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PubMed1985

The oligosaccharide moieties of the epidermal growth factor receptor in A-431 cells. Presence of complex-type N-linked chains that contain terminal N-acetylgalactosamine residues.

The receptor for epidermal growth factor (EGF) in the human epidermoid carcinoma cell line A-431 is a glycoprotein of apparent molecular weight = 170,000. During biosynthesis, the receptor is first detected as a precursor of apparent Mr = 160,000. In this report we describe our studies on the structures of the oligosaccharide moieties of the mature receptor and its precursor. A-431 cells were grown in medium containing radioactive sugars and the radiolabeled receptors were purified by immunoprecipitation and sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Radiolabeled glycopeptides were prepared from the purified receptor by proteolysis, and their structures were examined by a variety of techniques. The mature EGF receptor contains both complex-type and high mannose-type Asn-linked oligosaccharides in the approximate ratio of 2 to 1, while the precursor contains only high mannose-type chains. A number of experimental results demonstrate that the mature receptor does not contain oligosaccharides in O-linkage through N-acetylgalactosamine to either serine or threonine. The high mannose-type oligosaccharides in both precursor and mature receptor can be cleaved by endo-beta-N-acetylglucosaminidase H and occur in the mature receptor as Man9GlcNAc2 (6%), Man8GlcNAc2 (49%), Man7GlcNAc2 (25%), and Man6GlcNAc2 (20%), whereas, in the receptor precursor the high mannose chains occur primarily as Man8GlcNAc2 (70%). The complex-type oligosaccharides in the mature receptor are predominantly tri- or tetraantennary species and are unusual in several respects. (i) Many of the chains do not contain sialic acid, while the remaining chains contain 1-2 sialic acid residues. (ii) Half of the [3H] mannose-derived radioactivity was recovered as [3H] fucose and the remaining half as [3H] mannose, indicating that there may be an average of 3 fucose residues/chain. (iii) About one-third of the [3H] glucosamine-derived radioactivity in these glycopeptides was recovered as N-acetylgalactosamine and these residues are all alpha-linked and occur at the nonreducing termini. These data demonstrate that the complex-type Asn-linked oligosaccharides in the EGF receptor from A-431 cells contain sugar residues related to human blood type A. In light of other recent studies, these results suggest that in A-431 cells blood group determinants in surface glycoproteins are contained in Asn-linked but not O-linked oligosaccharides.

Carcinoma, Squamous Cell↗
PubMed1987

Structure determination of five sulfated oligosaccharides derived from tracheobronchial mucus glycoproteins.

The structure of five sulfated oligosaccharide units of highly anionic tracheobronchial mucous glycoproteins, isolated from a cystic fibrosis patient's sputum, were established. Reduced oligosaccharides (84%) were released under alkaline borohydride conditions, and the acidic oligosaccharides (63%) were isolated by Dowex 1-X2 chromatography. Following the removal of acidic oligosaccharides possessing N-acetylneuraminic acid and L-fucose by lectin affinity chromatography a heterogeneous mixture of sulfated oligosaccharides was obtained. From this fraction, five short chain monosulfated oligosaccharides (S-I to S-V) were purified by sequential separation by SynChroprep AX300 anion exchange high pressure liquid chromatography, gel filtration on Bio-Gel P-2, and high voltage paper electrophoresis. Based on the results of carbohydrate composition, sequential exoglycosidase degradation, permethylation analysis, lectin affinity chromatography, and periodate oxidation, the following structures (where GalNAcol is N-acetylgalactosaminitol) were proposed for these oligosaccharides. (formula; see text)

Adolescent↗
PubMed1988

Homogeneous, structurally defined heparin-oligosaccharides with low anticoagulant activity inhibit the generation of the amplification pathway C3 convertase in vitro.

This paper demonstrates that heparin-oligosaccharides with low anticoagulant activity have a high capacity to inhibit activation of the amplification pathway of complement in vitro. We prepared heparin-oligosaccharides by partial depolymerization of heparin using purified flavobacterial heparinase. The resulting oligosaccharide mixture was then fractionated using strong anion exchange-high pressure liquid chromatography to produce individual oligosaccharide components of this mixture, with degree of polymerization ranging from 2 to 16. These heparin-oligosaccharides were examined for both their anticoagulant activity and capacity to inhibit activation of the amplification pathway of complement. Although there was little difference among commercial heparins, a correlation between molecular weight and activity to inhibit convertase generation was clearly established for heparin-oligosaccharides between degree of polymerization 2 through 16. Heparin-oligosaccharides of degree of polymerization 10-16 (Mr 3888-5320) demonstrated up to 54% of heparin's activity on a molar basis (and up to 163% of heparin's activity on a weight basis) in inhibiting the amplification pathway of complement in vitro while showing almost no anticoagulant activity. These studies, for the first time, completely separate heparin's ability to inhibit complement activation from its anticoagulant activity.

