The structure of the O-glycosylically-linked oligosaccharide chains of LPG-I,a glycoprotein present in articular lubricating fraction of bovine synovial fluid

Periodate oxidation of LPG-1 established that N-acetylneuraminic acid residues are linked preponderantly α-(2→3) to D-galactose residues. The resistance of 2-acetamido-2-deoxyD-galactose residues to periodate oxidation suggests that they are linked at either O-3 or O-4 to D-galactose residues. After treatment of LPG-I with alkaline sulfite, ≈80% of 2-acetamido-2-deoxygalactose was recovered as the sulfonic acid derivative. The Gal→GalNAc disaccharide released from sialic-acid-free LPG-I by digestion with endo-2-acetamido-2-deoxy-α-D-galactosidase (which suggests an α-D-GalNAc→-L-Ser or -L-Thr linkage) gave a high color-yield in the Morgan—Elson reaction, indicating that 2-acetamido-2-deoxy-D-galactose residues are linked at C-3 to D-galactose residues. The migration of the released Gal-GalNAc disaccharide was the same as that of a standard sample of O-β-D-galactosyl-(1→3)-2-acetamido-2-deoxy-D-galactose. Treatment of sialic acid-free LPG-I with Streptococcus pneumoniae β-D-galactosidase, which hydrolyzes only galactosides linked β-D-(1→4) gave no free D-galactose, whereas treatment of LPG-I with bovine testes β-D-galactosidase released > 90% of D-galactose. These results provide evidence for β-D-Galp-(1→3)-α-D-GalNAcp-(1→3)-L-Ser or -L-Thr and α-NeuAc-(2→3)-β-D-Galp-(1→3)-α-D- GalNAcp-(1→3)-L-Ser or -L-Thr structures. The sensitivity of the methods used and the recovery of constituents following treatment of LPG-I do not rule out the occurrence of small amounts of other tri- or tetra-saccharide chains.     

Studies on a testosterone glucuronyltransferase from the cytosol fraction of human liver

An enzyme that conjugates the 16alpha-hydroxyl group of oestriol with glucuronic acid was found in the cytosol fraction of human liver. The enzymic activity could not be sedimented when the cytosol fraction was centrifuged at 158000g(av.) for 120min. The oestriol 16alpha-glucuronyltransferase was purified 100-fold by 0-30% saturation of the cytosol fraction with ammonium sulphate followed by filtration of the precipitate through Sephadex G-200. The activity was eluted at the void volume. The product of the reaction, oestriol 16alpha-monoglucuronide, was identified by paper chromatography and by crystallization of radioactive product to constant specific radioactivity. The optimum temperature was 37 degrees C, and the activation energy was calculated to be 11.1kcal/mol. The apparent Michaelis-Menten constants for oestriol and UDP-glucuronic acid were 13.3 and 100mum respectively. Cu(2+), Zn(2+) and Hg(2+) inhibited, whereas Mg(2+), Mn(2+) and Fe(2+) stimulated the enzyme. Substrate-specificity studies indicated that the amount of oestradiol-17beta, oestradiol-17alpha and oestrone conjugated was not more than about 5% of that found for oestriol. Oestriol 16alpha-monoglucuronide, a product of the reaction, did not inhibit the 16alpha-oestriol glucuronyltransferase; in contrast, UDP, another product of the reaction, inhibited the enzyme competitively with respect to UDP-glucuronic acid as the substrate, and non-competitively with respect to oestriol as the substrate. ATP and UDP-N-acetylglucosamine did not affect the oestriol 16alpha-glucuronyltransferase. 17-Epioestriol acted as a competitive inhibitor and 16-epioestriol as a non-competitive inhibitor of the glucuronidation of oestriol. 5alpha-Pregnane-3alpha,20alpha-diol also inhibited the enzyme non-competitively. It is most likely that the oestriol 16alpha-glucuronyltransferase described here is bound to the membranes of the endoplasmic reticulum.     

