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John S Schutzbach 《Glycoconjugate journal》1997,14(2):175-182
The enzymes in the dolichol pathway are membrane-proteins that utilize a combination of hydrophilic and extremely hydrophobic
substrates. The enzymes in this pathway that have been purified and characterized to any extent have either been shown to
be stabilized by mixed phospholipid/detergent micelles, or else require a lipid matrix for catalytic activity. Further understanding
of the mechanisms of these essential enzymes may require developing methods for the reconstitution of the glycosyltransferases
and their hydrophobic substrates in appropriate lipid matrices. Abbreviations: CHO, Chinese hamster ovary; Dol, dolichol;
DAG, diacylglycerol; DOPC, dioleolylphosphatidylcholine; DOPE, dioleolyphosphatidylethanolamine; ER, endoplasmic reticulum;
PC, phosphatidylcholine; PE, phosphatidylethanolamine; PG, phosphatidylglycerol; PI, phosphatidylinositol
This revised version was published online in November 2006 with corrections to the Cover Date. 相似文献
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Uridine diphospho glucose (UDP-Glc) and uridine diphospho N-acetylglucosamine (UDP-GlcNAc), modified in the uridine moiety by either periodate oxidation of the ribose ring or substitution at position 5 of the uracil ring with fluorine, have been tested as potential inhibitors of glucosyl monophosphoryl dolichol (Glc-P-Dol) or N,N-diacetylchitobiosyl pyrophosphoryl dolichol [GlcNAc)2-PP-Dol) assembly in chick embryo cell membranes. The periodate oxidised sugar nucleotides inhibited glycosyl transfer from their respective natural counterparts by 50% at 230 micron periodate oxidised UDP-Glc and 70 micron periodate oxidised UDP-GlcNAc respectively. Inhibition in both cases was irreversible and addition of exogenous Dol-P stimulated only the residual non-inhibited reaction. Periodate oxidised UDP-GlcNAc preferentially inhibited the transfer of GlcNAc to GlcNac-PP-Dol. The sugar nucleotide containing 5-fluorouridine were, on the other hand, alternative substrates for Glc-P-Dol or (GlcNAc)2-PP-Dol synthesis. FUDP-Glc was a good substrate for Glc-P-Dol formation; having Km and Vmax values equal to those of UDP-Glc, whereas FUDP-GlcNAc was a less efficient substrate for the formation of (GlcNAc)2-PP-Dol; having Km and Vmax values one half and one third respectively of those of UDP-GlcNAc. 相似文献
4.
Clara W. Hall April R. Robbins Sharon S. Krag 《Molecular and cellular biochemistry》1986,72(1-2):35-45
A novel screening procedure was developed for isolating Chinese hamster ovary cell mutants altered in the early steps of the biosynthesis of asparagine-linked glycoproteins. This procedure identifies cells with low intracellular levels of two lysosomal hydrolases, beta-glucuronidase and alpha-iduronidase. One mutant cell line isolated in this way, CHB 11-1-3, has low intracellular levels of seven lysosomal enzymes as compared to wild-type cells. Although CHB 11-1-3 synthesizes mannosylphosphoryldolichol and [Man]5[NAcG1cNH2]2-P-P-lipid, it fails to utilize these lipid intermediates to make normal amounts of [Glc]3[Man]9[NAcG1cNH2]2P-P-lipid. As a consequence of this glycosylation defect, this mutant transfers oligosaccharides of a different structure than wild type to the lysosomal enzyme beta-hexosaminidase. In addition, it underglycosylates its proteins. 相似文献
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The antiobiotics, diumycin, amphomycin, bacitracin, and showdomycin have been shown previously to block the synthesis of GlcNAc-P-P-dolichol and GlcNAc-GlcNAc-P-P-dolichol. In view of inconsistencies in the literature concerning the sites of inhibition, we have reinvestigated the influence of these drugs on the formation of these early intermediates of the dolichol pathway. Unexpectedly, when the individual products of the reactions were examined, instead of inhibition, showdomycin and bacitracin were found to stimulate the formation of GlcNAc-P-P-dolichol, and diumycin stimulated at low concentrations. Three derivatives of showdomycin were examined with similar results, showing stimulations of GlcNAc-P-P-dolichol formation of up to two-fold over controls. Amphomycin specifically inhibited GlcNAc-P-P-dolichol formation, an effect that was reversed by a high concentration of dolichyl phosphate. In contrast, with the exception of amphomycin, each compound directly inhibited the formation of GlcNAc-GlcNAc-P-P-dolichol. Using chemically synthesized GlcNAc-P-P-dolichol as substrate, the kinetics of inhibition of GlcNAc-GlcNAc-P-P-dolichol formation by showdomycin, bacitracin and diumycin was examined. The apparent Ki values calculated from these studies indicated that showdomycin was the most active inhibitor. These findings provide a new understanding of the action of these compounds on the GlcNAc-transferases of the dolichol pathway. © 1998 Rapid Science Ltd 相似文献
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Krishna Baksi Zhenbo Zhang Aditi Banerjee Dipak K. Banerjee 《Glycoconjugate journal》2009,26(6):635-645
Mannosylphospho dolichol synthase (DPMS) is a critical enzyme in the biosynthesis of lipid-linked oligosaccharide (LLO; Glc3Man9GlcNAc2-PP-Dol), a pre-requisite for asparagine-linked (N-linked) protein glycosylation. We have shown earlier that DPMS is important
for angiogenesis, i.e., endothelial cell proliferation. This is true when cAMP is used for intracellular signaling. During cAMP signaling, DPMS
is activated and ER stress is reduced. To understand the activation of DPMS at the molecular level we have isolated a cDNA
clone for the DPMS gene (bDPMS) from the capillary endothelial cells of bovine adrenal medulla. DNA sequencing and the deduced
amino acid sequence have established that bDPMS has a motif to be phosphorylated by cAMP-dependent protein kinase (PKA). Based
on the sequence information Serine 165 has been found to be the phosphorylation target in bDPMS. Hydropathy Index when plotted
against amino acid number indicates the presence of a hydrophobic region around the amino acid residues 120–160, supporting
that bDPMS has one membrane spanning region. The recombinant bDPMS has now been purified as His-tag protein with an apparent
molecular weight of M
r 33 kDa. Additionally, we show here that overexpression of DPMS is indeed angiogenic. The capillary endothelial cells proliferate
at a higher rate carrying the DPMS overexpression plasmid over the parental cells or the vector. 相似文献
8.