Animals↗
PubMed1986

Structures of O-linked oligosaccharides isolated from normal granulocytes, chronic myelogenous leukemia cells, and acute myelogenous leukemia cells.

O-Linked oligosaccharides were isolated from normal granulocytes, chronic myelogenous leukemia cells, and acute myelogenous leukemia cells by alkaline borohydride treatment. Oligosaccharides were fractionated by Sephadex G-50 gel filtration and QAE-Sephadex column chromatography, and their structures were elucidated by fast atom bombardment-mass spectrometry after permethylation and methylation analysis before and after specific exoglycosidase treatments. Results show that normal granulocytes and chronic myelogenous leukemia cells contain a series of O-linked oligosaccharides with the following structure, (formula: see text) where, in normal granulocytes n = 0 is major and n = 1 or 2, and thus polylactosaminyl oligosaccharides are present as minor components. However, these polylactosaminyl oligosaccharides were barely detectable in chronic myelogenous leukemia cells. On the other hand, acute myelogenous leukemia cells, which represent poorly differentiated myeloid cells, mainly contain short O-linked oligosaccharides with 2----6-linked sialic acid as follows. (formula: see text) These results suggest that structures of O-linked oligosaccharides vary in the different maturation stages along the same cell lineage.

Carbohydrate Conformation↗
PubMed1987

Structural study of the asparagine-linked oligosaccharides of lipophorin in locusts.

The complete structure of oligosaccharides from locust lipophorin was studied. The asparagine-linked oligosaccharides were first liberated from the protein moiety of lipophorin by digestion with almond glycopeptidase (N-oligosaccharide glycopeptidase, EC 3.5.1.52). Two major oligosaccharides (E and F), separated by subsequent thin-layer chromatography, were analyzed by methylation analysis and 1H-NMR. Based on the experimental data, the whole structure of oligosaccharide E was identified as Man alpha 1----2Man alpha 1----6(Man alpha 1----2Man alpha 1----3) Man alpha 1----6(Man alpha 1----2Man alpha 1----2Man alpha 1----3)Man beta 1----4GlcNAc beta 1----4GlcNAc. The data also revealed that oligosaccharide F is identical with oligosaccharide E in the structure, except for one glucose residue that is linked to the nonreducing terminal Man alpha 1----2 residue.

Animals↗
PubMed1979

Biosynthesis of yeast glycoproteins. Processing of the oligosaccharides transferred from dolichol derivatives.

The oligosaccharides previously bound to dolichol diphosphate were isolated from Saccharomyces cerevisiae cells incubated with [U-14C]glucose. Five compounds were obtained that migrated with RGlucose of 0.100, 0.120, 0.145, 0.180, and 0.215 on paper chromatography. All of them contained mannose and 2 N-acetylhexosamine residues. The substances that migrated with the three lower RGlucose values had, in addition, glucose units. The structure of the oligosacchardies was very similar if not identical with that of the oligosaccharides isolated from the dolichol diphosphate derivatives synthesized "in vitro" by yeast or rat liver particulate preparations or "in vivo" by dog thyroid or rat liver slices as judged by their migration on paper chromatography, monosaccharide composition, and degradation compounds produced by alpha-mannosidase treatment or acetolysis. The oligosaccharides previously bound to asparagine residues in proteins were isolated from yeast cells which had been pulsed with [U-14C]glucose and chased with medium containing the unlabeled monosaccharide. The samples taken after very short pulses contained four oligosaccharides that migrated with RGlucose of 0.100, 0.120, 0.145, and 0.180 on paper chromatography. The first three compounds contained glucose, mannose, and 2 N-acetylhexosamine residues whereas the one that migrated with a RGlucose of 0.180 was devoid of the former monosaccharide. Samples taken after short chase periods revealed that the compounds that migrated with the lower RGlucose values gradually disappeared and were converted to the oligosaccharide with the higher RGlucose value was they lost their glucose residues. Similar analysis as those mentioned above showed that the structures of these compounds were similar to those of the dolichol diphosphate-bound oligosaccharides. Samples taken after longer chase periods revealed that the oligosaccharide that migrated with a RGlucose of 0.180 was subsequently either enlarged by the addition of more mannose residues or trimmed to smaller sizes.

