Sugary Logistics Gone Wrong:Membrane Trafficking and Congenital Disorders of Glycosylation

Contains fulltext : 225799.pdf (publisher's version ) (Open Access)Glycosylation is an important post-translational modification for both intracellular and secreted proteins. For glycosylation to occur, cargo must be transported after synthesis through the different compartments of the Golgi apparatus where distinct monosaccharides are sequentially bound and trimmed, resulting in increasingly complex branched glycan structures. Of utmost importance for this process is the intraorganellar environment of the Golgi. Each Golgi compartment has a distinct pH, which is maintained by the vacuolar H(+)-ATPase (V-ATPase). Moreover, tethering factors such as Golgins and the conserved oligomeric Golgi (COG) complex, in concert with coatomer (COPI) and soluble N-ethylmaleimide-sensitive factor attachment protein receptor (SNARE)-mediated membrane fusion, efficiently deliver glycosylation enzymes to the right Golgi compartment. Together, these factors maintain intra-Golgi trafficking of proteins involved in glycosylation and thereby enable proper glycosylation. However, pathogenic mutations in these factors can cause defective glycosylation and lead to diseases with a wide variety of symptoms such as liver dysfunction and skin and bone disorders. Collectively, this group of disorders is known as congenital disorders of glycosylation (CDG). Recent technological advances have enabled the robust identification of novel CDGs related to membrane trafficking components. In this review, we highlight differences and similarities between membrane trafficking-related CDGs

Weak coupling between magnetically inequivalent spins: the deceptively simple, complicated spectrum of a C-13-labeled trimethylated amine

Microbial Biotechnolog

Weak coupling between magnetically inequivalent spins: the deceptively simple, complicated spectrum of a C-13-labeled trimethylated amine

Microbial Biotechnolog

Transgenic Rescue of the LARGEmyd Mouse: A LARGE Therapeutic Window?

LARGE is a glycosyltransferase involved in glycosylation of α-dystroglycan (α-DG). Absence of this protein in the LARGEmyd mouse results in α-DG hypoglycosylation, and is associated with central nervous system abnormalities and progressive muscular dystrophy. Up-regulation of LARGE has previously been proposed as a therapy for the secondary dystroglycanopathies: overexpression in cells compensates for defects in multiple dystroglycanopathy genes. Counterintuitively, LARGE overexpression in an FKRP-deficient mouse exacerbates pathology, suggesting that modulation of α-DG glycosylation requires further investigation. Here we demonstrate that transgenic expression of human LARGE (LARGE-LV5) in the LARGEmyd mouse restores α-DG glycosylation (with marked hyperglycosylation in muscle) and that this corrects both the muscle pathology and brain architecture. By quantitative analyses of LARGE transcripts we also here show that levels of transgenic and endogenous LARGE in the brains of transgenic animals are comparable, but that the transgene is markedly overexpressed in heart and particularly skeletal muscle (20–100 fold over endogenous). Our data suggest LARGE overexpression may only be deleterious under a forced regenerative context, such as that resulting from a reduction in FKRP: in the absence of such a defect we show that systemic expression of LARGE can indeed act therapeutically, and that even dramatic LARGE overexpression is well-tolerated in heart and skeletal muscle. Moreover, correction of LARGEmyd brain pathology with only moderate, near-physiological LARGE expression suggests a generous therapeutic window

Prenatal muscle development in a mouse model for the secondary dystroglycanopathies

The defective glycosylation of α-dystroglycan is associated with a group of muscular dystrophies that are collectively referred to as the secondary dystroglycanopathies. Mutations in the gene encoding fukutin-related protein (FKRP) are one of the most common causes of secondary dystroglycanopathy in the UK and are associated with a wide spectrum of disease. Whilst central nervous system involvement has a prenatal onset, no studies have addressed prenatal muscle development in any of the mouse models for this group of diseases. In view of the pivotal role of α-dystroglycan in early basement membrane formation, we sought to determine if the muscle formation was altered in a mouse model of FKRP-related dystrophy

Mutations in <i>N</i>-acetylglucosamine (<i>O</i>-GlcNAc) transferase in patients with X-linked intellectual disability

Contains fulltext : 177227.pdf (publisher's version ) (Open Access)N-Acetylglucosamine (O-GlcNAc) transferase (OGT) regulates protein O-GlcNAcylation, an essential and dynamic post-translational modification. The O-GlcNAc modification is present on numerous nuclear and cytosolic proteins and has been implicated in essential cellular functions such as signaling and gene expression. Accordingly, altered levels of protein O-GlcNAcylation have been associated with developmental defects and neurodegeneration. However, mutations in the OGT gene have not yet been functionally confirmed in humans. Here, we report on two hemizygous mutations in OGT in individuals with X-linked intellectual disability (XLID) and dysmorphic features: one missense mutation (p.Arg284Pro) and one mutation leading to a splicing defect (c.463-6T>G). Both mutations reside in the tetratricopeptide repeats of OGT that are essential for substrate recognition. We observed slightly reduced levels of OGT protein and reduced levels of its opposing enzyme O-GlcNAcase in both patient-derived fibroblasts, but global O-GlcNAc levels appeared to be unaffected. Our data suggest that mutant cells attempt to maintain global O-GlcNAcylation by down-regulating O-GlcNAcase expression. We also found that the c.463-6T>G mutation leads to aberrant mRNA splicing, but no stable truncated protein was detected in the corresponding patient-derived fibroblasts. Recombinant OGT bearing the p.Arg284Pro mutation was prone to unfolding and exhibited reduced glycosylation activity against a complex array of glycosylation substrates and proteolytic processing of the transcription factor host cell factor 1, which is also encoded by an XLID-associated gene. We conclude that defects in O-GlcNAc homeostasis and host cell factor 1 proteolysis may play roles in mediation of XLID in individuals with OGT mutations

B3GALNT2 mutations associated with non-syndromic autosomal recessive intellectual disability reveal a lack of genotype-phenotype associations in the muscular dystrophy-dystroglycanopathies.

