Human Congenital Disorders Of Glycosylation Research Articles

Reduction in N-Acetylglucosaminyltransferase-I Activity Decreases Survivability and Delays Development of Zebrafish.

A lack of complex and hybrid types of N-glycans in mice is embryonically lethal due to neural tube maldevelopment. N-acetylglucosaminyltransferase-I (GnT-I; Mgat1) catalyzes a required step for converting oligomannose N-glycans into hybrid and complex N-glycans. Unlike mice, zebrafish have two Mgat1a/b genes. Herein, CRISPR/Cas9 technology was used to knockdown GnT-Ib activity in zebrafish, referred to as Mgat1b-/-, to examine the impact of a decrease in complex types of N-glycans on survival and development, and sensory and motor functions. Genotyping verified the occurrence of edited Mgat1b, and LC-ESI-MS and lectin blotting identified higher levels of oligomannose and lower levels of complex N-glycans in Mgat1b-/- relative to Wt AB. The microscopic visualization of developmental stages and locomotor studies using an automated tracking unit and manual touch assays revealed reduced survivability, and delayed motor and sensory functions in Mgat1b-/-. Moreover, embryonic staging linked reduced survivability of Mgat1b-/- to disruption in brain anlagen formation. Birefringence measurements supported delayed skeletal muscle development, which corresponded with motor and sensory function impediments in Mgat1b-/-. Furthermore, GnT-Ib knockdown hindered cardiac activity onset. Collectively, Mgat1b-/- displayed incomplete penetrance and variable expressivity, such that some died in early embryonic development, while others survived to adulthood, albeit, with developmental delays. Thus, the results reveal that reducing the amount of complex-type N-glycans is unfavorable for zebrafish survival and development. Moreover, our results support a better understanding of human congenital disorders of glycosylation.

Evolutionary rescue of phosphomannomutase deficiency in yeast models of human disease.

The most common cause of human congenital disorders of glycosylation (CDG) are mutations in the phosphomannomutase gene <i>PMM2,</i> which affect protein <i>N</i>-linked glycosylation. The yeast gene <i>SEC53</i> encodes a homolog of human <i>PMM2</i>. We evolved 384 populations of yeast harboring one of two human-disease-associated alleles, <i>sec53-</i>V238M and <i>sec53</i>-F126L, or wild-type <i>SEC53</i>. We find that after 1000 generations, most populations compensate for the slow-growth phenotype associated with the <i>sec53</i> human-disease-associated alleles. Through whole-genome sequencing we identify compensatory mutations, including known <i>SEC53</i> genetic interactors. We observe an enrichment of compensatory mutations in other genes whose human homologs are associated with Type 1 CDG, including <i>PGM1</i>, which encodes the minor isoform of phosphoglucomutase in yeast. By genetic reconstruction, we show that evolved <i>pgm1</i> mutations are dominant and allele-specific genetic interactors that restore both protein glycosylation and growth of yeast harboring the <i>sec53</i>-V238M allele. Finally, we characterize the enzymatic activity of purified Pgm1 mutant proteins. We find that reduction, but not elimination, of Pgm1 activity best compensates for the deleterious phenotypes associated with the <i>sec53</i>-V238M allele. Broadly, our results demonstrate the power of experimental evolution as a tool for identifying genes and pathways that compensate for human-disease-associated alleles.

Dissecting the molecular organization of the translocon-associated protein complex

In eukaryotic cells, one-third of all proteins must be transported across or inserted into the endoplasmic reticulum (ER) membrane by the ER protein translocon. The translocon-associated protein (TRAP) complex is an integral component of the translocon, assisting the Sec61 protein-conducting channel by regulating signal sequence and transmembrane helix insertion in a substrate-dependent manner. Here we use cryo-electron tomography (CET) to study the structure of the native translocon in evolutionarily divergent organisms and disease-linked TRAP mutant fibroblasts from human patients. The structural differences detected by subtomogram analysis form a basis for dissecting the molecular organization of the TRAP complex. We assign positions to the four TRAP subunits within the complex, providing insights into their individual functions. The revealed molecular architecture of a central translocon component advances our understanding of membrane protein biogenesis and sheds light on the role of TRAP in human congenital disorders of glycosylation.

Conserved Oligomeric Golgi and Neuronal Vesicular Trafficking.

