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Pseudomonas aeruginosa is an opportunistic Gram-negative bacterium that causes severe infections in a number of hosts from plants to mammals. A-band lipopolysaccharide of P. aeruginosa contains d-rhamnosylated O-antigen. The synthesis of GDP-d-rhamnose, the d-rhamnose donor in d-rhamnosylation, starts from GDP-d-mannose. It is first converted by the GDP-mannose-4,6-dehydratase (GMD) into GDP-4-keto-6-deoxy-d-mannose, and then reduced to GDP-d-rhamnose by GDP-4-keto-6-deoxy-d-mannose reductase (RMD). Here, we describe the enzymatic characterization of P. aeruginosa RMD expressed in Saccharomyces cerevisiae . Previous success in functional expression of bacterial gmd genes in S. cerevisiae allowed us to convert GDP-d-mannose into GDP-4-keto-6-deoxy-d-mannose. Thus, coexpression of the Helicobacter pylori gmd and P. aeruginosa rmd genes resulted in conversion of the 4-keto-6-deoxy intermediate into GDP-deoxyhexose. This synthesized GDP-deoxyhexose was confirmed to be GDP-rhamnose by HPLC, matrix-assisted laser desorption/ionization time-of-flight MS, and finally NMR spectroscopy. The functional expression of P. aeruginosa RMD in S. cerevisiae will provide a tool for generating GDP-rhamnose for in vitro rhamnosylation of glycoprotein and glycopeptides. [ABSTRACT FROM AUTHOR]

Mannose analogues (2-deoxy-d-glucose, 2-deoxy-2-fluoro-d-glucose and 2-amino-2-deoxy-d-mannose) have been used to study glycosylphosphatidylinositol (GPtdIns) biosynthesis and GPtdIns protein anchoring in protozoal and mammalian systems. The effects of these analogues on GPtdIns biosynthesis and GPtdIns-protein anchoring of the human malaria parasite Plasmodium falciparum were evaluated in this study. At lower concentrations of 2-deoxy-d-glucose and 2-deoxy-2-fluoro-d glucose (0.2 and 0.1 mm, respectively), GPtdIns biosynthesis is inhibited without significant effects on total protein biosynthesis. At higher concentrations of 2-deoxy-d-glucose and 2-deoxy-2-fluoro-d-glucose (1.5 and 0.8 mm, respectively), the incorporation of [3H]glucosamine into glycolipids was inhibited by 90%, and the attachment of GPtdIns anchor to merozoite surface protein-1 (MSP-1) was prevented. However, at these concentrations, both sugar analogues inhibit MSP-1 synthesis and total protein biosynthesis. In contrast to 2-deoxy-2-fluoro-d-glucose and 2-amino-2-deoxy-d-mannose (mannosamine), the formation of new glycolipids was observed only in the presence of tritiated or nonradiolabelled 2-deoxy-d-glucose. Mannosamine inhibits GPtdIns biosynthesis at a concentration of 5 mm, but neither an accumulation of aberrant intermediates nor significant inhibition of total protein biosynthesis was observed in the presence of this analogue. Furthermore, the [3H]mannosamine-labelled glycolipid spectrum resembled the one described for [3H]glucosamine labelling. Total hydrolysis of mannosamine labelled glycolipids showed that half of the tritiated mannosamine incorporated into glycolipids was converted to glucosamine. This high rate of conversion led us to suggest that no actual inhibition from GPtdIns biosynthesis is achieved with the treatment with mannosamine, which is different to what has been observed for mammalian cells and other parasitic protozoa. [ABSTRACT FROM AUTHOR]

Characterizes the pig liver GDP-mannose pyrophosphorylase (GMPP). Expression of GMPP in Escherichia coli cells; Determination on the molecular mass of GMPP; Analysis on the properties of recombinant GMPP.

The surface antigens of the free-living protozoan Paramecium primaurelia belong to the family of glycosylphosphatidylinositol (GPtdIns)-anchored proteins. Using a cell-free system prepared from P. primaurelia, we have described the structure and biosynthetic pathway for GPtdIns glycolipids. The core glycans of the polar glycolipids are modified by a mannosyl phosphate side chain. The data suggest that the mannosyl phosphate side chain is added onto the core glycan in two steps. The first step involves the phosphorylation of the GPtdIns trimannosyl conserved core glycan via an ATP-dependent kinase, prior to the addition of the mannose linked to the phosphate group. We show that dolichol phosphate mannose is the donor of all mannose residues including the mannose linked to phosphate. Furthermore, we were able to identify in vitro a hydrophilic intermediate containing an additional N-acetylgalactosamine linked to the mannosyl phosphate side chain. The addition of this purified hydrophilic radiolabelled intermediate into the cell-free system leads to a loss of the GalNAc residue and its conversion to the penultimate intermediate having only mannosyl phosphate as a side chain. Together the data indicate that the GalNAc-containing intermediate is a transitional intermediate. We suggest that the GalNAc-containing intermediate is essential for biosynthesis and maturation of GPtdIns precursors. It is hypothesized that this oligosaccharide processing in the course of GPtdIns biosynthesis is required for the translocation of GPtdIns from the cytoplasmic side of the endoplasmic reticulum to the luminal side. [ABSTRACT FROM AUTHOR]

A particulate insoluble fraction from Candida albicans NIH B-792 (serotype B) strain cells was obtained as the residue after extracting a 105000×g pellet of cell homogenate with 1% Triton X-100 Incubation of this fraction with a mannopentaose, Manα1→3Manα1→2Manα1→Manα1→2Man, in the presence of GDP-mannose and Mn2- at pH 6.0 gave a branched mannohexaose, Manα1→3Manα1→2Manα1→2Manα1→2Man, ↑6 Manα1 the structure of which was identified by means of sequential NMR assigtmqent. However, the enzyme fraction obtained from Candida parapsilosis gave Manα1→2Manα1→3 Manα1→2Manα1→2Manα1→2 Man under the same conditions. These results demonstrate the finding that the structural difference in the mannans of these two species is due to the presence of α-1,6-linked branching mannose units in the C, albicans mennan [Shibata, N., Ikuta, K., Imai, T., Satoh, Y., Satoh, R., Suzuki, A,. Kojima, C., Kobayashi, H., Hisamichi, K, & Suzuki, S (1995) J. Biol. Chem. 270, 1113–1122]. The substrate-specificity study of the enzyme indicated that the structural requirement of the α-1,6-mannosyltransferase is Manα1→3-Manα1→. The α-1,6-mannosyltransferase also transferred the α-1,6-linked branching mannose unit to the mannan of Saccharomyces cerevisiae. The transformation of the mannan was detected by the appearance of antigenic factor 4 using an enzyme-linked immunosorbent assay and two-dimensional bomonuclear Hartmann-Hahn spectroscopy. [ABSTRACT FROM AUTHOR]

The mannose transporter of the bacterial phosphotransferase system mediates carbohydrate transport across the cytoplasmic membrane concomitant with carbohydrate phosphorylation. It also functions as a receptor for bacterial chemotaxis [ Adler, J & Epstein, W. (1974) Proc. Natl Acad, Sci. USA 71, 2895- 2899] and is required for infection of the cell by bacteriophage where it most likely functions as a pore for penetration of phage DNA [ElIiott, J. & Arber, W (1 978) Mol & Gen. Genet. 161, 1-8]. The transporter consists of two transmembrane subunits (27 kDa IICMan and 31 -kDa lIDMan) and a hydrophilic subunit (35-kDa lIABMan). Protein fusions of IICMan and IIDMan with β-gaIaetosidase (LacZ) and with alkaline phosphatase (PhoA) were analyzed to determine the membrane topology of the two proteins Protein fusions were obtained by progressively deleting the manY and manZ genes from their ends and ligating them to manZ and phoA that lack promotor and leader sequences. Based on the analysis of 30 HCMan PhoA, 10 IICMMan LacZ, 12 IIDMan- PhoA, and 30 lIDMan LacZ fusions, it is predicted that IICMan has six membrane-spanning segments with the N- and C-termini on the cytoplasmic face of the membrane. lIDMan is anchored in the membrane by a single membrane-spanning segment at the end of the C-terminus, while most of the protein (250 residues) protrudes into the cytoplasm. [ABSTRACT FROM AUTHOR]