BACKGROUND: The phenotypic severity of congenital muscular dystrophy-dystroglycanopathy (MDDG) syndromes associated with aberrant glycosylation of α-dystroglycan ranges from the severe Walker-Warburg syndrome or muscle-eye-brain disease to mild, late-onset, isolated limb-girdle muscular dystrophy without neural involvement. However, muscular dystrophy is invariably found across the spectrum of MDDG patients. METHODS: Using linkage mapping and whole-exome sequencing in two families with an unexplained neurodevelopmental disorder, we have identified homozygous and compound heterozygous mutations in B3GALNT2. RESULTS: The first family comprises two brothers of Dutch non-consanguineous parents presenting with mild ID and behavioral problems. Immunohistochemical analysis of muscle biopsy revealed no significant aberrations, in line with the absence of a muscular phenotype in the affected siblings. The second family includes five affected individuals from an Iranian consanguineous kindred with mild-to-moderate intellectual disability (ID) and epilepsy without any notable neuroimaging, muscle, or eye abnormalities. Complementation assays of the compound heterozygous mutations identified in the two brothers had a comparable effect on the O-glycosylation of α-dystroglycan as previously reported mutations that are associated with severe muscular phenotypes. CONCLUSIONS: In conclusion, we show that mutations in B3GALNT2 can give rise to a novel MDDG syndrome presentation, characterized by ID associated variably with seizure, but without any apparent muscular involvement. Importantly, B3GALNT2 activity does not fully correlate with the severity of the phenotype as assessed by the complementation assay

The reductive activation of CO2 across a Ti═Ti double bond: synthetic, structural, and mechanistic studies

[Image: see text] The reactivity of the bis(pentalene)dititanium double-sandwich compound Ti(2)Pn(†)(2) (1) (Pn(†) = 1,4-{Si(i)Pr(3)}(2)C(8)H(4)) with CO(2) is investigated in detail using spectroscopic, X-ray crystallographic, and computational studies. When the CO(2) reaction is performed at −78 °C, the 1:1 adduct 4 is formed, and low-temperature spectroscopic measurements are consistent with a CO(2) molecule bound symmetrically to the two Ti centers in a μ:η(2),η(2) binding mode, a structure also indicated by theory. Upon warming to room temperature the coordinated CO(2) is quantitatively reduced over a period of minutes to give the bis(oxo)-bridged dimer 2 and the dicarbonyl complex 3. In situ NMR studies indicated that this decomposition proceeds in a stepwise process via monooxo (5) and monocarbonyl (7) double-sandwich complexes, which have been independently synthesized and structurally characterized. 5 is thermally unstable with respect to a μ-O dimer in which the Ti–Ti bond has been cleaved and one pentalene ligand binds in an η(8) fashion to each of the formally Ti(III) centers. The molecular structure of 7 shows a “side-on” bound carbonyl ligand. Bonding of the double-sandwich species Ti(2)Pn(2) (Pn = C(8)H(6)) to other fragments has been investigated by density functional theory calculations and fragment analysis, providing insight into the CO(2) reaction pathway consistent with the experimentally observed intermediates. A key step in the proposed mechanism is disproportionation of a mono(oxo) di-Ti(III) species to yield di-Ti(II) and di-Ti(IV) products. 1 forms a structurally characterized, thermally stable CS(2) adduct 8 that shows symmetrical binding to the Ti(2) unit and supports the formulation of 4. The reaction of 1 with COS forms a thermally unstable complex 9 that undergoes scission to give mono(μ-S) mono(CO) species 10. Ph(3)PS is an effective sulfur transfer agent for 1, enabling the synthesis of mono(μ-S) complex 11 with a double-sandwich structure and bis(μ-S) dimer 12 in which the Ti–Ti bond has been cleaved

Congenital disorder of glycosylation caused by starting site-specific variant in syntaxin-5

Mutations in genes critical for proper intra-Golgi transport can cause human syndromes due to defects in glycosylation of proteins. Here, the authors identify a human variant of Syntaxin-5 that causes fatal multisystem disease and mislocalization of glycosyltransferases due to altered Golgi transport

From glycosylation disorders to dolichol biosynthesis defects: a new class of metabolic diseases

Polyisoprenoid alcohols are membrane lipids that are present in every cell, conserved from archaea to higher eukaryotes. The most common form, alpha-saturated polyprenol or dolichol is present in all tissues and most organelle membranes of eukaryotic cells. Dolichol has a well defined role as a lipid carrier for the glycan precursor in the early stages of N-linked protein glycosylation, which is assembled in the endoplasmic reticulum of all eukaryotic cells. Other glycosylation processes including C- and O-mannosylation, GPI-anchor biosynthesis and O-glucosylation also depend on dolichol biosynthesis via the availability of dolichol-P-mannose and dolichol-P-glucose in the ER. The ubiquity of dolichol in cellular compartments that are not involved in glycosylation raises the possibility of additional functions independent of these protein post-translational modifications. The molecular basis of several steps involved in the synthesis and the recycling of dolichol and its derivatives is still unknown, which hampers further research into this direction. In this review, we summarize the current knowledge on structural and functional aspects of dolichol metabolites. We will describe the metabolic disorders with a defect in known steps of dolichol biosynthesis and recycling in human and discuss their pathogenic mechanisms. Exploration of the developmental, cellular and biochemical defects associated with these disorders will provide a better understanding of the functions of this lipid class in human