The conserved oligomeric Golgi (COG) complex is an evolutionary conserved multi-subunit vesicle tethering complex essential for the majority of Golgi apparatus functions: protein and lipid glycosylation and protein sorting. COG is present in neuronal cells, but the repertoire of COG function in different Golgi-like compartments is an enigma. Defects in COG subunits cause alteration of Golgi morphology, protein trafficking, and glycosylation resulting in human congenital disorders of glycosylation (CDG) type II. In this review we summarize and critically analyze recent advances in the function of Golgi and Golgi-like compartments in neuronal cells and functions and dysfunctions of the COG complex and its partner proteins.

Immunological aspects of congenital disorders of glycosylation (CDG): a review.

Congenital disorders of glycosylation (CDG) are a rapidly growing family of genetic diseases comprising more than 85 known distinct disorders. They show a great phenotypic variability ranging from multi-organ/system to mono-organ/system involvement with very mild to extremely severe expression. Immunological dysfunction has a significant impact on the phenotype in a minority of CDG. CDG with major immunological involvement are ALG12-CDG, MAGT1-CDG, MOGS-CDG, SLC35C1-CDG and PGM3-CDG. This review discusses the variety of immunological abnormalities reported in human CDG. Understanding the immunological aspects of CDG may contribute to a better management/treatment of these pathologies and possibly of more common diseases, such as inflammatory diseases.

A mouse model of a human congenital disorder of glycosylation caused by loss of PMM2.

The most common congenital disorder of glycosylation (CDG), phosphomannomutase 2 (PMM2)-CDG, is caused by mutations in PMM2 that limit availability of mannose precursors required for protein N-glycosylation. The disorder has no therapy and there are no models to test new treatments. We generated compound heterozygous mice with the R137H and F115L mutations in Pmm2 that correspond to the most prevalent alleles found in patients with PMM2-CDG. Many Pmm2R137H/F115L mice died prenatally, while survivors had significantly stunted growth. These animals and cells derived from them showed protein glycosylation deficiencies similar to those found in patients with PMM2-CDG. Growth-related glycoproteins insulin-like growth factor (IGF) 1, IGF binding protein-3 and acid-labile subunit, along with antithrombin III, were all deficient in Pmm2R137H/F115L mice, but their levels in heterozygous mice were comparable to wild-type (WT) littermates. These imbalances, resulting from defective glycosylation, are likely the cause of the stunted growth seen both in our model and in PMM2-CDG patients. Both Pmm2R137H/F115L mouse and PMM2-CDG patient-derived fibroblasts displayed reductions in PMM activity, guanosine diphosphate mannose, lipid-linked oligosaccharide precursor and total cellular protein glycosylation, along with hypoglycosylation of a new endogenous biomarker, glycoprotein 130 (gp130). Over-expression of WT-PMM2 in patient-derived fibroblasts rescued all these defects, showing that restoration of mutant PMM2 activity is a viable therapeutic strategy. This functional mouse model of PMM2-CDG, in vitro assays and identification of the novel gp130 biomarker all shed light on the human disease, and moreover, provide the essential tools to test potential therapeutics for this untreatable disease.

Cardiomyocyte sialylation and complex N‐glycosylation protect against dilated cardiomyopathy and heart failure (697.2)

Human congenital disorders of glycosylation are characterized by variably reduced glycoprotein glycosylation resulting in multi‐system effects, including cardiac dysfunction. We seek to understand whether and how reduced glycosylation contributes to cardiac symptoms and reported previously that the reduced glycoprotein sialylation that results from the global gene deletion of a cardiac expressing sialyltransferase, ST3Gal4, contributed to pressure‐induced heart failure (FASEB J, 2013, 27:595.2). Here we questioned whether cardiomyocyte‐specific (αMHC‐Cre) deletion of mannosyl (alpha‐1,3‐)‐glycoprotein beta‐1,2‐N‐acetylglucosaminyltransferase, Mgat1, responsible for initiating synthesis of complex and hybrid N‐glycans, contributes to the onset of dilated cardiomyopathy. At age 16 weeks (earliest time point tested), homozygous floxed Mgat1 and αMHC‐Cre positive mice, Mgat1F/FαMHC‐Cre, showed significantly reduced ejection fraction (EF) and fractional shortening (FS) compared to littermate controls (in %, for controls (n=8), EF = 73.8 ± 2.1, FS = 42.5 ± 1.8; for Mgat1F/FαMHC‐Cre mice (n=7), EF = 61.4 ± 2.3; FS = 32.8 ± 1.6; p&lt;0.002). Systolic function of Mgat1F/FαMHC‐Cre mice, but not of control mice, continued to deteriorate with age (at 30 weeks, Mgat1F/FαMHC‐Cre mice (n=5) in %: EF = 38.2 ± 4.5; FS = 18.8 ± 2.5; p&lt;0.0005). Mgat1F/FαMHC‐Cre mice presented with apparent dilated cardiomyopathy indicated by reduced systolic function, a thinner wall and larger internal diameter of the left ventricle (p&lt;0.0005) with no apparent increase in left ventricular mass index, and marked right and left ventricular dilatation observed during necropsy. Together, our data suggest sialylation and complex N‐glycosylation are necessary for normal cardiac function, and reduced cardiomyocyte N‐glycosylation contributes to the onset of significant ventricular systolic dysfunction consistent with dilated cardiomyopathy leading to heart failure.Grant Funding Source: Supported by NSF grant # IOS‐1146882