46-kDa mannose-6-phosphate receptor forms homooligomers in cell membranes and in detergent solution. The quaternary structure of detergent-solubilized 46-kDa mannose 6-phosphate receptor is regulated by the presence of ligands, pH and receptor concentration [Waheed, A. & von Figura, K. (1990) Eur. J. Biochem. 193, 47-54). To find out whether the intracellular recycling of 46-kDa mannose 6-phosphate receptor is accompanied by changes in its quaternary structure, we have performed chemical cross-linking in membranes of intact cells. In all conditions tested, the dimer was the predominating form (more than 67% of total 46-kDa mannose 6-phosphate receptor). The amount of trimeric and tetrameric forms varied among cell lines and contributed up to 20% of total endogenous 46-kDa mannose 6-phosphate receptor in human and mouse fibroblasts. Within a given cell line, the ratio of the oligomers was not significantly changed upon elevating endosomal pH by bafilomycin A1, upon changes in receptor occupancy (treatment of cells with tunicamycin or use of mouse fibroblasts deficient in 300-kDa mannose 6-phosphate receptor), nor upon depletion of adaptors from clathrin-coated vesicles of the trans Golgi network by brefeldin A. At the cell surface, where 46-kDa mannose 6-phosphate receptor does not bind ligands, the percentage of dimer was similar to that observed intracellularly. thus, the oligometric state of 46-kDa mannose 6-phosphate receptor apparently does not change during recycling as well as binding and dissociation of ligands. In view of the abundance of the dimer of 46-kDa mannose 6-phosphate receptor in situ, our data sugest that it represents the main physiologically active form of the receptor, and therefore present indirect evidence that binding of ligands to 46-kDa mannose 6-phosphate receptor is probably regulated by conformational changes of receptor or ligand rather than by change in the quaternary structure. [ABSTRACT FROM AUTHOR]

Gaucher's disease is an inherited lysosomal storage disorder that is caused by a deficiency of glucocerebrosidase. The resulting accumulation of the substrate glucosylceramide in macrophages of liver, spleen, and bone marrow causes severe clinical symptoms. Gaucher's disease is treated by intravenous administration of a modified glucocerebrosidase (Alglucerase), which has exposed mannose residues to promote uptake by target macrophages. To evaluate the effectiveness of the targeting of Alglucerase, we studied the fate of the enzyme in the rat. Intravenously injected Alglucerase was rapidly cleared from the circulation (half-life 2.0 + 0.5 min). The liver was the main site of uptake. with 65.6 π 1.2% of the dose present at 10 min after injection. Smaller amounts (<3% of the dose) were taken up by spleen and bone marrow. Previous injection with mannan substantially increased the plasma half-life of the enzyme (14.8±3.2 min versus 1.7±0.3 min in solvent-preinjected controls) and uptake of the enzyme by liver. spleen and bone marrow was reduced by >90%. These findings indicate that the enzyme is taken up by these organs via mannose-specific receptors. Subcellular fractionation of the liver indicated that the enzyme is internalized and transported to the lysosomes. By isolating various liver cell types after injection of the Alglucerase. it was found that endothelial cells are the main site of uptake of the enzyme: 6O.8±3.4% of the total liver uptake. Parenchymal and Kupffer cells were responsible for 31.0± 3.1 % and 8.2±0.7% of the hepatic uptake. respectively We conclude that Alglucerase is rapidly cleared from the circulation by mannose-specific receptors in liver, spleen. and bone marrow. However, less than 10% of the enzyme taken up by the liver is accounted for by Kupffer cells, the hepatic target cells for therapeutic intervention. It is suggested that alterations of the formulation of the therapeutic enzyme may lead to a higher uptake by Kupffer cells and other macrophages, and thus to a more (cost)effective therapy of Gaucher's disease. [ABSTRACT FROM AUTHOR]

Two lectins were isolated from the bulbs of Tulipa cv. Apeldoorn and their corresponding cDNA clones analyzed. The first, called TxLMII (second mannose-binding Tulipa hybrid lectin), is a novel mannose-binding tulip lectin. Based on its molecular structure, carbohydrate-binding specificity and amino acid sequence, TxLMII belongs to the superfamily of mannose-binding monocot lectins which are also found in representatives of the plant families Amaryllidaceae, Alliaceae, Orchidaceae and Araceae. Molecular cloning of the second lectin, called TxLCI (first Tulipa hybrid lectin with complex specificity), allowed determination unambiguously of the molecular structure of this previously described protein. In addition, evidence is presented that each TxLCI subunit possesses a mannose-binding site and an N- acetylgalactosamine-binding site, which act independently of each other. Both binding sites are located in a separate domain of the lectin polypeptide. Since the first domain of TxLCI shows sequence similarity to TxLMII, it is suggested that their genes evolved from a common ancestor. [ABSTRACT FROM AUTHOR]

Describes the experimental procedure for the covalent cuopling of biotinamidohexanoyl hydrazide to the ManLAM whose functional and structural integrity was not significantly altered by the procedure. Dependence of the interaction on the presence of serum and divalent cations; Involvement of a protease-sensitive membrane receptor.

Targeted disruption of the Mr 46000 mannose 6-phosphate receptor (MPR 46) in mice is associated with normal levels of lysosomal enzymes in the circulation, while in MPR46-deficient cells an increased secretion of iysosomal enzymes is apparent [Köster. A., Saftig, P., Matzner. U., von Figura, K., Peters, C. & Pohlmann, R. (1993) EMBO J. 12, 5219-5223]. This points to the existence of mechanisms that prevent or compensate for mistargeting of lysosomal enzymes in vivo. In the present study, we have injected inhibitors of three carbohydrate-specific endocytotic receptors into MPR 46-deficient and control mice. Inhibition of these receptors was associated with a pronounced increase of three lysosomal enzymes in the serum of MPR 46-deficient mice. These results clearly show that lysosomal enzymes are mistargeted in MPR46-deficient mice and that carbohydratespecific endocytotic receptors are part of the mechanisms that compensate for the mistargeting of lysosomal enzymes in MPR 46-deficient mice. Moreover. evidence was obtained that, also in control mice, the steady-state level of some lysosomal enzyme is controlled by these receptors. [ABSTRACT FROM AUTHOR]