Biosynthesis of lipid-linked oligosaccharides. Isolation and structure of a second lipid-linked oligosaccharide in Chinese hamster ovary cells.

Previous work has shown that vesicular stomatitis virus-infected Chinese hamster ovary cells contain a major high molecular weight lipid-linked oligosaccharide which is transferred en bloc to protein during the formation of the asparagine-linked complex-type oligosaccharides of the vesicular stomatitis virus G protein (Tabas, I., Schlesinger, S., and Kornfeld, S. (1978) J. Biol. Chem. 253, 716-722). We now report the characterization of a second, lower molecular weight lipid-linked oligosaccharide. The oligosaccharide portion of this molecule was isolated and its structure was determined by methylation analysis, digestion with exoglycosidases, acetolysis and Smith periodate degradation to be: (formula: see text). Several lines of evidence are presented which indicate that this lipid-linked oligosaccharide is primarily involved in the assembly of the major lipid-linked oligosaccharide rather than in the direct glycosylation of proteins.     

Reconstitution of CMP-N-acetylneuraminic acid hydroxylation activity using a mouse liver cytosol fraction and soluble cytochrome b5 purified from horse erythrocytes.

The hydroxylation of CMP-N-acetylneuraminic acid (CMP-NeuAc) in the formation of CMP-N-glycolylneuraminic acid requires several components which comprise an electron transport system. A protein, which replaces one of the components, was purified to homogeneity from a horse erythrocyte lysate. Based on its partial amino acid sequence and immunological cross-reactivity, this protein was identified as soluble cytochrome b5 lacking the membrane domain of microsomal cytochrome b5. The electron transport system involved in CMP-NeuAc hydroxylation was reconstituted, and then characterized using the purified horse soluble cytochrome b5 and a fraction from mouse liver cytosol. The hydroxylation reaction requires a reducing reagent, DTT being the most effective. Either NADH or NADPH was used as an electron donor, but the activity with NADPH amounted to about 74% of that with NADH. The hydroxylation was inhibited by salts and azide due to interruption of the electron transport from NAD(P)H to cytochrome b5 and in the terminal enzyme reaction, respectively.     

Nondenaturing two-dimensional electrophoresis enzyme profile involving activity and sequence structure of cytosol proteins from mouse liver

After cytosol proteins in the mouse liver were separated by nondenaturing two-dimensional electrophoresis (2-DE), activities of several enzymes, such as fructose bisphosphatase, sorbitol dehydrogenase and malate dehydrogenase, transferase and sorbitol dehydrogenase, or several dehydrogenases, were analyzed on the same 2-D gel. Further, peptidase (or protease) activity can be examined by matrix-assisted laser desorption/ionization-time of flight-mass spectrometry (MALDI-TOF-MS) when peptides such as angiotensin and adenocorticotropic hormone are incubated in the presence of the cytosol protein separated by nondenaturing 2-DE. Sequence structures of proteins on the 2-D gel were analyzed by peptide mass fingerprinting using MALDI-TOF-MS or by peptide sequencing using electrospray ionization-tandem mass spectrometry (ESI-MS/MS). The combination of activity and sequence structure accurately verified the position and activity range of the separated enzymes on the nondenaturing 2-D gel. From these results, we created a nondenaturing 2-D enzyme profile involving activities and sequence structure of cytosol proteins from mouse liver. This profile can be used for checking whether activities of enzymes were specifically or nonspecifically inhibited by inhibitors.     

Arrangement of glucose residues in the lipid-linked oligosaccharide precursor of asparaginyl oligosaccharides.

The lipid-linked oligosaccharide synthesized in vitro, in the presence of 1.0 microM UDP-[3H]Glc, GDP-[14C]Man, and UDP-GlcNAc has been isolated and the structure of the oligosaccharide has been analyzed. The oligosaccharide contains 2 N-acetylglucosamine, 9 mannose, and 3 glucose residues. The N-acetylglucosamine residues are located at the reducing terminus. The 3 glucose residues are arranged in a linear order at one of the nonreducing termini in the sequence Glc 1,2--Glc 1,3--Glc--(Man)9 (GlcNAc)2. The structural analysis was made possible largely by the availability of glucosidase preparations of fungal anad microsomal origin which remove glucose residues from the oligosaccharide without releasing mannose residues.     

