Asparagine (N)-linked glycosylation begins with a stepwise synthesis of the dolichol-linked oligosaccharide (DLO) precursor, Glc3Man9GlcNAc2-PP-Dol, which is catalyzed by a series of endoplasmic reticulum membrane-associated glycosyltransferases. Yeast ALG1 (asparagine-linked glycosylation 1) encodes a β-1, 4 mannosyltransferase that adds the first mannose onto GlcNAc2-PP-Dol to produce a core trisaccharide Man1GlcNAc2-PP-Dol. ALG1 is essential for yeast viability, and in humans mutations in the ALG1 cause congenital disorders of glycosylation known as ALG1-CDG. Alg1 is difficult to purify because of its low expression level and as a consequence, has not been well studied biochemically. Here we report a new method to purify recombinant Alg1 in high yield, and a mass spectral approach for accurately measuring its β-1, 4 mannosyltransferase activity.
N-terminally truncated yeast His-tagged Alg1 protein was expressed in Escherichia coli and purified by HisTrap HP affinity chromatography. In combination with LC-MS technology, we established a novel assay to accurately measure Alg1 enzyme activity. In this assay, a chemically synthesized dolichol-linked oligosaccharide analogue, phytanyl-pyrophosphoryl-α-N, N\'-diacetylchitobioside (PPGn2), was used as the acceptor for the β-1, 4 mannosyl transfer reaction.
Using purified Alg1, its biochemical characteristics were investigated, including the apparent Km and Vmax values for acceptor, optimal conditions of activity, and the specificity of its nucleotide sugar donor. Furthermore, the effect of ALG1-CDG mutations on enzyme activity was also measured.
This work provides an efficient method for production of Alg1 and a new MS-based quantitative assay of its activity.

Glycosyltransferases are sugar-processing enzymes that require a specific metal ion cofactor for catalysis. In the presence of other ions the catalysis is often impaired. Here, for the first time, the enzymatic catalysis in the presence of various metal ions was modeled for a glycosyltransferase using a large enzymatic model. The catalytic mechanism of α-1,2-mannosyltransferase Kre2p/Mnt1p in the presence of Mn(2+) and other ions (Mg(2+), Zn(2+) and Ca(2+)) was modeled at the two hybrid DFT-QM/MM (M06-2X/OPLS2005 and B3LYP/OPLS2005) levels. Kinetic and structural parameters of transition states and intermediates, as well as kinetic isotope effects, were predicted and compared with available experimental and theoretical data. The catalysis in the presence of the metal ions is predicted as a stepwise SNi-like nucleophilic substitution reaction (DNint*AN(‡)DhAxh) via oxocarbenium ion intermediates. In the rate-determining step the leaving phosphate group of the donor substrate plays a role of the base catalyst. The predicted increased enzymatic reactivity (kcat: Zn(2+) ≈ Mg(2+) < Mn(2+) < Ca(2+)) correlated with the metal ion ability to polarize the Kre2p environment (Mg(2+) > Zn(2+) > Mn(2+) > Ca(2+)). The formation of the retained anomeric configuration in the product is controlled by a strict geometry of the active site of Kre2p. The 6-OH group of the attacking acceptor substrate may assist in protection of the anomeric carbon against unwanted hydrolysis by a through-space interaction with the electron deficient C1[double bond, length as m-dash]O5(+) moiety of the oxocarbenium-ion-like transition state.

Interactions of transcriptional activators are difficult to study using transcription-based two-hybrid assays due to potent activation resulting in false positives. Here we report the development of the Golgi two-hybrid (G2H), a method that interrogates protein interactions within the Golgi, where transcriptional activators can be assayed with negligible background. The G2H relies on cell surface glycosylation to report extracellularly on protein-protein interactions occurring within the secretory pathway. In the G2H, protein pairs are fused to modular domains of the reporter glycosyltransferase, Och1p, and proper cell wall formation due to Och1p activity is observed only when a pair of proteins interacts. Cells containing interacting protein pairs are identified by selectable phenotypes associated with Och1p activity and proper cell wall formation: cells that have interacting proteins grow under selective conditions and display weak wheat germ agglutinin (WGA) binding by flow cytometry, whereas cells that lack interacting proteins display stunted growth and strong WGA binding. Using this assay, we detected the interaction between transcription factor MyoD and its binding partner Id2. Interfering mutations along the MyoD:Id2 interaction interface ablated signal in the G2H assay. Furthermore, we used the G2H to detect interactions of the activation domain of Gal4p with a variety of binding partners. Finally, selective conditions were used to enrich for cells encoding interacting partners. The G2H detects protein-protein interactions that cannot be identified via traditional two-hybrid methods and should be broadly useful for probing previously inaccessible subsets of the interactome, including transcriptional activators and proteins that traffic through the secretory pathway.

