Gene/Protein Disease Symptom Drug Enzyme Compound
Pivot Concepts: Gene/Protein Disease Symptom Drug Enzyme Compound   Target Concepts: Gene/Protein Disease Symptom Drug Enzyme Compound

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Query: EC:3.6.3.1 (Mg2+-ATPase)
1,484 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

A 53,000-dalton intrinsic glycoprotein of the sarcoplasmic reticulum was separated from the Ca2+ + Mg2+-ATPase by dissolution with low concentrations of deoxycholate in the presence of 1 M KCl and purified in two successive gel filtration steps. It was aggregated and eluted at the void volume when subjected to gel filtration in the presence or absence of deoxycholate. When subsequently chromatographed in the presence of sodium dodecyl sulfate, the glycoprotein eluted in pure form as a monomer. The glycoprotein contained 48% nonpolar amino acids. It also contained 4 mol of glucosamine and 18 mol of mannose per mol of protein, suggesting that it contained two chains of (GlcNAc)2, (Man)9. The 53,000-dalton glycoprotein was completely removed from deoxycholate extracts of sarcoplasmic reticulum by affinity chromatography on concanavalin A Sepharose. Elution of glycoproteins with alpha-methyl-D-mannoside and deoxycholate resulted in co-purification of the 53,000-dalton glycoprotein and 160,000-dalton glycoprotein previously observed in sarcoplasmic reticulum. The apparent molecular weight of the glycoprotein was reduced from 53,000 to 49,000 after digestion with endo-beta-N-acetylglucosaminidase H (Endo H) and its reactivity with concanavalin A (Con A) was lost. There was no change in molecular weight of the glycoprotein and no diminution of its reactivity with Con A when sealed vesicles of sarcoplasmic reticulum were treated with Endo H. Endo H reduced the molecular weight and the Con A reactivity of the protein when the vesicles were made permeable by detergents. These observations, together with our previous demonstration that the glycoprotein reacts with a cycloheptaamylose-fluorescamine complex in sealed vesicles (Michalak, M., Campbell, K. P., and MacLennan, D. H. (1980) J. Biol. Chem. 255, 1317-1326), show that the glycoprotein is a transmembrane protein. A protein of approximately 53,000 daltons was labeled when the sarcoplasmic reticulum was reacted with the photoaffinity label [32P]8-N3-cAMP. The labeled protein was neither the glycoprotein nor the high affinity calcium-binding protein since it was not sensitive to Endo H and was sensitive to trypsin digestion.
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PMID:Purification and characterization of the 53,000-dalton glycoprotein from the sarcoplasmic reticulum. 626 Aug 6

This study reports the organization of the wbp gene cluster and characterization of a number of genes that are essential for B-band O antigen biosynthesis in the clinically prevalent Pseudomonas aeruginosa serotype 06. Twelve genes were identified that share homology with other LPS and polysaccharide biosynthetic genes. This cluster contains homologues of wzx (encoding the O antigen flippase/translocase) and wzz (which modulates O antigen chain length distribution) genes, typical of a wzy-dependent pathway. However, a complete wzy gene (encoding the O-polymerase) was not found within the cluster. Four biosynthetic genes, wbpO, wbpP, wbpV and wbpM, and four putative glycosyltransferase genes, wbpR, wbpT, wbpU and wbpL, were identified in the cluster. To characterize their roles in LPS biosynthesis, null mutants of wbpO, wbpP, wbpV, wbpL and wbpM were generated using a gene-replacement strategy. Mutations in each of these genes caused deficiency in B-band synthesis. The wbpL mutant was deficient in both A-band and B-band LPS. WbpL(O6) is a bi-functional enzyme which could initiate B-band synthesis through the addition of QuiNAc to undecaprenol phosphate, and A-band synthesis by transferring either a GalNAc or a GlcNAc residue. Another approach used to assign function to the wbp(O6) genes was by complementation analysis. Two genes from Salmonella typhi, wcdA and wcdB, responsible for the synthesis of a homopolymer of GalNAcA called Vi antigen were used in complementation experiments to verify the functions of wbpO and wbpP. wcdA and wcdB restored B-band synthesis in wbpO and wbpP mutants respectively, implying that wbpO and wbpP are involved in UDP-GalNAcA synthesis. Although wbpV has homology to wbpK of the serotype O5 B-band LPS synthesis cluster, complementation analysis using the respective null mutants showed that the genes are not interchangeable. A knockout mutation of wbpN (located downstream of wbpM) did not abrogate LPS synthesis in either 05 or 06; therefore, it has been renamed orf48.5. These results establish the organization of genes involved in P. aeruginosa B-band O antigen synthesis and provide the evidence to assign functions to a number of LPS biosynthetic genes.
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PMID:Functional analysis of genes responsible for the synthesis of the B-band O antigen of Pseudomonas aeruginosa serotype O6 lipopolysaccharide. 1062 48