Mannose-binding lectins were purified from the leaves of three Orchidaceae species, namely Listera ovata (twayblade), Epipactis helleborine (broad-leaved helleborine) and Cymbidium hybrid. using affinity chromatography on mannose-Sepharose-4B. Apparently, the Orchidaceae lectins are dimeric proteins composed of lectin subunits of 12-13 kDa. All or the isolated lectins exhibit exclusive specificity towards mannose. A eDNA library constructed from poly(A) rich RNA isolated from leaves of L. ovata was screened for eDNA clones encoding the lectin using colony hybridization. Since N-terminal sequence analysis of the twayblade lectin revealed some sequence similarity to the previously cloned mannose-binding lectin from Hippeastrum hybrid (amaryllis) ovaries, the amaryllis lectin eDNA clone was used as a probe to screen the L. ovata library. Subsequently, the eDNA clone encoding the L. ovata lectin was used to screen the eDNA libraries from the taxonomically related orchid species Cymbidium hybrid and E. helleborine. Sequence analysis of the lectin cDNA clones from different Orchidaceae species revealed approximately 50% sequence similarity both at the nucleotide and amino acid level. The Orchidaceae lectins are apparently translated from mRNAs consisting of approximately 800 nucleotides. The primary translation products are preproproteins which are converted into the mature lectms following post-translational modifications. Southern blot analysis of genomic DNA has shown that the lectins are most probably encoded by a family of closely related genes which is in good agreement with the sequence heterogeneity found between different lectin cDNA clones of one species. [ABSTRACT FROM AUTHOR]

All possible N-glycosylation sites of the σ subunit of the nicotinic acetylcholine receptor from Torpedo californica electric tissue are utilized. By a combination of microsequencing and mass spectrometry, it was shown that a high-mannose-type oligosaccharide is bound at Asn143 of the σ subunit. The oligosaccharide at positions Asn70 and Asn208 of the σ subunit are probably of the complex type. The utilized glycosylation sites pose restrictions on possible transmembrane folding models of the subunit. [ABSTRACT FROM AUTHOR]

Phosphomannose isomerase catalyses the interconversion of fructose-6-P and mannose-6-P and has a critical role in the supply of D-mannose derivatives required for many eukaryotic glycosylation reactions. Three classes of enzymes processing phosphomannose-isomerase activity have been identified in bacteria and lower eukaryotes. We have purified human phosphomannose isomerase to homogeneity from placental tissue. Protein sequence information obtained from internal fragments of the protein was used to design degenerate oligonucleotides which were used to amplify a fragment of a human phosphomannose-isomerase cDNA. A full-length cDNA was isolated from a human testes λgt11 library using this garment as a probe. The cDNA encoded a protein with significant sequence identity to fungal and some bacterial phosphomannose isomerases but was unrelated to those from other bacteria. Based on amino acid sequence identity we propose a classification system for enzymes with phosphomannose-isomerase activity. The cDNA, under the control of the GALI promoter, was expressed in a Saccharomyces cerevisiae strain from which the native gene encoding phophomannose isomerase had been deleted. The human enzyme was found to be able to functionally substitute for the yeast enzyme. phosphomannose-isomerase mRNA was found in all human tissues tested but was more highly expressed in heart, brain and skeletal muscle. The cDNA was expressed in Escherichia coli permitting the isolation of pure recombinant protein which will be used for kinetic and structural studies. [ABSTRACT FROM AUTHOR]

The structure N-linked oligosaccarides, especially the distribution of sialic acid species, present on porcine plasma vitronectin were elucidated. Oligosaccharides were released from the vitronectin by N-glycosidase F digestion and tagged with 2-aminopyridine, and the pyridylamino­oligosaccharides were fractionated by anion­exchange and reverse­phase HPLC. Nine major pyridylamino­oligosaccharides were isolated. The linkages and locations of sialic acids were determined by a novel approach involving desialylation with Salmonella sialidase in combination with acid desialylation. After desialylation, the asialo­forms were analyzed by two-dimensional sugar mapping, component sugar analysis and 4-MHz ¹H-NMR spectroscopy. The major oligosaccharides of porcine vitronectin were of the fucosylated biantennary type, with a small amount of the triantennary N-acetyllactosamine type, to which 1–3 mol sialic acids was linked. Sialic acids were linked predominantly through α2–6 linkages, although α2–3 linkages were also present,and fucose was linked to the innermost N-acetylglucosamine through an α1–6 linkage. It was found that every pyridylamino­shy;oligosaccharide population contained N-glycolylneuraminic acid and N-acetylneuraminic acid in a molar ration of 1:2–9, and that N-glycolneuraminic acids were located predominantly on the Man &alpha1–6 arm. [ABSTRACT FROM AUTHOR]

The fate of a mutant form of each of the two yeast vauolar enzymes proteinase yscA (PrA) and carboxypeptidase yscY (CPY) has been investigated. Both mutant proteins are rapidly degraded after entering the secretory pathway. Mutant PrA is deleted in 37 amino acids spanning the processing site region of the PrA pro-petide. The mutant enzyme shows no activity towards maturation of itself or other vacuolar hydrolases, a function of wild-type PrA. Mutant CPY carries and Arg instead of a Gly residue in a highly conserved region, two positions distant from the active-site Ser. In contrast to wild-type CPY, the mutant form was quickly degraded by trypsin in vitro, indicating an altered structure. Using antisera specific for α-1→6 and α-1→3 outer-chain mannose linkages, no Golgi-specific carbohydrate modification could be detected on either mutant protein Subcellular fractionation studies located both mutant enzymes in the endoplasmic reticulum. Degradation kinetics of both proteins show the same characteristics, indicating similar degradation pathways. The degradation process was shown to be independent of a functional sec18 gene product and takes place before Golgi-specific carbohydrate modifications occur. The proteasome, the major proteolytic activity of the cytoplasm, is not involved in this degradation event. All degradation characteristics of the two mutant proteins are consistent with a degradation process within the endoplasmic reticulum (‘ER degradation’). [ABSTRACT FROM AUTHOR]

Characterizes lectin cDNA clones encoding the two mannose-binding lectins from the bulbs of ramsons, Allium ursinum L. Sequence comparison of the different cDNA clones; Occurrence of glycosylated lectin precursors in the organelle fraction of radioactively labeled ramson bulbs; Difference in the organization of the lectin genes in A. ursinum and A. sativum.

Presents a structural study of a cell-wall mannan of Saccharomyces kluyveri IFO 1685 strain. Structures of mannopentaose and mannohexaose; Presence of a branched side chain and Β-1,2-linkage.

A protein-O-D-mannosyltransferase (PMT) assay was optimized using a microsomal membrane preparation from Candida albicans and a peptide acceptor, YNPTSV. [14C]Mannose was transferred from dolichyl phosphate [14C]mannose to the threonine or serine residues of the peptide. During the assay, the peptide was highly susceptible to proteolysis. A blocked peptide Ac YNPTSV-NH2 was resistant to proteolysis and was apparently a better acceptor for o-mannosylation. This peptide had a Km value of 4.3 mM in the assay. A number of other peptides were tested with altered sequences. Maximum incorporation of [14C]mannose was obtained with a pentapeptide YATAV (Km 2.2 mM) which was further improved by blocking both ends: Ac-YATAV-NH2 (Km 0.25 mM). Finally, and unexpectedly, an improvement was noted if the acetyl group on the N terminus was replaced by a biotin residue. Biotin-YATAV-NH2 had a km of 0.075 nM. The biotin residue may be important in increasing the lipophilicity of the peptide and thus aid its adhesion to the Candida membranes. The simplest peptide that could act as an efficient mannose acceptor was Ac-ATA-NH2 whilst no incorporation was observed with AC-GTG-NH2. [ABSTRACT FROM AUTHOR]