Carbohydrates↗
PubMed1979

Structural studies of the membrane-derived oligosaccharides of Escherichia coli.

Membrane-derived oligosaccharides are a novel class of glucose-containing oligosaccharides found in the cell envelope of Escherichia coli and other Gram-negative organisms (Schulman, H., AND Kennedy, E.P. (1979) J. Bacteriol. 137, 686-688). Previous work has shown that these oligosaccharides contain sn-1-glycero-P and smaller amounts of phosphoethanolamine, derived from membrane phospholipids, attached to position 6 of certain of the glucose residues. The structure of the parent oligosaccharides (obtained by reduction with borohydride followed by alkaline hydrolysis) has now been studied. The oligosaccharide was permethylated, followed by hydrolysis and conversion of the products to methylated glucitol acetates, which were then analyzed and identified by gas-liquid chromatography and mass spectrometry. The membrane oligosaccharides contain 10 to 12 D-glucopyranoside residues/mol, linked solely by 1 yields 2 and 1 yields 6 bonds. They are highly branched structures, with four nonreducing termini per mol. Glucose units at the branch points are doubly substituted at positions 2 and 6. The low specific rotation of the oligosaccharide (+8.3 degrees) indicates that the glycosidic bonds are predominantly or entirely beta.

Carbohydrate Conformation↗
PubMed1985

N-Acetylneuraminic acid and N-glycolylneuraminic acid in the O-linked oligosaccharides of a tumor cell glycoprotein. Incorporation and distribution.

The MAT-B1 and MAT-C1 ascites sublines of the 13762 rat mammary adenocarcinoma, which differ in several cell surface properties, contain a major mucin-type glycoprotein, termed ASGP-1. The sialic acid content of MAT-C1 ASGP-1 is 2-3-fold greater than MAT-B1 ASGP-1 (Sherblom, A. P., Buck, R. L., and Carraway, K. L. (1980) J. Biol. Chem. 255, 783-790). Sialic acid analysis demonstrated that, whereas MAT-C1 ASGP-1 contained approximately equal amounts of N-acetylneuraminic acid (NeuAc) and N-glycolylneuraminic acid (NeuGl), MAT-B1 ASGP-1 was devoid of NeuGl. MAT-B1 microsomes also did not contain NeuGl. MAT-B1 cells incubated with [3H]N-acetylmannosamine did not synthesize either labeled CMP-NeuGl or free NeuGl, even though the CMP-sialic acid synthetase was active with the substrate NeuGl. Thus, MAT-B1 cells may be deficient in the enzyme N-acetylneuraminate monooxygenase. The O-linked oligosaccharides from both MAT-B1 and MAT-C1 ASGP-1 have been shown to contain a core tetrasaccharide Gal(beta 1-4)GlcNAc(beta 1-6)(Gal(beta 1-3]GalNAc in which both galactose residues may be linked to additional sugars (Hull, S. R., Laine, R. A., Kaizu, T., Rodriquez, I., and Carraway, K. L. (1984) J. Biol. Chem. 259, 4866-4877). The distribution of NeuAc and NeuGl between the two galactose termini of the core tetrasaccharide was examined for MAT-C1 ASGP-1. Oligosaccharides were released by alkaline-borohydride treatment of MAT-C1 ASGP-1 which had been labeled with [14C]glucosamine and galactose oxidase/B3H4. Following fractionation by Bio-Gel P-4, DEAE-Sephadex, and high-performance liquid chromatography, oligosaccharides were analyzed for NeuAc and NeuGl and for susceptibility to digestion with beta-galactosidase. Three disialylated oligosaccharides were identified containing 2 mol of NeuAc (5.5% recovery), 2 mol of NeuGl (4.5%), or 1 mol each of NeuAc and NeuGl (11.1%). For monosialylated oligosaccharides, NeuGl appeared preferentially associated with the Gal(beta 1-4)GlcNAc terminus (9.0%), whereas significant amounts of oligosaccharide containing NeuAc at both the Gal(beta 1-3)GalNAc (2.6%) and Gal(beta 1-4)GlcNAc (4.5%) termini were detected. Each of the major qualitative differences between MAT-B1 and MAT-C1 oligosaccharides, including the presence of NeuGl (MAT-C1), sulfate (MAT-B1), and alpha-linked galactose (MAT-B1), occurs at the Gal(beta 1-4)GlcNAc terminus.