Shigella is a well-known human pathogen causing dysentery and their typing is solely based on the O antigens. We investigated the chemical structure and gene cluster of Shigella boydii type 16 O antigen. As judged by sugar and methylation analyses along with NMR spectroscopy data, the O antigen has an O-acetylated branched pentasaccharide repeating O unit, which consists of two D-mannose residues (D-Man), one residue each of d-glucuronic acid (D-GlcA), N-acetylglucosamine (D-GlcNAc) and D-galactose (D-Gal), and the structure of the O unit was established. The O antigen gene cluster of S. boydii type 16 was identified and shown to contain putative genes for the synthesis of GDP-D-Man, genes encoding sugar transferases, O unit flippase (Wzx) and O antigen polymerase (Wzy) as expected. The function of the wzy gene was characterized by mutation test. Genes specific to S. boydii type 16 O antigen gene cluster were identified by screening 186 Escherichia coli and Shigella type strains, and can be used to develop PCR assays for detection of type 16 strains.
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PMID:Structural and molecular characterization of Shigella boydii type 16 O antigen. 1685 42

During protein N-glycosylation, dolichyl pyrophosphate (Dol-P-P) is discharged in the lumenal monolayer of the endoplasmic reticulum (ER). Dol-P-P is then cleaved to Dol-P by Dol-P-P phosphatase (DPPase). Studies with the yeast mutant cwh8Delta, lacking DPPase activity, indicate that recycling of Dol-P produced by DPPase contributes significantly to the pool of Dol-P utilized for lipid intermediate biosynthesis on the cytoplasmic leaflet. Whether Dol-P formed in the lumen diffuses directly back to the cytoplasmic leaflet or is first dephosphorylated to dolichol has not been determined. Incubation of sealed ER vesicles from calf brain with acetyl-Asn-Tyr-Thr-NH(2), an N-glycosylatable peptide, to generate Dol-P-P in the lumenal monolayer produced corresponding increases in the rates of Man-P-Dol, Glc-P-Dol, and GlcNAc-P-P-Dol synthesis in the absence of CTP. No changes in dolichol kinase activity were observed. When streptolysin-O permeabilized CHO cells were incubated with an acceptor peptide, N-glycopeptide synthesis, requiring multiple cycles of the dolichol pathway, occurred in the absence of CTP. The results obtained with sealed microsomes and CHO cells indicate that Dol-P, formed from Dol-P-P, returns to the cytoplasmic leaflet where it can be reutilized for lipid intermediate biosynthesis, and dolichol kinase is not required for recycling. It is possible that the flip-flopping of the carrier lipid is mediated by a flippase, which would provide a mechanism for the recycling of Dol-P derived from Man-P-Dol-mediated reactions in N-, O-, and C-mannosylation of proteins, GPI anchor assembly, and the three Glc-P-Dol-mediated reactions in Glc(3)Man(9)GlcNAc(2)-P-P-Dol (DLO) biosynthesis.
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PMID:Recycling of dolichyl monophosphate to the cytoplasmic leaflet of the endoplasmic reticulum after the cleavage of dolichyl pyrophosphate on the lumenal monolayer. 1807 51

