequency of c.391T>C (p.Tyr131His) in the US population, is 0.0214, suggesting that homozygotes would occur at a rate of& tilde;1:2,200. We identified one patient with typical CDG-Ic symptoms and a homozygous p.Tyr131His alteration in ALG6. However, in contrast to most CDG patients, her LLO and plasma transferrin glycosylation appeared normal. Thus, it is unclear whether c.391T>C causes CDG-Ic or contributes to the symptoms. Genotyping additional patients with CDG-like symptoms will be required to resolve this issue.     

CDG-Id caused by homozygosity for an ALG3 mutation due to segmental maternal isodisomy UPD3(q21.3-qter)

We report on a patient with a congenital disorder of glycosylation type Id (CDG-Id) caused by a homozygous mutation in the ALG3 gene, which results from a de novo mutation in combination with a segmental maternal uniparental isodisomy (UPD). The patient presented with severe psychomotor delay, primary microcephaly, and opticus atrophy, compatible with a severe form of CDG. Isoelectric focusing of transferrin showed a type I pattern and lipid-linked oligosaccharide analysis showed an accumulation of dol-PP-GlcNAc2Man5 in patient's fibroblasts suggesting a defect in the ALG3 gene. A homozygous ALG3 missense mutation p.R266C (c.796C > T) was identified. Further evaluation revealed that neither the mother nor the father were carrier of the p.R266C mutation. Marker analysis revealed a segmental maternal isodisomy for the chromosomal region 3q21.3-3qter. UPD for this region has not been described before. More important, the combination of UPD with a de novo mutation is an exceptional coincidence and an extraordinary observation.     

Molecular and clinical description of the first US patients with congenital disorder of glycosylation Ig

In this report we describe the first two US patients with congenital disorder of glycosylation type Ig (CDG-Ig). Both patients presented with symptoms indicating CDG, including developmental delay, hypotonia and failure to thrive, and tested positive for deficient glycosylation of transferrin. Labeling of the patients' lipid-linked oligosaccharides suggested mutations in the hALG12 gene, encoding a mannosyltransferase. Both patients were shown to carry previously unpublished hALG12-mutations. Patient 1 has one allele with a deletion of G29, resulting in a premature stop codon, and another allele with an 824G>A mutation yielding an S275N amino acid change. Patient 2 carries two heterozygous mutations (688T>G and 931C>T), resulting in two amino acid exchanges, Y230D and R311C. An adenoviral vector expressing wild type hALG12 corrects the abnormal lipid-linked oligosaccharide pattern of the patients' cells. In addition to common CDG symptoms, these patients also presented with low IgG and genital hypoplasia, symptoms previously described in CDG-Ig patients. We therefore conclude that a combination of developmental delay, low IgG, and genital hypoplasia should prompt CDG testing.     

The oligosaccharides of influenza virus hemagglutinin expressed in insect cells by a baculovirus vector

The hemagglutinin of fowl plague virus has been expressed in Spodoptera frugiperda (SF) cell cultures using a baculovirus vector. To elucidate the structure of the carbohydrate side chains, radioactively labeled oligosaccharides were liberated by treatment with endoglucosaminidase H and glycopeptidase F. Sequential degradation with exoglycosidases and chromatographic analyses revealed the presence of oligomannosidic side chains, predominantly of the structures Man5-9GlcNAc2, and the truncated oligosaccharide cores Man3GlcNAc2 and Man3[Fuc]GlcNAc2. Polyacrylamide gel electrophoresis of endoglycosidase-treated hemagglutinin showed that most side chains of the HA1 subunit are truncated, whereas the HA2 subunit has one oligomannosidic and one truncated oligosaccharide. Comparison of these results with the glycosylation pattern of hemagglutinin obtained from vertebrate cells allowed a tentative allocation of the oligosaccharides to individual glycosylation sites. The results indicate that SF cells have the capacity to trim N-glycans to trimannosyl cores and to further process these by the addition of fucose. Thus, the complex oligosaccharides found on hemagglutinin from vertebrate hosts are replaced on hemagglutinin derived from insect cells by small truncated side chains.     