Apoptosis of mouse liver nuclei induced in the cytosol of carrot cells

We report here the apoptosis of mouse liver nuclei induced in the cytosol of carrot cells by cytochrome c. Several typical characteristics of apoptosis, such as chromatin condensation, margination and apoptotic bodies, were detected. The result of DNA gel electrophoresis showed that DNA was degraded into nucleosomal fragments. The terminal deoxynucleotidyl transferase-mediated dUTP-digoxigenin nick end labelling procedure was also performed to detect the breakage of 3'-OH ends of a DNA strand. Furthermore, we found that nuclear lamins were degraded from 88 kDa and 66 kDa to 37 kDa and 47 kDa fragments. The DNA fragmentation could be inhibited by AC-DEVD-CHO and AC-YVAD-CHO. The results indicate that the apoptosis in plant cells may share some similar pathways to apoptosis in animal cells.     

Man2C1, an alpha-mannosidase, is involved in the trimming of free oligosaccharides in the cytosol

The endoplasmic-reticulum-associated degradation of misfolded (glyco)proteins ensures that only functional, correctly folded proteins exit from the endoplasmic reticulum and that misfolded ones are degraded by the ubiquitin-proteasome system. During the degradation of misfolded glycoproteins, they are deglycosylated by the PNGase (peptide:N-glycanase). The free oligosaccharides released by PNGase are known to be further catabolized by a cytosolic alpha-mannosidase, although the gene encoding this enzyme has not been identified unequivocally. The findings in the present study demonstrate that an alpha-mannosidase, Man2C1, is involved in the processing of free oligosaccharides that are formed in the cytosol. When the human Man2C1 orthologue was expressed in HEK-293 cells, most of the enzyme was localized in the cytosol. Its activity was enhanced by Co2+, typical of other known cytosolic alpha-mannosidases so far characterized from animal cells. The down-regulation of Man2C1 activity by a small interfering RNA drastically changed the amount and structure of oligosaccharides accumulating in the cytosol, demonstrating that Man2C1 indeed is involved in free oligosaccharide processing in the cytosol. The oligosaccharide processing in the cytosol by PNGase, endo-beta-N-acetylglucosaminidase and alpha-mannosidase may represent the common 'non-lysosomal' catabolic pathway for N-glycans in animal cells, although the molecular mechanism as well as the functional importance of such processes remains to be determined.     

The enzymatic defluorination of fluoroacetate in mouse liver cytosol: the separation of defluorination activity from several glutathione S-transferases of mouse liver

The liberation of free fluoride ion from fluoroacetate (FAc) proceeds as an enzyme-catalyzed dehalogenation reaction in the soluble fractions of several organs of the CFW Swiss mouse. Liver contained the highest FAc defluorinating activity. The enzyme activity in other organs decreased in the order kidney greater than lung greater than heart greater than testes. No activity was detected in the brain. Experiments were designed to characterize and identify the enzyme species responsible for FAc metabolism in liver. Enzyme activity was dependent on the concentration of glutathione (GSH) in the assay mixture, with maximal activity occurring above 5 mM. The dehalogenation of FAc had an apparent Km of 7.0 mM when measured in the presence of a saturating concentration of GSH. An increase in the pH of the assay mixture enhanced fluoride release in both phosphate and borate buffer. The defluorination activity was reduced to negligible levels when stored for 24 h at 4 degrees C. The addition of either GSH, dithiothreitol, or 2-mercaptoethanol increased stability, with the latter providing protection for greater than 150 h at a concentration of 15 mM. DEAE anion-exchange chromatography separated the defluorinating activity from 90% of the soluble GSH S-transferase activity measured with 1-chloro-2,4-dinitrobenzene. FAc defluorination activity did not bind to a GSH affinity column which selectively separates it from a group of anionic GSH S-transferases. The GSH-dependent enzyme which dehalogenates FAc has unique properties and can be separated from the liver GSH S-transferases previously described in the literature.     

delta-Aminolevulinate synthetases in the liver cytosol fraction and mitochondria of mice treated with allylisopropylacetamide and 3,5-dicarbethoxyl-1,4-dihydrocollidine.