N-Glycosylation Site Analysis of Proteins from Saccharomyces cerevisiae by Using Hydrophilic Interaction Liquid Chromatography-Based Enrichment, Parallel Deglycosylation, and Mass Spectrometry

N-Glycosylation site analysis of baker's yeast Saccharomyces cerevisiae is of fundamental significance to elucidate the molecular mechanism of human congenital disorders of glycosylation (CDG). Here we present a mass spectrometry (MS)-based workflow for the profiling of N-glycosylated sites in S. cerevisiae proteins. In this workflow, proteolytic glycopeptides were enriched by using a hydrophilic material named Click TE-Cys to improve the glycopeptide selectivity and coverage. To enhance the reliability of the identified results, the enriched glycopeptides were subjected to parallel deglycosylation by using two endoglycosidases (i.e., PNGase F and Endo Hf), respectively, prior to LC-MS/MS analysis. On the basis of the workflow, a total of 135 N-glycosylated sites including 6 known, 93 potential, and 36 novel sites were identified and mapped to 79 proteins. Among the novel-type sites, nine sites from eight proteins, which were simultaneously identified via PNGase F and Endo Hf deglycosylation, are believed to possess high confidence. The established workflow, together with the profile of N-glycosylated sites, will contribute to the improvement of S. cerevisiae model for revealing the pathogenesis of CDG.

The Sialyltransferase, ST3Gal4, Protects Against Pressure‐Induced Cardiac Hypertrophy

There are more than 40 types of human congenital disorders of glycosylation (CDG) characterized by a relatively modest but variable reduction in glycoprotein glycosylation that leads to dramatic multi‐system effects, including cardiac dysfunction. Nearly all CDG patients suffer from reduced protein sialylation. We sought to question whether and how reduced sialylation contributes to the observed cardiac symptoms that include dilated or hypertrophic cardiomyopathies. Trans‐aortic constriction (TAC) of a sialyltransferase knockout strain, ST3Gal4, that produces glycoproteins with fewer sialic acids attached was used to question whether ST3Gal4 expression protected against heart failure. Post‐TAC hypertrophy was observed in all mice, but survival and cardiac function of the post‐TAC ST3Gal4−/− mice were significantly compromised compared to wild type (WT) animals. Survival at one week post‐TAC was ~50% of WT (WT, 74%; ST3Gal4−/−, 38%), with ejection fraction (EF) and fraction shortening (FS) severely reduced (WT to ST3Gal4−/− in %: EF, 54 ± 2 to 29 ± 4; FS, 28 ± 1 to 14 ± 2), and ventricles and myocytes were significantly larger than WT. The calcineurin signaling pathway was upregulated in the ST3Gal4−/− ventricle. Together, the data suggest that ST3Gal4 gene deletion results in reduced protection against pressure‐induced hypertrophy and heart failure, likely through increased calcineurin activity.

The Drosophila Neurally Altered Carbohydrate Mutant Has a Defective Golgi GDP-fucose Transporter

Studying genetic disorders in model organisms can provide insights into heritable human diseases. The Drosophila neurally altered carbohydrate (nac) mutant is deficient for neural expression of the HRP epitope, which consists of N-glycans with core α1,3-linked fucose residues. Here, we show that a conserved serine residue in the Golgi GDP-fucose transporter (GFR) is substituted by leucine in nac(1) flies, which abolishes GDP-fucose transport in vivo and in vitro. This loss of function is due to a biochemical defect, not to destabilization or mistargeting of the mutant GFR protein. Mass spectrometry and HPLC analysis showed that nac(1) mutants lack not only core α1,3-linked, but also core α1,6-linked fucose residues on their N-glycans. Thus, the nac(1) Gfr mutation produces a previously unrecognized general defect in N-glycan core fucosylation. Transgenic expression of a wild-type Gfr gene restored the HRP epitope in neural tissues, directly demonstrating that the Gfr mutation is solely responsible for the neural HRP epitope deficiency in the nac(1) mutant. These results validate the Drosophila nac(1) mutant as a model for the human congenital disorder of glycosylation, CDG-IIc (also known as LAD-II), which is also the result of a GFR deficiency.