Adenocarcinoma↗
PubMed1986

N-linked high mannose-type oligosaccharides in the protozoa Crithidia fasciculata and Crithidia harmosa contain galactofuranose residues.

Incubation of Crithidia fasciculata cells with [U-14C] glucose led to the synthesis of Man-P-dolichol but not of Glc-P-dolichol. The main and largest dolichol-P-P-linked oligosaccharide formed was Man7GlcNAc2 whether labeling was performed in 5 mM sodium pyruvate or 5.5 mM glucose. The protein-linked, endo-beta-N-acetylglucosaminidase H-sensitive oligosaccharides isolated from mature glycoproteins were Man7GlcNAc and Gal1Man6GlcNAc, the latter being a mixture of two isomers. All the galactose residues were present in the furanose configuration, as judged by their extreme lability to acid hydrolysis, by the products obtained upon mild periodate oxidation, and by their sensitivity to beta-galactofuranosidase. Labeling cells for short times or at low temperature yielded a protein-bound, endo-beta-N-acetylglucosaminidase H-sensitive oligosaccharide whose composition was Glc1Man7GlcNAc, of transient existence, and that was mainly labeled in the glucose residue. The latter oligosaccharide was detected on paper chromatography only as a smearing of Man7GlcNAc and Gal1Man6GlcNAc when cells were labeled with [2-3H] mannose, thus indicating that it was only present in minute amounts. Protein-bound endo beta-N-acetylglucosaminidase H-resistant oligosaccharides liberated, upon a mild acid treatment, galactose residues and an unidentified substituent. The treatment rendered the oligosaccharides sensitive to endo beta-N-acetylglucosaminidase H, which liberated Man7GlcNAc and two isomers of Man6GlcNAc. An almost similar mechanism of protein N-glycosylation, including the existence of galactofuranose residues in N-linked oligosaccharides, was found to occur in Crithidia harmosa.

Animals↗
PubMed1985

Oligosaccharide composition of an influenza virus hemagglutinin with host-determined binding properties.

We have previously reported that the binding properties of the hemagglutinin (HA) of the WSN-F strain of influenza A are affected by the cells in which the virus is grown (Crecelius, D. M., Deom, C. M., and Schulze, I.T. (1984) Virology 139, 164-177); at 37 degrees C chick embryo fibroblast-grown F virus has a greater affinity for host cells than does the same virus grown in Madin-Darby bovine kidney (MDBK) cells. In an attempt to explain this host-determined property, we have characterized the carbohydrate put onto the viral HA by these two cells. Experiments using tunicamycin indicate that the HA made by MDBK cells contains about 4000 daltons of carbohydrate in excess of that on the HA from chick embryo fibroblast. Serial lectin affinity chromatography of the asparagine-linked oligosaccharides on the HA subunits, HA1 and HA2, detected a number of host-dependent differences in the complex oligosaccharides. Both HA1 and HA2 from MDBK cells contained more highly branched (i.e. tri- and tetraantennary) complex oligosaccharides than did the subunits from chick embryo fibroblasts. In addition, the HA subunits from the two sources differed in the amount of galactose-containing "bisected" complex oligosaccharides and in the presence of certain fucosylated triantennary oligosaccharides. Profiles of the asparagine-linked oligosaccharides from the host cells did not show these differences, indicating that the HA subunit profiles were not necessarily representative of the structures found on the cellular glycoproteins. The data support the conclusion that bulky oligosaccharides on the MDBK-HA subunits of WSN-F reduce the affinity of the virus for cellular receptors.

Animals↗
PubMed1979

A concanavalin A-resistant Chinese hamster ovary cell line is deficient in the synthesis of [3H]glucosyl oligosaccharide-lipid.