The N-linked oligosacchafides of C-reactive protein (CRP) from the arachnid Limulus polyphemus, the horseshoe crab, were characterized after their release by hydrazinolysis, re-N-acetylation, and reduction with NaB3H4. High-voltage paper electrophoresis of the reduced oligosaccharides revealed only neutral species. Gel-permeation chromatography on Bio-Gel P4 yielded five tractions. The oligosaccharide fractions were further fractionated using high-voltage borate paper electrophoresis and Dionex BioLC ion-exchange chromatography. The oligosaccharides were structurally characterized by sequential exoglycosidase digestion, fragmentation by acetolysis and methylation analysis. Three major structures were found, of which two were the biantennary oligomannose type with compositions Man5GlcNAc2 (B-1), Man4GlcNAc2 (C-3) and one was the monoantennary structure Man3GlcNAc2 (D-1). The biantennary oligomannose structures B-1 and C-3 contained the structural unit Manα6Manα6R. This unusual arrangement of mannose linkages suggests a biosynthetic pathway in Limulus which differs from that reposed in mammals, plants and the parasitic protozoa. [ABSTRACT FROM AUTHOR]

Thyroglobulin, the major secretory product of thyrocytes, is the macromolecular precursor of thyroid hormones. After its synthesis, thyroglobulin follows a complex secretion, storage and recapture pathway to lysosomes. Porcine thyroglobulin was shown to carry the mannose 6-phosphate- (Man6P)-recognition marker on its N-linked glycans. Since the cation-independent Man6P receptor could also be found on the apical plasma membrane of porcine thyrocytes, we examined the significance of the Man6P signal for the transport of thyroglobulin. Here, we present data implying that Man6P receptors are not relevant for endocytosis of thyroglobulin in thyrocytes. Instead, we provide evidence for the existence of specific, low-affinity-binding sites for thyroglobulin on the apical plasma membrane of thyrocytes responsible for endocytosis of thyroglobulin. Binding studies with intact, polar-organized porcine thyrocytes grown on collagen-coated filters revealed cooperative and saturable binding of thyroglobulin to the apical-plasma-membrane domain at relatively high concen- trations of thyroglobulin (20 μM). These observations show that low-affinity interactions between thyroglobulin and the apical plasma membrane play a key role in endocytosis of thyroglobulin and hormone formation in the thyroid. The data in this publication have been published as an abstract [Lemansky, P. and Herzog, V. (1991) J. Cell Biol. 115, 261a]. [ABSTRACT FROM AUTHOR]

Mitochondrial dolichyl-phosphate mannose synthase has been purified to homogeneity using an original procedure, reconstitution into specific phospholipid vesicles and sedimentation on a sucrose gradient as final step. The enzyme has an apparent molecular mass of 30 kDa on an SDS/polyacrylamide gel. Increased enzyme activity could be correlated with this polypeptide band. A specific antibody was raised in rabbits against this transferase. Specific IgG obtained from the immune serum removed enzymatic activity from a detergent extract of mitochondrial outer membrane and reacted specifically with the 30-kDa band on immunoblots. Furthermore, an immunocytochemical experiment proved the localization of dolichyl-phosphate mannose synthase on the cytosolic face of the outer membrane of mitochondria. [ABSTRACT FROM AUTHOR]

After treatment with swainsonine, an inhibitor of both lysosomal α-mannosidase and Golgi α-mannosidase-II activities, analysis of [³H]mannose-labeled glycans showed that HT-29 cells, derived from a human colonic adenocarcinoma, displayed distinct patterns of N-glycan expression, depending upon their state of enterocytic differentiation. In differentiated HT-29 cells hybrid-type chains were detected, whereas undifferentiated HT-29 cells accumulated high-mannose-type oligosaccharide, despite our demonstration of Golgi α-mannosidase-II activity in both cell populations. Pulse/chase experiments carried out in the presence of swainsonine revealed that the persistence of high-mannose-type chains in undifferentiated HT-29 cells was the result of the stabilization of glycoproteins substituted with these glycans. These data suggest that in undifferentiated HT-29 cells, glycoproteins with high-mannose-type oligosaccharides are delivered to a degradative compartment containing swainsonine-sensitive α-mannosidase(s), whereas in differentiated HT-29 cells glycoproteins enter a compartment in which α-mannosidase II (Golgi apparatus) is present. Thus, this apparent dual effect of swainsonine on N-glycan trimming may reflect differences in the intracellular traffic of glycoproteins as a function of the state of enterocytic differentiation of HT-29 cells. [ABSTRACT FROM AUTHOR]

Mucins in human amniotic fluid are represented by two distinct molecular species, FM-1 and FM-2, with apparent molecular masses of 700 and 570 kDa, respectively, in SDS/polyacrylamide gradient gels. FM-1 and FM-2 were isolated by preparative SDS/PAGE to apparent homogeneity and subjected to structural studies on their carbohydrate portions. The carbohydrate compositions of the mucin species differed only marginally and exhibited significant amounts of mannose. O-linked core-region glycans on human amniotic mucin-derived pronase-stable glycopeptides were analyzed after reductive β-elimination and purification on HPLC by a combination of methylation analysis, electron-impact mass spectrometry of permethylated oligosaccharide alditols and fastatom-bombardment mass spectrometry of acetylated or methylated alditols (positive-ion mode) or aiditol-derived neoglycolipids (negative-ion TLC-MS). The primary structures of major monosaccharides to tetrasaccharides have been established which exhibit at their reducing termini core 1. [ABSTRACT FROM AUTHOR]

Human transferrin receptor was isolated from placenta and from the hepatocarcinoma cell line Hep G2. Asparagine-linked oligosaccharides were released by treatment of tryptic glycopeptides with endo-β-N-acetylglucosaminidase H or peptide-N4-(N-acetyl-β-glucosaminyl)asparagine amidase F. Oligosaccharide alditols were fractionated by anion-exchange high-performance liquid chromatography and by high-pH anion-exchange chromatography. Glycans from placental transferrin receptor were further characterized, after desialylation, by methylation analysis and, in part, by liquid secondary-ion mass spectrometry. Sialylation of placental transferrin receptor was examined by lectin affinity blotting with Sambucus nigra agglutinin and Maackia amurensis agglutinin. In order to trace possible inter-individual differences in N-glycosylation of the receptor, two preparations of placental transferrin receptor purified from two donors were compared. The results demonstrate that human transferrin receptor from placenta predominantly carries diantennary and triantennary N-acetyllactosaminic glycans as well as hybrid-type species, the galactose residues of which being almost completely substituted with (α2–3)-linked sialic acid residues. Distinct differences were noted in the glycosylation pattern of the receptor from different individuals. Transferrin receptor from donor A carried predominantly diantennary and triantennary complex-type glycans, in part fucosylated at the innermost N-acetylglucosamine residue, in addition to small amounts of bisected and of incomplete diantennary species. Placental transferrin receptor from donor B predominantly carried triantennary N-acetyllactosaminic glycans without fucose and hybrid-type oligosaccharides with four or five mannose residues. Distinct from placental transferrin receptor, the receptor from Hep G2 cells contained larger amounts of oligomannosidic glycans with six to nine mannose residues and tetrasialylated complex-type oligosaccharides apart from mono-, di- and trisialylated species. [ABSTRACT FROM AUTHOR]

An overlay and a solid-phase assay are presented which allow the specific detection of 9-Oacetylated sialic acids on sialogtycoconjugates immobilized on microtiter plates, nitrocellulose or separated on thin-layer chromatograms. The assay takes advantage of two different biological properties of influenza C virus, its high-affinity binding to 9-O-acetylated sialic acids and its sialate 9-0 acetylesterase that is used for detection of bound virus with fluorogenic or chromogenic substrates. Though simple and rapid, the assay is highly sensitive with a detection limit of 65 fmol 9-O-acetylated sialic acid in 9-O-acetylated ganglioside GD1a. Influenza C virus is able to bind to a wide spectrum of sialoglycoconjugates like mucins, serum glycoproteins or gangliosides containing naturally or synthetically O-acetylated sialic acids. 9-0 Acetyl-N-glycoloylneuraminic acid can also function as a high-affinity receptor determinant for influenza C virus. While the acetyl ester at the 9 position is essential for virus binding in all cases, a 4-O-acetyl group is not recognized. In addition to α(2,3) or α(2,6) bonds, 9-O-acetyl-N-acetylneuraminic acid in α(2,8) linkage to N-acetylneuraminic acid is also functionally active. [ABSTRACT FROM AUTHOR]