The DNA sequence of the genome of Staphylococcus haemolyticus JCSC1435 revealed a putative capsule operon composed of 13 genes in tandem. The first seven genes (capABCDEFG(Sh)) showed > or = 57% similarity with the Staphylococcus aureus cap5 or cap8 locus. However, the capHIJKLM(Sh) genes are unique to S. haemolyticus and include genes encoding a putative flippase, an aminotransferase, two glycosyltransferases, and a transcriptional regulator. Capsule-like material was readily apparent by immunoelectron microscopy on bacteria harvested in the postexponential phase of growth. Electron micrographs of a JCSC1435 mutant with a deleted cap region lacked the capsule-like material. Both strains produced small amounts of surface-associated material that reacted with antibodies to polyglutamic acid. S. haemolyticus cap genes were amplified from four of seven clinical isolates of S. haemolyticus from humans, and three of these strains produced a serologically cross-reactive capsular polysaccharide. In vitro assays demonstrated that the acapsular mutant strain showed greater biofilm formation but was more susceptible to complement-mediated opsonophagocytic killing than the parent strain. Structural characterization of capsule purified from S. haemolyticus strain JCSC1435 showed a trisaccharide repeating unit: -3-alpha-L-FucNAc-3-(2-NAc-4-N-Asp-2,4,6-trideoxy-beta-D-Glc)-4-alpha-D-GlcNAc-. This structure is unique among staphylococcal polysaccharides in that its composition includes a trideoxy sugar residue with aspartic acid as an N-acyl substituent.
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PMID:Structural and biological characterization of a capsular polysaccharide produced by Staphylococcus haemolyticus. 1816 9

Protein N-glycosylation requires flipping of the glycolipid Man(5)GlcNAc(2)-diphosphate dolichol (Man(5)GlcNAc(2)-PP-Dol) across the endoplasmic reticulum (ER). Helenius et al. report genetic evidence suggesting that Rft1, an essential ER membrane protein in yeast, is required directly to translocate Man(5)GlcNAc(2)-PP-Dol. We now show that a specific ER protein(s), but not Rft1, is required to flip Man(5)GlcNAc(2)-PP-Dol in reconstituted vesicles. Rft1 may have a critical accessory role in translocating Man(5)GlcNAc(2)-PP-Dol in vivo, but the Man(5)GlcNAc(2)-PP-Dol flippase itself remains to be identified.
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PMID:Does Rft1 flip an N-glycan lipid precursor? 1180 58

Caulobacter crescentus cells adhere to surfaces by using an extremely strong polar adhesin called the holdfast. The polysaccharide component of the holdfast is comprised in part of oligomers of N-acetylglucosamine. The genes involved in the export of the holdfast polysaccharide and the anchoring of the holdfast to the cell were previously discovered. In this study, we identified a cluster of polysaccharide biosynthesis genes (hfsEFGH) directly adjacent to the holdfast polysaccharide export genes. Sequence analysis indicated that these genes are involved in the biosynthesis of the minimum repeat unit of the holdfast polysaccharide. HfsE is predicted to be a UDP-sugar lipid-carrier transferase, the glycosyltransferase that catalyzes the first step in polysaccharide biosynthesis. HfsF is predicted to be a flippase, HfsG is a glycosyltransferase, and HfsH is similar to a polysaccharide (chitin) deacetylase. In-frame hfsG and hfsH deletion mutants resulted in severe deficiencies both in surface adhesion and in binding to the holdfast-specific lectin wheat germ agglutinin. In contrast, hfsE and hfsF mutants exhibited nearly wild-type levels of adhesion and holdfast synthesis. We identified three paralogs to hfsE, two of which are redundant to hfsE for holdfast synthesis. We also identified a redundant paralog to the hfsC gene, encoding the putative polysaccharide polymerase, and present evidence that the hfsE and hfsC paralogs, together with the hfs genes, are absolutely required for proper holdfast synthesis.
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PMID:Characterization of the Caulobacter crescentus holdfast polysaccharide biosynthesis pathway reveals significant redundancy in the initiating glycosyltransferase and polymerase steps. 1875 30

