Sweet solution: sugars to the rescue

Sugar pills are usually placebos, but Smith et al. (2002, this issue) use one to rescue designer mice unable to make GDP-Fucose. Dietary fucose enters a salvage pathway and spares the mice. Sound simple? Not so. Unknown genetic factors determine life or death

Molecular basis of carbohydrate-deficient glycoprotein syndromes type I with normal phosphomannomutase activity

AbstractCarbohydrate deficient glycoprotein syndromes (CDGS) are inherited disorders in glycosylation. Isoelectric focusing of serum transferrin is used as a biochemical indicator of CDGS; however, this technique cannot diagnose the molecular defect. Even though phosphomannomutase (PMM) deficiency accounts for the great majority of known CDGS cases (CDGS type Ia), newly discovered cases have significantly different clinical presentations than the PMM-deficient patients. These differences arise from other defects affecting the biosynthesis of N-linked oligosaccharides in the endoplasmic reticulum and in the Golgi compartment. The most notable is the loss of phosphomannose isomerase (PMI) (CDGS type Ib). It causes severe hypoglycemia, protein-losing enteropathy, vomiting, diarrhea, and congenital hepatic fibrosis. In contrast to PMM-deficiency, there is no developmental delay nor neuropathy. Most symptoms in the PMI-deficient patients can be successfully treated with dietary mannose supplements. Another defect is the lack of glucosylation of the lipid-linked oligosaccharide precursor. The clinical features of this form of CDGS are milder, but similar to, PMM-deficient patients. Yeast genetic and biochemical techniques were critical in unraveling these disorders since many of the defective genes were known in yeast and corresponding mutants were available for complementation. Yeast strains carrying mutations in the homologous genes are likely to provide conclusive identification of the primary defects in novel CDGS types that affect the synthesis and transfer of precursor oligosaccharides

Insufficient ER-stress response causes selective mouse cerebellar granule cell degeneration resembling that seen in congenital disorders of glycosylation

BACKGROUND: Congenital disorders of glycosylation (CDGs) are inherited diseases caused by glycosylation defects. Incorrectly glycosylated proteins induce protein misfolding and endoplasmic reticulum (ER) stress. The most common form of CDG, PMM2-CDG, is caused by deficiency in the cytosolic enzyme phosphomannomutase 2 (PMM2). Patients with PMM2-CDG exhibit a significantly reduced number of cerebellar Purkinje cells and granule cells. The molecular mechanism underlying the specific cerebellar neurodegeneration in PMM2-CDG, however, remains elusive. RESULTS: Herein, we report that cerebellar granule cells (CGCs) are more sensitive to tunicamycin (TM)-induced inhibition of total N-glycan synthesis than cortical neurons (CNs). When glycan synthesis was inhibited to a comparable degree, CGCs exhibited more cell death than CNs. Furthermore, downregulation of PMM2 caused more CGCs to die than CNs. Importantly, we found that upon PMM2 downregulation or TM treatment, ER-stress response proteins were elevated less significantly in CGCs than in CNs, with the GRP78/BiP level showing the most significant difference. We further demonstrate that overexpression of GRP78/BiP rescues the death of CGCs resulting from either TM-treatment or PMM2 downregulation. CONCLUSIONS: Our results indicate that the selective susceptibility of cerebellar neurons to N-glycosylation defects is due to these neurons’ inefficient response to ER stress, providing important insight into the mechanisms of selective neurodegeneration observed in CDG patients

Pathogenic Variants in Fucokinase Cause a Congenital Disorder of Glycosylation

FUK encodes fucokinase, the only enzyme capable of converting L-fucose to fucose-1-phosphate, which will ultimately be used for synthesizing GDP-fucose, the donor substrate for all fucosyltransferases. Although it is essential for fucose salvage, this pathway is thought to make only a minor contribution to the total amount of GDP-fucose. A second pathway, the major de novo pathway, involves conversion of GDP-mannose to GDP-fucose. Here we describe two unrelated individuals who have pathogenic variants in FUK and who presented with severe developmental delays, encephalopathy, intractable seizures, and hypotonia. The first individual was compound heterozygous for c.667T>C (p.Ser223Pro) and c.2047C>T (p.Arg683Cys), and the second individual was homozygous for c.2980A>C (p.Lys994Gln). Skin fibroblasts from the first individual confirmed the variants as loss of function and showed significant decreases in total GDP-[3H] fucose and [3H] fucose-1-phosphate. There was also a decrease in the incorporation of [5,6-3H]-fucose into fucosylated glycoproteins. Lys994 has previously been shown to be an important site for ubiquitin conjugation. Here, we show that loss-of-function variants in FUK cause a congenital glycosylation disorder characterized by a defective fucose-salvage pathway

Selective inhibition of N-linked glycosylation impairs receptor tyrosine kinase processing

Global inhibition of N-linked glycosylation broadly reduces glycan occupancy on glycoproteins, but identifying how this inhibition functionally impacts specific glycoproteins is challenging. This limits our understanding of pathogenesis in the congenital disorders of glycosylation (CDG). We used selective exo-enzymatic labeling of cells deficient in the two catalytic subunits of oligosaccharyltransferase - STT3A and STT3B - to monitor the presence and glycosylation status of cell surface glycoproteins. We show reduced abundance of two canonical tyrosine receptor kinases - the insulin receptor and insulin-like growth factor 1 receptor (IGF-1R) - at the cell surface in STT3A-null cells, due to decreased N-linked glycan site occupancy and proteolytic processing in combination with increased endoplasmic reticulum localization. Providing cDNA for Golgi-resident proprotein convertase subtilisin/kexin type 5a (PCSK5a) and furin cDNA to wild-type and mutant cells produced under-glycosylated forms of PCSK5a, but not furin, in cells lacking STT3A. Reduced glycosylation of PCSK5a in STT3A-null cells or cells treated with the oligosaccharyltransferase inhibitor NGI-1 corresponded with failure to rescue receptor processing, implying that alterations in the glycosylation of this convertase have functional consequences. Collectively, our findings show that STT3A-dependent inhibition of N-linked glycosylation on receptor tyrosine kinases and their convertases combines to impair receptor processing and surface localization. These results provide new insight into CDG pathogenesis and highlight how the surface abundance of some glycoproteins can be dually impacted by abnormal glycosylation

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

Mosaicism of the UDP-Galactose Transporter SLC35A2 Causes a Congenital Disorder of Glycosylation

Biochemical analysis and whole-exome sequencing identified mutations in the Golgi-localized UDP-galactose transporter SLC35A2 that define an undiagnosed X-linked congenital disorder of glycosylation (CDG) in three unrelated families. Each mutation reduced UDP-galactose transport, leading to galactose-deficient glycoproteins. Two affected males were somatic mosaics, suggesting that a wild-type SLC35A2 allele may be required for survival. In infancy, the commonly used biomarker transferrin showed abnormal glycosylation, but its appearance became normal later in childhood, without any corresponding clinical improvement. This may indicate selection against cells carrying the mutant allele. To detect other individuals with such mutations, we suggest transferrin testing in infancy. Here, we report somatic mosaicism in CDG, and our work stresses the importance of combining both genetic and biochemical diagnoses

Symbol Nomenclature for Graphical Representations of Glycans

ISSN:0959-665