The specificity of the Golgi carrier for CMP-sialic-acids and the lumenal sialylation of glycoconjugates in mechanically permeabilized cells (semi-intact CHO 15B cells) was studied with CM P-activated fluorescent sialic acids as sensitive markers. Semi-intact cells represent a well-established cellular model for studies on the constitutive secretion pathway because the perforated plasma membrane allows membrane-impermeable CMP-sialicacids to gain access to cellular organdies. The subcellular structures of semi-intact cells remain morphologically intact and hence synthetic CMP-sialic-acids can be assayed as substrates for the corresponding Golgi sugar-nucleotide transporter. The results prove that the CMP-sialic-acid carrier is able to translocate fluorescent CMPglycosides, despite the bulky fluoresceinyl residue located at position C5 or C9 of the sialic-acid moiety; the data suggest a slightly higher affinity of the carrier for the C9-substituted CMP-glycoside, whereas the affinity of cellular sialyltransferases is fourfold higher for CMP-5-N-fluoresceinylaminoacetylneuraminic acid (5-FTIUNeuAc; 5-N-fluoresceinylaminoneuraminic acid). Using CMP-9-fluoresceinylthioureido-N-acetylneuraminic acid (CMP-9-FTIUNeuAc), an easy and sensitive fluorometric assay was established for the lumenal sialylation in semi-intact cells. Cellular proteins and gangliosides are both labelled by covalent incorporation of the fluorescent N-acetylneuraminic acid analogue. The assay allows rapid screening for small biomolecules or proteins that influence cellular sialyl transport and sialyl transfer; the lumenal fluorescence incorporation does not require ATP or cytosolic compounds. The suitability of fluorescent CMP-glycosides as markers for intracellular sialylation, proven in this paper, introduces the use of synthetic sialic acids for visualisation of cellular sialic acid pathways by fluorescence microscopy. Based on the data presented here, specific CMP-N-acetylneuraminicacid analogues can be produced and used for the characterization of the Golgi CMP-sialic-acid carrier. [ABSTRACT FROM AUTHOR]

Interleukin 6 (IL-6) is a glycosylated cytokine which is important in exerting cell-specific growthinducing, growth-inhibiting and differentiation-inducing effects. IL-6 produced in mammalian cell lines is heterogeneous, reflecting specific cell-type-dependent post-translational modifications. Native IL-6 was purified from human blood mononuclear cells and the oligosaccharides released, radiolabelled and sequenced by a combination of sequential exoglycosidase digestion using Bio-Gel P-4 high-resolution gel chromatography and acetolysis. N- and O-linked glycans were found. The Nlinked glycans were sialylated di- and tri-antennary complex-type and oligomannose-type structures. However, the most predominant N-linked oligosaccharide was a small tetrasaccharide with the sequence Manα6Manβ4GlcNAc/54GlcNAc. This is the first report of this structure on a circulating glycoprotein. This structure has only previously been reported to be present on the syncytiotrophoblast of human placenta. The presence of the oligomannose structures and the mannose-terminating tetrasaccharide on I L-6 may be important in maintaining a high local concentration of the cytokine while limiting its systemic serum level via interaction with soluble mannose-binding serum lectins. [ABSTRACT FROM AUTHOR]

Sialosyl-lactosylceramide, GM3, is the major ganglioside of human liver, where it constitutes more than 90% of the total lipid-bound sialic acid. When analyzed by thin-layer chromatography, human liver GM3 migrates as two main spots. They are representative of ganglioside molecular species which differ in the acyl moiety. The faster running spot is mainly composed of molecular species with non-hydroxylated C22-C24 acyl chains; the other contains mainly molecular species bearing nonhydroxylated C16-C18 and α-hydroxylated C16-C24 acyl chains. In this study the content of the two GM3 molecular species groups was investigated in 31 subjects ranging from 19 to 85 years of age. By thin-layer chromatography we observed that the group of molecular species containing non-hydroxylatcd C22-C24 acyl chains, decreased linearly with subject age, while that of non-hydroxylated C16-C18 acyl chains and hydroxylated C16-C25 acyl chains increased linearly. Fast-atom-bombardment mass spectrometry performed on seven samples from subjects ranging from 21 to 78 years of age demonstrated that the age-dependent increase of the lower spot is caused by an increase in the hydroxylated fatty acid form of GM3, the content of non-hydroxylated C16C18 fatty acid species remaining constant with age. [ABSTRACT FROM AUTHOR]

The ability of peptide-N4-(N-acetyl-β-glucosaminyl)asparagine amidase F (PNGase F) from Flavobacterium meningosepticum and PNGase A from sweet almonds to deglycosylate N-glycopeptides and N-glycoproteins from plants was compared. Bromelain glycopeptide and horseradish peroxidase-C glycoprotein, which contain xylose linked β1 →2 to β-mannose and fucose linked α1→3 to the innermost N-acetylglucosamine, were used as substrates. In contrast to PNGase A, the enzyme from F. meningosepticum did not act upon these substrates even at concentrations 100-fold higher than required for complete deglycosylation of commonly used standard substrates. After removal of α1→3-1inked fucose from the plant glycopeptide and glycoprotein by mild acid hydrolysis, they were readily degraded by PNGase F at moderate enzyme concentrations. Hence we conclude that α1→3 fucosylation of the inner N-acetylglucosamine impedes the enzymatic action of PNGase F. Knowledge of this limitation of the deglycosylation potential of PNGase F may turn it from a pitfall into a useful experimental tool. [ABSTRACT FROM AUTHOR]

From a genomic library in EMBL 3, two overlapping clones for the human 46-kDa mannose 6-phosphate receptor (MPR46) were isolated, which span the entire coding sequence. The human MPR46 gene is distributed over 12 kb and is divided into seven exons (110-1573 bp). All the intron/exon borders agree with the consensus sequences of splice junctions. Exon 1 codes for a 5' untranslated sequence. The ATG initiation codon begins with the second nucleotide in exon 2. A signal sequence of 26 amino acid residues is followed by the extracytoplasmic (luminal) domain, which extends to exon 5. The transmembrane domain of the receptor spans exons 5 and 6 and the cytoplasmic domain is encoded by exons 6 and 7. The latter domain also codes for an extended 3' untranslated sequence. The transcription-initiation site was defined by primer extension. The sequence upstream of the cap site has strong promoter activity and contains structural elements characteristic of promoters found in housekeeping genes. No correlation between the genomic organization and known protein domains of the MPR46 was apparent. Moreover, the sequence of about 150 amino acids within the luminal domain of MPR46, which is homologous to the 15 repeats that constitute the luminal domain of the 300-kDa mannose 6-phosphate receptor (MPR300), does not correlate with intron/exon borders. MPR46 and MPR300 have therefore diverged from a common ancestral gene before introduction of the present intron sequences. [ABSTRACT FROM AUTHOR]