The oligosaccharide donor for protein N-glycosylation, Glc(3)Man(9)GlcNAc(2)-PP-dolichol, is synthesized via a multistep pathway that starts on the cytoplasmic face of the endoplasmic reticulum (ER) and ends in the lumen where the glycosylation reaction occurs. This necessitates transbilayer translocation or flipping of the lipid intermediate Man(5)GlcNAc(2)-PP-dolichol (M5-DLO) across the ER membrane. The mechanism by which M5-DLO-or any other lipid-is flipped across the ER is unknown, except that specific transport proteins or flippases are required. We recently demonstrated M5-DLO flipping activity in proteoliposomes reconstituted from detergent-solubilized ER membrane proteins and showed that it was ATP-independent and required a trypsin-sensitive protein that sedimented at approximately 4S. By using an activity-enriched fraction devoid of glycerophospholipid flippase activity, we now report that M5-DLO is rapidly flipped in the reconstituted system with a time constant tau <2 min, whereas its triantennary structural isomer is flipped slowly with tau >200 min. DLOs larger than M5-DLO are also poorly translocated, with tau ranging from approximately 10 min to >200 min. We conclude that (i) the number and arrangement of mannoses in the DLO glycan has a profound effect on the ability of the DLO to be translocated by the flippase, (ii) glycan size per se does not dictate whether a DLO will be flipped, and (iii) the flippase is highly specific for M5-DLO. Our results suggest a simple structural model for the interaction between the DLO head group and the flippase.
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PMID:Specific transbilayer translocation of dolichol-linked oligosaccharides by an endoplasmic reticulum flippase. 1912 92

Very recently, Haeuptle and colleagues described a new glycosylation defect due to RFT1 deficiency (CDG In). Accumulation of intracellular DolPP-GlcNAc(2)Man(5) with absence of cytosolic GlcNAc(2)Man(5) resembled the profile of a yeast mutant deficient in RFT1, a protein that is thought to have a role as a flippase. This is the first detailed description of the clinical phenotype of this patient. It was a severe disorder affecting intrauterine development and movement, and leading to intrauterine growth retardation. The child was born with several musculoskeletal abnormalities including arthrogryposis. Postnatally, severe reflux and irregular bowl movements contributed to failure to thrive. The patient showed very little development and no vision and suffered from drug-resistant epilepsy. Abnormal coagulation resulted in thrombosis and the patient died at the age of 4 years from a pulmonary embolus.
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PMID:Comprehensive description of the phenotype of the first case of congenital disorder of glycosylation due to RFT1 deficiency (CDG In). 1926 16

To further evaluate the role of Rft1 in the transbilayer movement of Man(5)GlcNAc(2)-P-P-dolichol (M5-DLO), a series of experiments was conducted with intact cells and sealed microsomal vesicles. First, an unexpectedly large accumulation (37-fold) of M5-DLO was observed in Rft1-depleted cells (YG1137) relative to Glc(3)Man(9)GlcNAc(2)-P-P-Dol in wild type (SS328) cells when glycolipid levels were compared by fluorophore-assisted carbohydrate electrophoresis analysis. When sealed microsomes from wild type cells and cells depleted of Rft1 were incubated with GDP-[(3)H]mannose or UDP-[(3)H]GlcNAc in the presence of unlabeled GDP-Man, no difference was observed in the rate of synthesis of [(3)H]Man(9)GlcNAc(2)-P-P-dolichol or Man(9)[(3)H]GlcNAc(2)-P-P-dolichol, respectively. In addition, no difference was seen in the level of M5-DLO flippase activity in sealed wild type and Rft1-depleted microsomal vesicles when the activity was assessed by the transport of GlcNAc(2)-P-P-Dol(15), a water-soluble analogue. The entry of the analogue into the lumenal compartment was confirmed by demonstrating that [(3)H]chitobiosyl units were transferred to endogenous peptide acceptors via the yeast oligosaccharyltransferase when sealed vesicles were incubated with [(3)H]GlcNAc(2)-P-P-Dol(15) in the presence of an exogenously supplied acceptor peptide. In addition, several enzymes involved in Dol-P and lipid intermediate biosynthesis were found to be up-regulated in Rft1-depleted cells. All of these results indicate that although Rft1 may play a critical role in vivo, depletion of this protein does not impair the transbilayer movement of M5-DLO in sealed microsomal fractions prepared from disrupted cells.
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PMID:Suppression of Rft1 expression does not impair the transbilayer movement of Man5GlcNAc2-P-P-dolichol in sealed microsomes from yeast. 1949 7


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