Hepatic delta-aminolevulinate (ALA) synthetase was induced in mice by the administration of allylisopropylacetamide (AIA) and 3,5-dicarbethoxy-1,4-dihydrocollidine (DDC). In both cases, a significant amount of ALA synthetase accumulated in the liver cytosol fraction as well as in the mitochondria. The apparent molecular weight of the cytosol ALA synthetase was estimated to be 320,000 by gel filtration, but when the cytosol ALA synthetase was subjected to sucrose density gradient centrifugation, it showed a molecular weight of 110,000. In the mitochondria, there were two different sizes of ALA synthetase with molecular weights of 150,000 and 110,000, respectively; the larger enzyme was predominant in DDC-treated mice, whereas in AIA-treated mice and normal mice the enzyme existed mostly in the smaller form. When hemin was injected into mice pretreated with DDC, the molecular size of the mitochondrial ALA synthetase changed from 150,000 to 110,000. The half-life of ALA synthetase in the liver cytosol fraction was about 30 min in both the AIA-treated and DDC-treated mice. The half-life of the mitochondrial ALA synthetase in AIA-treated mice and normal mice was about 60 min, but in DDC-treated mice the half-life was as long as 150 min. The data suggest that the cytosol ALA synthetase of mouse liver is a protein complex with properties very similar to those of the cytosol ALA synthetase of rat liver, which has been shown to be composed of the enzyme active protein and two catalytically inactive binding proteins, and that ALA synthetase may be transferred from the liver cytosol fraction to the mitochondria with a size of about 150,000 daltons, followed by its conversion to enzyme with a molecular weight of 110,000 within the mitochondria. The process of intramitochondrial enzyme degradation seems to be affected in DDC-treated animals.     

Direct analysis of the mini-exon donor RNA of Trypanosoma brucei: detection of a novel cap structure also present in messenger RNA.

The mini-exon, a short segment found at the 5' end of trypanosome mRNAs, is contributed by a small RNA, the mini-exon donor (medRNA). In vivo 32P-labeled medRNA, a set of smaller RNAs related to it, and mRNA, were purified from Trypanosoma brucei by hybrid selection and gel electrophoresis. Using RNA fingerprinting and sequencing techniques, mini-exon oligonucleotides were identified and characterized. We detected a novel 5' terminal capped oligonucleotide present in both medRNA and mRNA. This structure contained m7G and at least four modified nucleotides, not identified previously. If the T. brucei mini-exon has exactly four transcribed nucleotides upstream from its originally designated 5' end, it would begin with the sequence: m7GpppA*A*C*U*AA*CG (asterisks denote modification) and medRNA would be 140 nucleotides long, excluding the m7G residue. The mini-exon contains, and retains during its transfer to mRNA, a novel 5' terminal structure whose presence could confer unique functional attributes.     

Transport of free polymannose-type oligosaccharides from the endoplasmic reticulum into the cytosol is inhibited by mannosides and requires a thapsigargin-sensitive calcium store