The enzyme dolichyl-phosphate-D-mannose:protein O-D-mannosyltransferase has been solubilized from Saccharomyces cerevisiae membranes and its mannosyltransferase activity demonstrated using short peptides. The specific activity of the protein was enriched 130-fold before it was further purified by native and SDS gel chromatography. A 92-kDa band correlated well with the enzyme activity; an antibody raised against this protein precipitated the mannosyltransferase. The 92-kDa band was hydrolysed to 84 kDa after treatment with endoglycosidase F, indicating that the protein is a glycoprotein which may contain four carbohydrate chains. The purified mannosyltransferase is distinctly influenced in transfer specificity by amino acids next to serine and threonine within the acceptor peptides. Thus acidic amino acids strongly inhibit acceptor activity as do glycine and proline residues as amino-terminal and carboxy-terminal neighbours, respectively. [ABSTRACT FROM AUTHOR]

At 4°C, detergent-solubilized 46-kDa mannose 6-phosphate receptor (MPR 46) exists as a mixture of dimeric and tetrameric forms IA. Waheed, A. Hille, U. Junghans & K. yon Figura (1990) Biochemistry 29, 2449-2455]. At 37°C, MPR 46 exists as a mixture of monomeric, dimeric and tetrameric forms, which can be separated by sucrose density centrifugation or chromatography on a mannose-6-phosphate affinity matrix. Monomeric MPR 46 did not bind to the affinity matrix, while dimeric and tetrameric receptors were eluted with increasing concentrations of mannose 6-phosphate. Depending on the incubation temperature, dimeric MPR 46 preferentially associates to tetramers (≤16°C) or dissociates to monomers (≥ 25 °C). The presence of mannose 6-phosphate shifts the equilibrium to higher quaternary structure, with a maximal effect at 37°C. Incubation of dimeric or tetrameric receptor at pH 4.0 induces a rapid dissociation of about a third of the receptor with t½ of < 2 min, followed by a phase of slow dissociation. Readjusting the pH to 7.5 induces a rapid formation of dimers and tetramers, which is promoted by the presence of mannose 6-phosphate or an increase of the receptor concentration. These observations may indicate that the recycling of the receptor between the Golgi apparatus, where it binds ligands at near-neutral pH, and the acidic prelysosomes, where it releases the ligands, is associated with a change of its quaternary structure, which in turn may affect the recycling kinetics. In keeping with this assumption, we observed in baby hamster kidney cells over-expressing MPR 46 and in membrane prepared from these cells monomeric, dimeric and tetrameric forms of the receptor. [ABSTRACT FROM AUTHOR]

The insulin-like growth-factor-II/mannose-6-phosphate (IGF-II/Man6P) receptor binds two classes of ligands. insulin-like growth factors and lysosomal enzymes. We have examined the ability of the lysosomal enzyme, βgalactosidase, to modulate the binding of 125I-IGF-II to the receptor. β-Galactosidase purified from bovine testis was fractionated on a DEAE-Sephacel ion-exchange column. Column fractions were assayed for enzymatic activity and for ability to inhibit the binding of 125I-IGF-II to the IGF-II/Man6P receptor. Enzyme fractions eluting at higher NaCl concentrations which had previously been shown to exhibit greater uptake by cells in culture. exhibited greater potency in inhibiting the binding of 125I-IGF-II to the receptor. A pool of these fractions from the DEAE-Sephacel column inhibited 125I-IGF-II binding to pure receptor by 80% with the concentration required for half-maximal inhibition being 25 nM. The inhibition of binding by β-galactosidase was completely blocked by simultaneous incubation with Man6P. Inhibition of the enzymatic activity of β-galactosidase with Dgalactonic acid γ-lactone did not affect the ability of β-galactosidase to inhibit the binding of 125I-IGF-II to the receptor. Scatchard analysis of IGF-II binding to pure receptor in the presence and absence of β-galactosidase showed that β-galactosidase decreased the binding affinity for IGF-II (Kd 0.26 nM versus 1.0 nM in the presence of 57 nM β-galactosidase). We confirmed the observations of others that Man6P alone actually increases the binding of 125I-IGF-II to the IGF-II/Man6P receptor, but we found that this phenomenon was dependent upon the method of preparation of the IGF-II/Man6P receptor. Microsomal membrane preparations, solubilized membranes, and receptors purified on an IGF-II-Sepharose column all exhibited stimulation of 125I-IGF-II binding by Man6P, whereas receptors purified on lysosomal enzyme affinity columns showed little or no stimulation of 125I-IGF-II binding by Man6P. We conclude that β-galactosidase decreases the binding affinity of the IGF-II/Man-6-P receptor for IGF-II by binding with high affinity to the Man6P-recognition site. [ABSTRACT FROM AUTHOR]

The activity of GDPmannose:dolichyl monophosphate mannosyltransferase in inner mitochondrial membranes can be triggered by dolichyl-monophosphate incorporation mediated through phospholipids or fatty acids. The efficiency of this incorporation and the efficiency of the enzyme activity are not equivalent. Among a variety of amphiphiles which were tested, the highest mannosyltransferase activity was obtained with the mixture of lipids extracted from the outer mitochondrial membranes. The results presented here appear consistent only with a mechanism involving collisional contacts of the phospholipid vesicles and fusion with the membranes, ESR spectroscopy confirms that (a) the incorporation process is followed by solubilization of dolichyl monophosphate molecules in the lipid phase and (b) the general organization of the inner mitochondrial membranes is not perturbed by the addition of dolichyl monophosphate. [ABSTRACT FROM AUTHOR]

The enzyme GDP mannose:dolichyl-phosphate O-β-D-mannosyltransferase (GDP-Man:DolP mannosyltransferase) catalyzing the reaction: GDP-man + DolP &lrarr; DolP-Man + GDP has been purified from Saccharomyces cerevisiae to homogeneity. The purification was achieved using a combination of column chromatographic methods with preparative gel electrophoresis. The enzyme has an apparent molecular mass of 30 kDa on SDS/polyacrylamide gels. Enzymatic activity could be correlated directly with this band. Antibodies against the transferase were raised in rabbits. The immune serum obtained removed enzymatic activity from a detergent extract of yeast membranes and reacted specifically with the 30-kDa band on immunoblots. Experiments addressing the orientation of this enzyme in the endoplasmic reticulum membrane are presented by using selective trypsin and N-ethylmaleimide treatment. [ABSTRACT FROM AUTHOR]

The initial plasma clearance and organ distribution of α1-acid glycoprotein and α2-macroglobulin carrying different types of oligosaccharide, side chains was studied in rats. The differently glycosylated proteins were synthesized by rat hepatocytes in culture in the presence of tunicamycin (unglycosylated form), swainsonine (hybrid type), or 1-deoxymannojirimycin (high-mannose type). Deglycosylated glycoproteins (Asn-Glc-NAc) were obtained by endoglucosaminidase H treatment of high-mannose-type glycoproteins. Ten minutes after intravenous injection 3% of complex type, 26% of hybrid type, 84% of high-mannose type. 64% of unglycosylated and 80% of deglycosylated α1-acid glycoprotein disappeared from the plasma. The respective values for α2-macroglobulin were 26%, 42%, 59% and 67%. When the clearance of total hepatic secretory proteins was examined, major differences between glycosylated and unglycosylated (glyco)proteins were found, particularly in the case of low-molecular-mass polypeptides. Whereas complex-type α1-acid glycoprotein and α2-macroglobulin showed no accumulation in various organs, hybrid-type α1-acid glycoprotein and α2-macroglobulin were present in spleen and liver. High-mannose-type α1-acid glycoprotein and α2-macroglobulin also accumulated mainly in spleen and liver. Spleen had the highest specific activity; liver, due to its larger organ mass, represented the major organ for the uptake of high-mannosetype glycoproteins. Competition experiments with mannan and GlcNAc-bovine-serum-albumin showed a mannose/GlcNAc receptor-mediated removal. Whereas unglycosylated α1-acid glycoprotein was taken up by the kidney, unglycosylated α2-macroglobulin was found in the spleen. Deglycosylated glycoproteins (Asn-GlcNAc) were removed from the plasma via two different mechanisms: firstly, clearance by the kidney similar to the unglycosylated glycoproteins; secondly, clearance by a mannose/GlcNAc receptor-mediated uptake mainly into the spleen. We conclude that N-linked oligosaccharide side chains are important for the plasma survival of hepatic secretory glycoproteins and that unphysiologically glycosylated forms are cleared by different mechanisms. [ABSTRACT FROM AUTHOR]

