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Various proteins are involved in the generation and maintenance of the membrane complex known as the Golgi apparatus. We have used mutant Chinese hamster ovary (CHO) cell lines Lec4 and Lec4A lacking N-acetylglucosaminyltransferase V (GlcNAcT-V, MGAT5) activity and protein in the Golgi apparatus to study the effects of the absence of a single glycosyltransferase on the Golgi apparatus dimension. Quantification of immunofluorescence in serial confocal sections for Golgi α-mannosidase II and electron microscopic morphometry revealed a reduction in Golgi volume density up to 49 % in CHO Lec4 and CHO Lec4A cells compared to parental CHO cells. This reduction in Golgi volume density could be reversed by stable transfection of Lec4 cells with a cDNA encoding Mgat5. Inhibition of the synthesis of β1,6-branched N-glycans by swainsonine had no effect on Golgi volume density. In addition, no effect on Golgi volume density was observed in CHO Lec1 cells that contain enzymatically active GlcNAcT-V, but cannot synthesize β1,6-branched glycans due to an inactive GlcNAcT-I in their Golgi apparatus. These results indicate that it may be the absence of the GlcNAcT-V protein that is the determining factor in reducing Golgi volume density. No dimensional differences existed in cross-sectioned cisternal stacks between Lec4 and control CHO cells, but significantly reduced Golgi stack hits were observed in cross-sectioned Lec4 cells. Therefore, the Golgi apparatus dimensional change in Lec4 and Lec4A cells may be due to a compaction of the organelle. [ABSTRACT FROM AUTHOR]

Mucolipidosis Type III, or pseudo-Hurler polydystrophy, is a rare genetic abnormality, the result of a mutation to one of two genes that encode the hexameric protein N-acetylglucosaminyl-l-phosphotransferase (Glc-NAc-PT). The abnormality results in the accumulation of unprocessed macromolecules in cell and tissue compartments throughout the body. In this case report, we describe the clinical and radiographic findings of a 15-year-old male with this disorder. He presented with bilateral ectopically developing mandibular molar teeth with enlarged follicles and multiple joint involvement, including the temporomandibular joints. The patient underwent surgical removal of the molar teeth and curettage of the associated follicles. The subsequent histopathological examination of the tissues revealed hyperplastic follicles suggestive of dentigerous cysts. This report presents the plain film and cone beam CT examinations of the patient. [ABSTRACT FROM AUTHOR]

Mucolipidosis (ML) II (I-cell disease) is a lysosomal storage disorder caused by a deficiency of UDP- N-acetylglucosamine:lysosomal enzyme N-acetylglucosamine-1-phosphotransferase. MLII is an autosomal recessive disease with a carrier rate estimated at 1/39 in Saguenay–Lac-Saint-Jean (SLSJ) (Quebec, Canada), which is the highest frequency documented worldwide. To identify the causing mutation, we sequenced GNPTAB exons in 27 parents of 16 MLII-deceased children from the SLSJ region as obligatory and potential carriers. We also performed a genealogical reconstruction for each parent to evaluate consanguinity levels and genetic contribution of ancestors. Our goal was to identify which parameters could explain the high MLII frequency observed in the SLSJ population. A single mutation (c.3503_3504delTC) was found in all obligatory carriers. In addition, 11 apparent polymorphisms were identified. The mutation was not detected in genomic DNA of 50 unrelated controls. Genealogical data show six founders (three couples) with a higher probability of having introduced the mutation in the population. The frequency of the mutation was increased as a consequence of this founder effect and of the resulting population structure. We suggest that c.3503_3504delTC is the allele causing MLII in the SLSJ population, and its high carrier rate is most likely explained by a founder effect. [ABSTRACT FROM AUTHOR]

Defects in the multimeric enzyme, UDP- N-acetylglucosamine:lysosomal enzyme N-acetylglucosamine-1-phosphotransferase (GNPT), result in the diseases of mucolipidosis (ML). This enzyme generates the mannose 6-phosphate residues on newly synthesized lysosomal enzymes for the efficient receptor-mediated transport to lysosomes. The enzyme contains alpha/beta and gamma subunits. Mutations in the alpha/beta subunit result in the classical ML II and IIIA, while defects in the gamma subunit results in the clinically milder ML IIIC. I-cells, a distinct histological feature characterized by the presence of abnormal cytoplasmic vacuoles, are detected in many cell types, most noticeably, in ML II patients. In this study, we investigated the interactions of the alpha/beta and gamma subunits in the pathogenesis of I-cells. We noted low and deranged alpha/beta subunit expressions in human mucolipidosis cell lines. Unexpectedly, high gamma subunit expressions were also observed. In normal mouse fibroblasts, when alpha/beta subunit was suppressed, abnormal cytoplasmic vacuoles were induced, and up-regulation of the gamma subunit was also observed. On the other hand, suppressing the gamma subunit resulted in biphasic responses of the alpha/beta subunit, while abnormal cytoplasmic vacuoles were not formed, regardless of the expression levels of the alpha/beta subunit. Our data suggest reciprocal feedback mechanisms between alpha/beta and the gamma subunits. A fine balance of the expressions of these subunits may play an important role in the formation of I-cells in this group of lysosomal storage disorders. [ABSTRACT FROM AUTHOR]

Visualization of the reaction coordinate undertaken by glycosyltransferases has remained elusive but is critical for understanding this important class of enzyme. Using substrates and substrate mimics, we describe structural snapshots of all species along the kinetic pathway for human O-linked ?-N-acetylglucosamine transferase (O-GlcNAc transferase), an intracellular enzyme that catalyzes installation of a dynamic post-translational modification. The structures reveal key features of the mechanism and show that substrate participation is important during catalysis. [ABSTRACT FROM AUTHOR]

Background: Mucolipidosis II (ML II; I-cell disease) is caused by a deficiency of N-acetylglucosamine-1-phosphotransferase (GNPTAB; EC 2.7.8.17), which leads to a failure to internalize acid hydrolases into lysosomes for proper catabolism of various substances. This is an autosomal recessive lysosomal storage disease and causes severe progressive neuropathy and oculoskeletal dysfunction in humans (OMIM 252500). A naturally occurring disease model has been reported in juvenile domestic cats (OMIA 001248–9685) with clinical signs similar to human patients. We investigated the molecular genetic basis of ML II in a colony of affected cats by sequencing the coding and regulatory regions of GNPTAB from affected and clinically healthy related and unrelated domestic cats and compared the sequences to the published feline genome sequence (NCBI-RefSeq accession no. XM_003989173.4, Gene ID: 101100231). Results: All affected cats were homozygous for a single base substitution (c.2644C > T) in exon 13 of GNPTAB. This variant results in a premature stop codon (p.Gln882*) which predicts severe truncation and complete dysfunction of the GNPTAB enzyme. About 140 GNPTAB variants have been described in human ML II patients, with 41.3% nonsense/missense mutations, nine occurring in the same gene region as in this feline model. Restriction fragment length polymorphism and allelic discrimination real-time polymerase chain reaction assays accurately differentiated between clear, asymptomatic carriers and homozygous affected cats. Conclusion: Molecular genetic characterization advances this large animal model of ML II for use to further define the pathophysiology of the disease and evaluate novel therapeutic approaches for this fatal lysosomal storage disease in humans. [ABSTRACT FROM AUTHOR]