Walker-Warburg syndrome (WWS) is a rare, lethal genetic disease associated with a cobblestone-type lissencephaly, eye abnormalities, and a type of muscular dystrophy. There is a wide spectrum of brain and eye defects associated with this diagnosis; therefore, this diagnosis may not initially be considered. This diagnosis is especially difficult for families because there is no treatment available and management of the condition is supportive only. Parents of an infant with WWS need to be shown support and empathy while they are dealing with the sorrow of a terminal illness. Use of a nursing model, \"Middle range theory of chronic sorrow,\" will be instrumental in assisting staff as they care for the patient and the patient\'s family. They also need to be guided toward receiving genetic counseling to weigh their options for future family planning as the risk of another WWS pregnancy is 25%.

BACKGROUND: Congenital muscular dystrophies (CMD) with reduced glycosylation of alpha-dystroglycan (alpha-DG) are a heterogeneous group of conditions associated with mutations in six genes encoding proven or putative glycosyltransferases.
OBJECTIVE: The aim of the study was to establish the prevalence of mutations in the six genes in the Italian population and the spectrum of clinical and brain MRI findings.
METHODS: As part of a multicentric study involving all the tertiary neuromuscular centers in Italy, FKRP, POMT1, POMT2, POMGnT1, fukutin, and LARGE were screened in 81 patients with CMD and alpha-DG reduction on muscle biopsy (n = 76) or with a phenotype suggestive of alpha-dystroglycanopathy but in whom a muscle biopsy was not available for alpha-DG immunostaining (n = 5).
RESULTS: Homozygous and compound heterozygous mutations were detected in a total of 43/81 patients (53%), and included seven novel variants. Mutations in POMT1 were the most prevalent in our cohort (21%), followed by POMT2 (11%), POMGnT1 (10%), and FKRP (9%). One patient carried two heterozygous mutations in fukutin and one case harbored a new homozygous variant in LARGE. No clear-cut genotype-phenotype correlation could be observed with each gene, resulting in a wide spectrum of clinical phenotypes. The more severe phenotypes, however, appeared to be consistently associated with mutations predicted to result in a severe disruption of the respective genes.
CONCLUSIONS: Our data broaden the clinical spectrum associated with mutations in glycosyltransferases and provide data on their prevalence in the Italian population.

Mutations in POMT1 and POMT2 genes were originally identified in Walker-Warburg syndrome (WWS) and subsequently reported in patients with milder phenotypes characterised by mental retardation with or without brain abnormalities and without ocular malformations. As part of a multicentric Italian study we screened the POMT1 and POMT2 genes in 61 congenital muscular dystrophy (CMD) patients with alpha-dystroglycan reduction on muscle biopsy and/or clinical and radiological findings suggestive of the known forms of CMD with alpha-dystroglycan deficiency. The aim of the study was to establish how frequently mutations in POMT1 and POMT2 occur in CMD patients in the Italian population and to evaluate the spectrum of associated phenotypes. Thirteen patients showed mutations in POMT1 and five harboured mutations in POMT2, accounting for a total of 20 different mutations, eight of which were novel (two in POMT1 and six in POMT2). Normal brain MRI associated with mental retardation and microcephaly was the most frequent finding in patients with mutations in POMT1 (six out of 13), but was also found in a patient with POMT2 mutations. Predominant cerebellar hypoplasia was also frequent both in patients with POMT1 (three out of 13) and POMT2 (three out of 5) mutations. A MEB phenotype with frontal cortical dysplasia and pons abnormalities was found in two patients with POMT1 and in one with POMT2 mutations, while a WWS phenotype was only found in a case with mutations in POMT1. Mutations causing frameshifts and stop codons were responsible for the more severe phenotypes. Our results provide further evidence that, as previously reported for FKRP, the array of mutations in POMT1 and POMT2 is ample and the spectrum of associated phenotypes is wider than initially thought.