The transport of free polymannose-type oligosaccharides from the lumen of the endoplasmic reticulum into the cytosol has been recently demonstrated (Moore,S.E.H., et al., 1995, EMBO J., 14, 6034-6042), but at present little is known of the characteristics of this process. Here, it is shown that inhibition of the transport of endogenously synthesized metabolically radiolabeled free oligosaccharides out of the endoplasmic reticulum into the cytosol of permeabilized HepG2 cells occurs when assays are conducted in the presence of mannose (IC50, 4.9 mM), or its derivatives modified at the first carbon (C1) of the sugar ring; alpha-methyl mannoside (IC50, 2.0 mM), mannoheptulose (IC50, 1.6 mM), and alpha-benzyl mannoside (IC50, 0.8 mM), whereas other monosaccharides (50 mM), differing from mannose at position; C2 (glucose), C3 (altrose), C4 (talose), C5 (l-rhamnose), and C6 (mannoheptose), have little effect. N-Acetylglucosamine does not inhibit oligosaccharide transport and, furthermore, although mannobioses and a mannotriose inhibit free oligosaccharide transport, di-N-acetylchitobiose is without effect. It is also shown that if the transport assay buffer is either depleted of calcium ions, or supplemented with the Ca2+/Mg2+ATPase inhibitor, thapsigargin, or with calcium ionophores, free oligosaccharide transport out of the endoplasmic reticulum is inhibited. These results demonstrate that the terminal nonreducing mannosyl residues of free polymannose-type oligosaccharides and not their N-acetylglucosamine-containing reducing termini, play an important role in the interaction of the free oligosaccharide with the transport machinery, and that this transport process requires the presence of calcium sequestered in the lumen of the endoplasmic reticulum.      

Purification and characterization of a dimer form of the cAMP-dependent protein kinase from mouse liver cytosol

A protein kinase that phosphorylates histones and polysomal proteins was partially purified from mouse liver cytosol. The active enzyme has a molecular mass of 100 kDa and a phosphorylatable subunit of 54 kDa. Biochemical as well as immunological data suggest that the enzyme is a heterodimer composed of the catalytic subunit of cyclic AMP-dependent protein kinase and the R_II regulatory subunit. This RC form does not seem to dissociate upon activation with 3, 5 cyclic AMP and exhibits identical specificity as the classical cAMP-dependent protein kinase (2.7.1.37). The enzyme is affected by the 3, 5 cyclic phosphates of adenosine mainly, but also of guanosine, uridine and cytidine in a substrate-dependent manner. Cyclic nucleotides slightly stimulate phosphate incorporation into histones, while phosphorylation of polysomal proteins in intact polysomes is dramatically increased. The substrate- specific stimulatory effects of 3, 5 cyclic nucleotides are due to repression of the inhibition exerted upon the reaction, by negatively charged macromolecules such as RNA, DNA and to a lesser extent heparin.     

Cytosol-to-lysosome transport of free polymannose-type oligosaccharides. Kinetic and specificity studies using rat liver lysosomes

In hepatocellular carcinoma HepG2 cells, free polymannose-type oligosaccharides appearing in the cytosol during the biosynthesis and quality control of glycoproteins are rapidly translocated into lysosomes by an as yet poorly defined process (Saint-Pol, A., Bauvy, C., Codogno, P., and Moore, S. E. H. (1997) J. Cell Biol. 136, 45-59). Here, we demonstrate an ATP-dependent association of [2-3H]mannose-labeled Man5GlcNAc with isolated rat liver lysosomes. This association was only observed in the presence of swainsonine, a mannosidase inhibitor, which was required for the protection of sedimentable, but not nonsedimentable, Man5GlcNAc from degradation, indicating that oligosaccharides were transported into lysosomes. Saturable high affinity transport (Kuptake, 22.3 microM, Vmax, 7.1 fmol/min/unit of beta-hexosaminidase) was dependent upon the hydrolysis of ATP but independent of vacuolar H+/ATPase activity. Transport was inhibited strongly by NEM and weakly by vanadate but not by sodium azide, and, in addition, the sugar transport inhibitors phloretin, phloridzin, and cytochalasin B were without effect on transport. Oligosaccharide import did not show absolute specificity but was selective toward partially demannosylated and dephosphorylated oligosaccharides, and, furthermore, inhibition studies revealed that the free reducing GlcNAc residue of the oligosaccharide was of critical importance for its interaction with the transporter. These results demonstrate the presence of a novel lysosomal free oligosaccharide transporter that must work in concert with cytosolic hydrolases in order to clear the cytosol of endoplasmic reticulum-generated free oligosaccharides.