Human factor V was subjected to desialation and deglycosylation to investigate the function of the molecular carbohydrate moiety. Removal of 90% of the sialic acid residues resulted in a 1.5-2-fold increase in clotting activity, and up to 70% deglycosylation in a concurrent decrease in clotting activity. Desialation had no effect on thrombin-induced activation, whereas deglycosylated factor V activation was impaired. Lectin-blot experiments with sialic-acidaspecific Limaxflavus agglutinin (LFA), galactose-specific Ricinus communis agglutinin (RCA-II) and mannose-specific concanavalin A on thrombin-induced factor V fragments revealed the presence of carbohydrate residues in fragments B, C1, D and F1F2. Interestingly, sialic acid was present in C1 whilst galactose was not detectable. Fragment F1F2 contained terminal galactose residues. LFA and RCA-Il inhibited the procoagulant activity of native factor V and of desialated factor V respectively. These investigations distinctly indicate the important role of the human factor V carbohydrate moiety in the process of blood coagulation. [ABSTRACT FROM AUTHOR]

A new lectin has been isolated from the coral Gerardia savaglia by affinity chromatography, using locust gum as an absorbent, and D-mannose as eluant. Final purification was achieved by Bio-Gel P300 gel filtration. The agglutinin is a protein composed of two polypeptide chains with a Mr of 14800; the two subunits are not linked by disulfide bond(s). The isoelectric point is 4.8, the amino acid composition is rich in the acidic amino acids aspartic acid and glutamic acid. The absorption maximum for the protein was at 276 nm; with a molar absorption coefficient of 1.27 × 105 M-1 cm-1. The lectin precipitated erythrocytes from humans (A, B and 0), sheep, rabbit and carp with a titer between 25 and 1010; the affinity constant for lectin binding to sheep red blood cells was 2.8 x 108 M1 and the number of binding sites, 3.2 × 105/cell. Ca2+ ions are required for full activity; the pH optimum lies in the range between 6 and 11. Inhibition experiments revealed that the lectin is specific for D-mannose. The lectin is mitogenic only for those spleen lymphocytes from mice which had been activated by lipopolysaccharide. An interesting feature of this lectin is its ability to bind to glycoproteins present in nuclei from CV-1 monkey kidney cells. The fluorescein-isothiocyanate-labelled lectin reacted with six polypeptides in the nuclear envelope from rat liver (Mr 190000, 115000,80000,62000,56000 and 42000) and with two polypeptides in the nuclear matrix or pore complex lamina fraction (Mr 190000 and 62000). The lectin inhibited the nuclear envelope mRNA translocation system in vitro, it is suggested that this effect is due to an interaction of the lectin with the nuclear glycoproteins gp190 and/or gp62. [ABSTRACT FROM AUTHOR]

Glycoproteins were isolated from the ovary of the starfish Asterias rubens (L.). After delipidation, sugar analysis revealed the presence of mannose, glucose and N-acetylglucosamine in a molar ratio of 9.0:1.3:2.3. Subsequently, hydrazinolysis, re-N-acetylation, reduction and high-voltage paper electrophoresis were carried out, resulting in a mixture of neutral oligosaccharide alditols which was fractionated on Bio-Gel P-4. The alditols, investigated by 500-MHz ¹H-NMR spectroscopy, turned out to be of the oligomannose type or of the glucooligomannose type containing 9 mannose and 1-3 glucose residues. The most abundant compounds were established to be: Manα1 → 2Manα1 → 6 Manα1 →6 6 Manα1 → 2Manα1 → 3 Manβ1→ 4GlcNAcβ1 → 4GlcNAc-ol Manα1 → 2Manα1 → 2Manα1 → 3 and Manα1 → 2Manα1 → 6Man&alpha :1→ 6 Man&alpha:1 &rarr:2M anα1→3 Manβ1→ β1 → 4GlcNAc·ol Glcα1 → 3Manα1 → 2Manα1 → 2Manα1 → 3. [ABSTRACT FROM AUTHOR]

Metabolic labelling of mouse splenocytes with radioactive mannose indicates that the glycosylation process is accompanied by the release of soluble oligomannoside material. Chase experiments with an excess of unlabelled mannose indicate that the radioactivity is mainly chased from oligosaccharide-PP-Dol (PP-Dol = diphosphodolichol): 10% is recovered as (Man)9(GlcNAc)2-P, (Man)9(GlcNAc)2, (Man)9GlcNAc and (Man)5GlcNAc, and 90% is rapidly degraded further. Tunicamycin inhibits both oligosaccharide-PP-Dol synthesis and the formation of the oligosaccharide material to the same extent. The results thus indicate that these soluble oligomannoside structures represent the main steps of the oligosaccharide-PP-Dol catabolic pathway, starting with the cleavage of the diphosphate bond. However, it cannot be excluded that part of this material is released from newly formed glycoproteins. The soluble oligomannoside material does not contain glucose residues despite the fact that part of the oligosacharide-PP-Dol is glucosylated and it was shown, by the use of glucosidase I inhibitors (castanospermine, deoxynojirimycin) that, after cleavage, the glycan moiety of glucosylated oligosaccharide-PP-Dol is first rapidly deglucosylated. These experiments provide a physiological basis to our previous results obtained in vitro and allow the definition of further steps in the catabolic pathway of oligosaccharide-PP-Dol. [ABSTRACT FROM AUTHOR]

Two types of linkages between the carbohydrate and the peptide moiety in the glycopeptide from Ascobolus furfuraceus are described. Treatment with mild alkali produced β-elimination of a small oligosaccharide. Evidence for the O-glycosidic linkage was provided by (a) increase in absorbance at 240 nm, (b) decrease in threonine and serine content after the alkaline treatment and (c) detection of tritiated oligosaccharide following alkaline NaB³H4 reduction. Mannose is the sugar involved in the O-glycosidic linkage. The remaining glycopeptide was branched by galactofuranose units, which were selectivity released by mild acid hydrolysis. The N-glycosidic linkage of the sugar chain was conclusively proved by cleavage with endo-β-N-acetyl-glucosaminidase. Sequential NaB³H4 reduction and acid hydrolysis gave [³H]glucosaminitol. The structure of the sugar chain was studied by 13C NMR spectroscopy and by methylation analysis. [ABSTRACT FROM AUTHOR]

The capsular polysaccharide of Klebsiella serotype K40 contained D-mannose, D-glucuronic acid, D-galactose, and L-rhamnose in the approximate molar ratios 1 : 1 : 1 : 2. The primary structure of the capsular polysaccharide has been investigated mainly by methylation analysis, periodate oxidation, characterization of oligosaccharides, base degradation reaction, and ¹H and 13CNMR spectroscopy. The polysaccharide does not contain any pyruvic acetal or O-acetyl substitution. It has a pentasaccharide repeating unit of the following primary structure: ... [ABSTRACT FROM AUTHOR]