Meredith B. Jones Julian N. Rosenberg Michael J. Betenbaugh Sharon S. Krag 《Biochimica et Biophysica Acta (BBA)/General Subjects》2009
N-linked protein glycosylation was originally thought to be specific to eukaryotes, but evidence of this post-translational modification has now been discovered across all domains of life: Eucarya, Bacteria, and Archaea. In all cases, the glycans are first assembled in a step-wise manner on a polyisoprenoid carrier lipid. At some stage of lipid-linked oligosaccharide synthesis, the glycan is flipped across a membrane. Subsequently, the completed glycan is transferred to specific asparagine residues on the protein of interest. Interestingly, though the N-glycosylation pathway seems to be conserved, the biosynthetic pathways of the polyisoprenoid carriers, the specific structures of the carriers, and the glycan residues added to the carriers vary widely. In this review we will elucidate how organisms in each basic domain of life synthesize the polyisoprenoids that they utilize for N-linked glycosylation and briefly discuss the subsequent modifications of the lipid to generate a lipid-linked oligosaccharide. 相似文献
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CWH8/YGR036c of Saccharomyces cerevisiae has been identifiedas a dolichylpyrophosphate (Dol-PP) phosphatase that removesa phosphate from the Dol-PP generated by the oligosaccharyltransferase(OST), while it adds N-glycans to nascent glycoproteins in theendoplasmic reticulum (ER). Lack of CWH8 was proposed to interruptthe so called dolichol (Dol) cycle by trapping Dol in the formof Dol-PP in the ER lumen. Indeed, cwh8D mutants display a severedeficiency in N-glycosylation. We find that cwh8D mutants havestrongly reduced levels of inositolphosphorylceramide (IPC),whereas its derivative, mannosyl-(inositol-P)2-ceramide (M(IP)2C)is not affected. Microsomes of cwh8D contain normal ceramidesynthase and IPC synthesis activities. Within a large panelof mutants affecting Dol dependent pathways such as N- or O-glycosylation,or glycosylphosphatidyl inositol (GPI)-anchoring, only the mutantshaving a deficiency of N-glycan addition show the defect inIPC biosynthesis. By mutating genes required for the additionof N-glycans or by treating cells with tunicamycin (Tm) onecan similarly reduce the steady state level of IPC and exactlyreproduce the phenotype of cwh8D cells. Some potential mechanismsby which the lack of N-glycans could lead to the sphingolipidabnormality were further explored. 相似文献
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Liliana Surmacz Danuta Plochocka Magdalena Kania Witold Danikiewicz Ewa Swiezewska 《Biochimica et Biophysica Acta (BBA)/Molecular and Cell Biology of Lipids》2014,1841(2):240-250
cis-Prenyltransferases (CPTs) comprise numerous enzymes synthesizing isoprenoid hydrocarbon skeleton with isoprenoid units in the cis (Z) configuration. The chain-length specificity of a particular plant CPT is in most cases unknown despite the composition of the accumulated isoprenoids in the tissue of interest being well established. In this report AtCPT6, one of the nine Arabidopsis thaliana CPTs, is shown to catalyze the synthesis of a family of very short-chain polyisoprenoid alcohols of six, seven, and eight isoprenoid units, those of seven units dominating. The product specificity of AtCPT6 was established in vivo following its expression in the heterologous system of the yeast Saccharomyces cerevisiae and was confirmed by the absence of specific products in AtCPT6 T-DNA insertion mutants and their overaccumulation in AtCPT6-overexpressing plants. These observations are additionally validated in silico using an AtCPT6 model obtained by homology modeling. AtCPT6 only partially complements the function of the yeast homologue of CPT-Rer2 since it restores the growth but not protein glycosylation in rer2Δ yeast. This is the first in planta characterization of specific products of a plant CPT producing polyisoprenoids. Their distribution suggests that a joint activity of several CPTs is required to produce the complex mixture of polyisoprenoid alcohols found in Arabidopsis roots. 相似文献