In this report we present an initial determination of the biochemical defect present in a Chinese hamster ovary cell line selected for resistance to concanavalin A. Membranes of this mutant, B211, incorporated at least 10-fold less mannose from GDP-[14C]mannose into oligosaccharide-lipid than membranes of the wild type. In the presence of dolichol phosphate, membranes of the mutant and wild type exhibited similar rates of synthesis of number of early intermediates, namely, mannosylphosphoryldolichol, N-acetylglucosaminyl- and N,N'-diacetylchitobiosylpyrophosphoryldolichol, glucosylphosphoryldolichol, and mannosyloligosaccharide-lipid. The membranes of B211 did not incorporate glucose from UDP-[3H]glucose into oligosaccharide-lipid or protein. Comparison by gel filtration chromatography of oligosaccharides derived from the oligosaccharide-lipids of B211 and wild type cells labeled with [2-3H]mannose revealed that B211 cells incorporated little if any label into an oligosaccharide corresponding to the most excluded oligosaccharide labeled by wild type cells. This concanavalin A-resistant cell line appears to lack the ability to glucosylate oligosaccharide-lipid.

Animals↗
PubMed1979

Inhibition of protein synthesis also inhibits synthesis of lipid-linked oligosaccharides.

Studies were initiated to determine whether the formation of lipid-linked oligosaccharides was coupled to the synthesis of protein. Canine kidney cells were grown with [2-3H]mannose or [3H]leucine in the presence of cycloheximide or puromycin and the effect of these inhibitors on the synthesis of proteins and lipid-linked oligosaccharides was measured. In all cases, the inhibition of protein synthesis resulted in a substantial inhibition in the incorporation of mannose into the lipid-linked oligosaccharides, although the synthesis of mannosyl-phosphoryl-dolichol was only slightly inhibited. Cycloheximide had no effect on the in vitro incorporation of mannose into lipid-linked oligosaccharides when GDP-[14C]mannose was incubated with aorta microsomal preparations. The inhibition of lipid-linked oligosaccharides was apparently not due to a decrease in the amount of glycosyltransferases as a result of protein degradation in the absence of protein synthesis, nor was it the result of a more rapid degradation of lipid-linked oligosaccharides. The inhibition also did not appear to be due to limitations in the available dolichyl-phosphate. The results suggest that the formation of lipid-linked oligosaccharides may be regulated by end product inhibition.

Animals↗
PubMed1983

Structure of oligosaccharides and the linkage region between keratan sulfate and the core protein on proteoglycans from monkey cornea.

Structural analyses were performed on the intact glycopeptides and on the linkage region oligosaccharide-peptides derived from the keratan sulfate proteoglycan from monkey cornea (Nakazawa, K., Newsome, D.A., Nilsson, B., Hascall, V.C., and Hassell, J.R. (1983) J. Biol. Chem. 258, 6051-6055) using trifluoroacetolysis, Smith degradation, chromium trioxide oxidation, and gas-liquid chromatography-mass spectrometry. The following structure was found for the linkage region (formula; see text) The following structures were found for the intact oligosaccharide peptides (formula; see text) and (formula; see text) The structure of the linkage region for keratan sulfate on corneal proteoglycans is clearly derived from a complex type of N-linked glycoprotein oligosaccharide precursor, indicating that only the oligosaccharides that have been processed to the complex type are used as primers for synthesizing keratan sulfate chains. The high mannose oligosaccharide in Formula 3 is an intermediate in the normal pathway for biosynthesis of complex type oligosaccharides. The structure in Formula 2, in which a single Man alpha 1-2 is retained on the Man alpha 1-3 branch while the Man alpha 1-6 branch is unsubstituted, can be an intermediate for an alternate, presumably minor pathway for complex oligosaccharide formation (Kornfeld, S., Gregory, W., and Chapman, A. (1979) J. Biol. Chem. 254, 11649-11654) in certain cases. This structure has not previously been shown to be present on normal glycoproteins.

Animals↗
PubMed1983

New oligosaccharides from heparin and heparan sulfate and their use as substrates for heparin-degrading enzymes.