The major urinary trypsin inhibitor (Mr 44000), isolated from human urine, contains 35% carbohydrate. In addition to N-acetylglucosamine and neutral sugars (primarily mannose and galactose), the carbohydrate moiety contains hexuronic acid and N-acetylgalactosamine and corresponds to a glycosaminoglycan. This carbohydrate chain is an integral component of the inhibitor: it does not dissociate from the inhibitor when using dissociative conditions such as sodium dodecyl sulfate, guanidinium chloride, or by increasing ionic strength or mixing with cetylpyridinium chloride. This glycosaminoglycan chain is sensitive to chondroitinase ABC or testicular hyaluronidase digestion and corresponds to slightly sulfated chondroitin 4-sulfate or 6-sulfate. After treatment by these enzymes, the urinary inhibitor has a lower molecular mass (Mr 26000) but still inhibits trypsin. [ABSTRACT FROM AUTHOR]

Reports on structure of carbohydrates in acetylcholine receptor (AChR) from Torpedo californica. Fractionation of oligosaccharides released from the whole molecule by N-oligosaccharide glycopeptidase digestion using thin-layer chromatography; Finding that more than 70 percent of the total oligosaccharide chains in Torpedo AChR are of the high-mannose type.

The enzyme GDP-D-mannose 4,6-dehydratase has been purified 1500-fold from porcine thyroid tissue. The enzyme exhibits a molecular mass of 251 000 Da as determined by sedimentation techniques. Its subunit size was determined as 41 500 Da by dodecyl sulfate gel electrophoresis. The enzyme has a Km of 3.3 µM with respect to GDP-D-mannose and appears specific with respect to this substrate. The enzyme appears to be inhibited by guanine nucleotides and by guanine nucleotide sugars. It is particularly susceptible to inhibition by GDP-D-fucose. It is suggested that this compound may have a physiological function as an end-product feedback inhibitor. [ABSTRACT FROM AUTHOR]

The synthesis, transport and processing of cathepsin C was studied in Morris hepatoma 7777 cells by metabolic labelling, immunoprecipitation and characterization of labelled polypeptides by gel electrophoresis and fluorography. The largest detectable precursor of cathepsin C was a polypeptide of Mε = 92 500. Even 3 min after synthesis this precursor was accompanied by four polypeptides with Mε values ranging from 63 000 to 54 000, indicating cleavage of the precursors within the endoplasmic reticulum. The early forms of cathepsin C were associated with low-buoyant-density organelles containing the markers of endoplasmic reticulum and Golgi complex. About 30% of these early forms were secreted within 3 h after synthesis. The remaining 70% were transferred into dense lysosomes and processed between 2 and 3 h after synthesis to a mixture of the least five major and nine minor polypeptides with Mx values ranging from 73 000 to 12 000. These forms remained stable for at least 3 days. In freshly isolated hepatocytes cathepsin C was processed to forms closely related to those found in the hepatoma cells. Cathepsin C was synthesized in Morris hepatoma 7777 cells as a glycoprotein with mannose-6-phosphate residues that mediated mannose-6-phosphate-specific receptor-dependent uptake in human skin fibroblasts. In contrast to hepatocytes, synthesis of mannose-6-phosphate receptors in Morris hepatoma 7777 cells was below the limit of detection. The hepatoma cells did not express at the cell surface these or other receptors mediating endocytosis of lysosomal enzymes. Further, processing and transport of newly synthesized cathepsin C was largely resistant to NH4Cl. Apparently, cathepsin C is transferred in Morris hepatoma 7777 cells by a mechanism independent of mannose-6-phosphate-specific receptors. [ABSTRACT FROM AUTHOR]

Dolichyl-phospho-mannose synthase, or GDPmannose:dolichyl-phosphate mannosyltransferase (EC 2.4.1.83), was solubilized from rat liver microsomes with 1.0% Nonidet P-40 and the enzyme was further purified by column chromatography on DEAE-cellulose in the presence of 0.1% Nonidet P-40. The purified enzyme preparation (880-fold over microsomes) was unstable in the presence of detergent and had no activity in the presence of Nonidet P-40, Triton X-100, octyl β-glucoside, or deoxycholate. Detergent-free enzyme was active in the presence of phosphatidylethanolamine (PtdEtn) and in the presence of phospholipid mixtures of PtdEtn and phosphatidylcholine (PtdCho) when the molar proportion of PtdCho was 70% or less. The enzyme was inactive in the presence of PtdCho alone. Unsaturated species of PtdEtn have a tendency to destabilize membrane bilayers [Cullis, P. R. & de Kruijff, B. (1978) Biochim. Biophys. Acta 507, 207-218] and we have shown that dolichol promotes the destabilizing effect of PtdEtn on membranes composed of PtdCho and PtdEtn [Jensen, J. W. & Schutzbach, J. S. (1984) Biochemistry 23, 1115-1119]. These results suggest that dolichyl-P-mannose synthase is optimally active in a phospholipid matrix that contains some component phospholipids that prefer non-bilayer structural organization in isolation. Heat-inactivation and sedimentation experiments demonstrated that the synthase associated with PtdEtn in the presence of dolichyl-P. The PtdEtn-reconstituted enzyme catalyzed the reversible transfer of mannose from GDP-mannose to dolichyl-P. The Km for GDP-mannose was found to be 0.69 µM and the apparent Km for dolichyl-P was 0.3 µM, GMP, GDP, and GTP inhibited mannosyltransfer 50% at concentrations of 16 µM, 1.3 µM and 3 µM respectively. [ABSTRACT FROM AUTHOR]

Human plasma low-density lipoproteins were purified by flotation followed by gel filtration. The protein moiety of the lipoproteins, apolipoprotein-B, which was detected by polyacrylamide gel electrophoresis as the only protein component, contained 4.4% (by weight) carbohydrate. Glycopeptides liberated from apolipoprotein-B by pronase were fractionated by affinity chromatography on concanavalin-A—Sepharose. The results indicated that high-mannose glycopeptides interacting strongly with the lectin comprise about 37% of the total monosaccharides of apolipoprotein-B. Thus, as compared to the total serum glycoproteins having about 5% of their monosaccharides in high-mannose glycopeptides, low-density lipoproteins are relatively enriched in these structures amounting up to about 10% of the total high-mannose oligosaccharides in serum. The rest of the carbohydrates in low-density lipoproteins are suggested to be mainly biantennary acidic oligosaccharides interacting weakly with concanavalin A. The oligomannosidic chains from native low-density lipoproteins and isolated glycopeptides were released by digestion with endo-β-N-acetylglucosaminidase H. [ABSTRACT FROM AUTHOR]

The human asialoglycoprotein receptor is a 46-kDa membrane glycoprotein. It is initially synthesized as a 40-kDa precursor species possessing two N-linked high-mannose oligosaccharides which is subsequently converted to the 46-kDa mature product upon modification of its oligosaccharides of the complex form [Schwartz, A. L. & Pup, D. (1983) J. Biol. Chem. 258, 11 249–11 255]. To investigate further the biosynthesis of the human asialoglycoprotein receptor, we have utilized a cell-free wheat germ translation system supplemented with dog pancreatic microsomal membranes and programmed with HepG2 and human liver RNA. The primary translation product of the human receptor is a single 34-kDa species and this species is expressed throughout human fetal and adult development. The primary translation product possesses no cleavable signal peptide and is cotranslationally glycosylated to form the 40-kDa precursor species. In addition, the human asialoglycoprotein receptor is co-translationally inserted into microsomal membranes such that a 4-kDa cytoplasmic tail is susceptible to trypsin digestion. [ABSTRACT FROM AUTHOR]