ALG11‐CDG syndrome: Expanding the phenotype

ALG11‐Congenital Disorder of Glycosylation (ALG11‐CDG, also known as congenital disorder of glycosylation type Ip) is an inherited inborn error of metabolism due to abnormal protein and lipid glycosylation. We describe two unrelated patients with ALG11‐CDG due to novel mutations, review the literature of previously described affected individuals, and further expand the clinical phenotype. Both affected individuals reported here had severe psychomotor disabilities and epilepsy. Their fibroblasts synthesized truncated precursor glycan structures, consistent with ALG11‐CDG, while also showing hypoglycosylation of a novel biomarker, GP130. Surprisingly, one patient presented with normal transferrin glycosylation profile, a feature that has not been reported previously in patients with ALG11‐CDG. Together, our data expand the clinical and mutational spectrum of ALG11‐CDG.     

Structural analysis of the asparagine-linked glycans from the procyclic Trypanosoma brucei and its glycosylation mutants resistant to Concanavalin A killing

The variant surface glycoprotein of the bloodstream form of Trypanosoma brucei is known to be glycosylated with a range of structures including high mannose and complex types. In contrast, glycosylation in the procyclic form of the parasite appears to be restricted to a single Man(5)GlcNAc(2) structure, as found on its procyclin. To gain a better insight into the developmentally regulated glycosylation pattern, we have structurally defined the full range of N-linked glycans made by the procyclic trypanosomes, as well as two previously described glycosylation mutants generated under Con A selection. It was found that the wild type procyclic cells could synthesize a full range of high mannose type structures from Man(5)GlcNAc(2) to Man(9)GlcNAc(2), with Man(5)GlcNAc(2) as the major component. In contrast, the two mutants mainly synthesized a truncated Man(4)GlcNAc(2) structure, Man alpha 1-3Man alpha 1-6(Man alpha1-3)Man be ta 1-4 GlcNAc beta 1-4GlcNAc, a significant portion of which was further extended by a single GlcNAc to form GlcNAc-Man(4)GlcNAc(2) and a single N-acetyllactosamine unit at the 3-arm position to form Gal beta 1-4GlcNAc beta 1-2Man alpha 1-3(Man al pha 1- 3Man alpha 1-6)Man beta 1-4G lcNAc beta 1-4GlcNAc. The results suggest that the procyclic trypanosomes could be induced by Con A selection to synthesize limited hybrid type structures, but in general do not further process their N-linked glycans into multiantennary complex types as the blood stream forms do.     

Structural characterization of murine Ia antigen N-linked oligosaccharides and localization of specific structures to two unique alpha-chain glycosylation sites

The sequence of N-linked oligosaccharides of differentially glycosylated murine I-Ak alpha-(alpha 2- and alpha 3-) and beta-chains was determined. I-Ak beta-chains predominantly bear a biantennary complex oligosaccharide with a core fucose, and with the peripheral sequence SA----Gal----GlcNAc----Man. The I-Ak alpha-chain has two N-linked glycosylation sites at Asn-82 and Asn-122. When Lubrol-insoluble alpha 3-chains are examined they are found to bear high-mannose oligosaccharides of either the Man9GlcNAc2 or Man8GlcNAc2 type at both sites. When Lubrol-soluble alpha 2-chains are examined, in about 85% of the molecules the Asn-82 site bears a biantennary complex oligosaccharide with core fucose, and with the peripheral sequence SA----Gal----GlcNAc----Man. Interestingly, the Asn-122 site bears a variety of structures. In about 50% of the molecules, the structure at Asn-122 is a biantennary complex oligosaccharide without core fucose and with the peripheral sequence SA----Gal----GlcNAc----Man. In addition, it can bear other complex structures which we did not define further. The apparently restricted addition of fucose to the oligosaccharide at the alpha-Asn-82 site, even when both alpha-sites bear biantennary complex structures with the same peripheral sequence, is a feature unique to this system. The unusual variety of structures present at the alpha-Asn-122 site may indicate differential processing in different cell types.     

CDG-Id in two siblings with partially different phenotypes

We present two sibs with congenital disorder of glycosylation (CDG) type Id. Each shows severe global delay, failure to thrive, seizures, microcephaly, axial hypotonia, and disaccharidase deficiency. One sib has more severe digestive issues, while the other is more neurologically impaired. Each is compound heterozygous for a novel point mutation and an already known mutation in the ALG3 gene that leads to the synthesis of a severely truncated oligosaccharide precursor for N-glycans. The defect is corrected by introduction of a normal ALG3 cDNA. CDG should be ruled out in all patients with severe seizures and failure to thrive. (c) 2007 Wiley-Liss, Inc.