Neural and Synaptic Defects in slytherin, a Zebrafish Model for Human Congenital Disorders of Glycosylation

Congenital disorder of glycosylation type IIc (CDG IIc) is characterized by mental retardation, slowed growth and severe immunodeficiency, attributed to the lack of fucosylated glycoproteins. While impaired Notch signaling has been implicated in some aspects of CDG IIc pathogenesis, the molecular and cellular mechanisms remain poorly understood. We have identified a zebrafish mutant slytherin (srn), which harbors a missense point mutation in GDP-mannose 4,6 dehydratase (GMDS), the rate-limiting enzyme in protein fucosylation, including that of Notch. Here we report that some of the mechanisms underlying the neural phenotypes in srn and in CGD IIc are Notch-dependent, while others are Notch-independent. We show, for the first time in a vertebrate in vivo, that defects in protein fucosylation leads to defects in neuronal differentiation, maintenance, axon branching, and synapse formation. Srn is thus a useful and important vertebrate model for human CDG IIc that has provided new insights into the neural phenotypes that are hallmarks of the human disorder and has also highlighted the role of protein fucosylation in neural development.

Genetic complementation reveals a novel human congenital disorder of glycosylation of type II, due to inactivation of the Golgi CMP–sialic acid transporter

AbstractWe have identified a homozygous G>A substitution in the donor splice site of intron 6 (IVS6 + 1G>A) of the cytidine monophosphate (CMP)–sialic acid transporter gene of Lec2 cells as the mutation responsible for their asialo phenotype. These cells were used in complementation studies to test the activity of the 2 CMP–sialic acid transporter cDNA alleles of a patient devoid of sialyl-Lex expression on polymorphonuclear cells. No complementation was obtained with either of the 2 patient alleles, whereas full restoration of the sialylated phenotype was obtained in the Lec2 cells transfected with the corresponding human wild-type transcript. The inactivation of one patient allele by a double microdeletion inducing a premature stop codon at position 327 and a splice mutation of the other allele inducing a 130–base pair (bp) deletion and a premature stop codon at position 684 are proposed to be the causal defects of this disease. A 4-base insertion in intron 6 was found in the mother and is proposed to be responsible for the splice mutation. We conclude that this defect is a new type of congenital disorder of glycosylation (CDG) of type IIf affecting the transport of CMP–sialic acid into the Golgi apparatus.

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

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

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

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

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.

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.

Balancing N-linked glycosylation to avoid disease

Complete loss of N-glycosylation is lethal in both yeast and mammals. Substantial deficiencies in some rate-limiting biosynthetic steps cause human congenital disorders of glycosylation (CDG). Patients have a range of clinical problems including variable degrees of mental retardation, liver dysfunction, and intestinal disorders. Over 60 mutations in phosphomannomutase (encoded by PMM2) diminish activity and cause CDG-Ia. The severe mutation R141H in PMM2 is lethal when homozygous, but heterozygous in about 1/70 Northern Europeans. Another disorder, CDG-Ic, is caused by mutations in ALG6, an α1,3glucosyl transferase used for lipid-linked precursor synthesis, yet some function-compromising mutations occur at a high frequency in this gene also. Maintenance of seemingly deleterious mutations implies a selective advantage or positive heterosis. One possible explanation for this is that production of infective viruses such as hepatitis virus B and C, or others that rely heavily on host N-glycosylation, is substantially inhibited when only a tiny fraction of their coat proteins is misglycosylated. In contrast, this reduced glycosylation does not affect the host. Prevalent functional mutations in rate-limiting glycosylation steps could provide some resistance to viral infections, but the cost of this insurance is CDG. A balanced glycosylation level attempts to accommodate these competing agendas. By assessing the occurrence of a series of N-glycosylation-compromising alleles in multi-genic diseases, it may be possible to determine whether impaired glycosylation is a risk factor or a major determinant underlying their pathology.