Derivatives of dolichyl phosphate (Dol-P) with 2-aminopyridine or 1-aminonaphtalene fluorophore groups at the omega-end of the chain were synthesized. These products serve as substrates for recombinant yeast Dol-P-mannose synthase. Fluorescence resonance energy transfer between a Trp residue of the enzyme and the 1-aminonaphtalene group of the Dol-P analogue was demonstrated.

We have investigated the specificity of the enzymes Q-insertase and mannosyl-Q transferase that replace the guanosine at position 34 (wobble base) in the anticodon of several tRNAs by Q or mannosyl-Q derivatives. We have restructured in vitro the normal anticodon of yeast tRNA-Asp-GUC, yeast tRNAArgICG and yeast tRNALeuUAG. With yeast tRNA-Asp-GUC, we have replaced one or several nucleotides in the vicinity of G34 by one of the four canonical nucleotides or by pseudouridylic acid; we have also constructed a tRNAAsp with eight bases instead of seven in the anticodon loop. With yeast tRNAArgICG and yeast tRNALeuUAG, we have replaced their anticodon by the trinucleotide GUC, coding for aspartic acid. The chimerical tRNAs were microinjected into the cytoplasm of Xenopus laevis oocytes and after 72 h the amount of Q34 and mannosyl-Q34 incorporated was measured. Our results show that the U33G34U35 sequence, within an anticodon loop of seven bases in chimerical yeast tRNA-Asp-GUC, tRNAArgGUC or tRNALeuGUC, is the main determinant for Q-insertase activity at position 34; the rest of the tRNA sequence has only a slight influence. For mannosyl-Q transferase, however, a much broader structural feature of the tRNA than just the U33G34U35 sequence is important for the efficiency of Q34 transformation into mannosyl-Q34.

The trypsin sensitivity of the mitochondrial N-acetylglucosaminyl and mannosyltransferase activities involved in the N-glycoprotein biosynthesis through dolichol intermediates as well as the N-acetylglucosaminyl-transferase activity involved in direct N-glycosylation were examined in mitochondria and isolated outer mitochondrial membrane preparations. The trypsin action on mitochondrial membrane was checked by measuring the activities of marker enzymes (rotenone-insensitive NADH cytochrome c reductase, adenylate kinase, and monoamine oxidase). Glycosyl-transferase activities of both N-glycosylation pathways were insensitive to trypsin action and consequently were located in the outer mitochondrial membrane. Based on the activator effect of the trypsin on these enzyme activities, the results suggested two distinct orientations of their active sites. As regards the N-glycoprotein biosynthesis pathway through dolichol intermediates, the dolicholphosphoryl-mannose and dolichol-pyrophosphoryl-di-N-acetylchitobiose synthases would be oriented outside while the oligomannosyl-synthase and the oligomannosyl-transferase would be rather oriented inside in the outer membrane. The N-acetylglucosaminyl-transferase involved in the direct transfer of N-acetylglucosamine from its nucleotide donor to a proteinic acceptor would be oriented outside in the outer membrane.

Yeast protoplasts were coated with different amounts of concanavalin A. Upon subsequent lysis and centrifugation in isopycnic density gradients, it was found that the buoyant density of plasma membranes was progressively increased from 1.125 to 1.21, according to the amount of bound concanavalin A. Enzymes that are attached to the plasma membrane showed the same density modifications and could thus be distinguished from constituents of intracellular membranes and organelles. With this methodology, it was confirmed that about two-thirds of yeast chitin synthetase is associated with the plasma membrane. The remainder of the enzyme was found in a peak at a lower density. Vanadate-sensitive ATPase showed a similar pattern, whereas GDP-mannose dolichyl-phosphate mannosyltransferase, an enzyme attached to the endoplasmic reticulum, remained in the same position in the gradients, irrespective of the amount of concanavalin A associated with the plasma membrane. Potential applications of this technique to the determination of plasma membrane markers and to the separation of subcellular organelles are discussed.