Oligosaccharides were isolated from heparin and heparan sulfate by a procedure consisting of three major steps: (a) acid hydrolysis; (b) gel chromatography; and (c) cation exchange chromatography on an amino acid analyzer. To date, six new oligosaccharides have been isolated by this procedure and have been sequenced by a combination of NaB3H4-labeling and deaminative cleavage with nitrous acid. The structures of these oligosaccharides were as follows: 1. GlcN-GlcUA-GlcN 2. GlcN-IdUA-GlcN 3. GlcN-GlcUA-GlcN-GlcUA-GlcN 4. GlcN-IdUA-GlcN-GlcUA-GlcN 5. GlcN-GlcUA-GlcN-IdUA-GlcN 6. GlcN-IdUA-GlcN-IdUA-GlcN The linkage positions and anomeric configurations were assumed to be the same as in the polysaccharides from which the oligosaccharides originated. The usefulness of some of these oligosaccharides as enzyme substrates was tested after appropriate modifications and radioactive labeling. Oligosaccharides 2 and 3 were N-[35S]sulfated and were found to serve as substrates for heparan N-sulfate sulfatase (heparin sulfamidase), with a homogenate of cultured skin fibroblasts as enzyme source. Similarly, reduction of oligosaccharide 2 with NaB3H4 yielded a substrate for acetyl-CoA:alpha-D-glucosaminide N-acetyltransferase. Finally, the previously known disaccharide, 4-O-alpha-D-glucosaminyl-L-iduronic acid, which was isolated in the course of this work, was N-acetylated with [3H] acetic anhydride and was shown to be a substrate for N-acetyl-alpha-D-glucosaminidase.

Acetylglucosaminidase↗
PubMed1983

Biosynthesis of N- and O-linked oligosaccharides of the low density lipoprotein receptor.

In human fibroblasts, the receptor for low density lipoprotein (LDL) is synthesized as a precursor of apparent Mr = 120,000 which is converted to a mature form of apparent Mr = 160,000, as determined by migration in sodium dodecyl sulfate (SDS)-polyacrylamide gels (Tolleshaug, H., Goldstein, J. L., Schneider, W. J., and Brown, M. S. (1982) Cell 30, 715-724). The current paper describes the relationship of N- and O-glycosylation to this post-translational modification. Oligosaccharides were analyzed from precursor and mature forms of LDL receptors that had been immunoprecipitated from cells grown in media containing radioactive sugars. In human epidermoid carcinoma A-431 cells, the receptor precursor appears to contain one N-linked high mannose oligosaccharide and approximately 6-9 N-acetylgalactosamine residues linked O-glycosidically to Ser/Thr residues. In the mature receptor, the O-linked oligosaccharides are mono- and disialylated species having the core structure of galactose leads to N-acetylgalactosamine leads to Ser/Thr. The single N-linked oligosaccharide of the mature receptor can either be a tri- or tetraantennary complex-type species. Similar results were obtained with normal human fibroblast receptor except that the O-linked oligosaccharides on the precursor are neutral disaccharides, of which one component is GalNAc and the N-linked complex type unit on the mature receptor is less branched. Since the addition of GalNAc residues to Ser/Thr residues precedes the conversion of N-linked high mannose-type oligosaccharides to complex-type structures, the transfer of N-acetylgalactosamine must occur prior to the entry of glycoproteins into the region of the Golgi containing the processing enzyme alpha-mannosidase I. We also studied the receptor from tunicamycin-treated cells and after treatment with neuraminidase. In addition, we analyzed the receptor synthesized by a lectin-resistant clone of Chinese hamster ovary cells that is deficient in adding galactose residues to both N- and O-linked oligosaccharides. These studies suggest that the apparent differences in molecular weight between the precursor and mature forms of the LDL receptor are largely, if not entirely, due to the addition of sialic acid and galactose residues to the O-linked GalNAc residues.(ABSTRACT TRUNCATED AT 400 WORDS)

Adrenal Cortex↗
PubMed1984

Regulation of the synthesis of membrane-derived oligosaccharides in Escherichia coli. Assay of phosphoglycerol transferase I in vivo.

Membrane-derived oligosaccharides are periplasmic constituents of Escherchia coli and other Gram-negative bacteria. Oligosaccharides in this family may be variously substituted with O-succinyl ester residues, and with sn-1-phosphoglycerol and phosphoethanolamine residues derived from membrane phospholipids. Membrane-derived oligosaccharides appear to be important in osmoregulation, because their synthesis is under strict control (Kennedy, E.P. (1982) Proc. Natl. Acad. Sci. U.S.A. 79, 1092-1095). Maximum rate of synthesis is at very low osmolarity of the medium. Phosphoglycerol residues are transferred from phosphatidylglycerol to membrane-derived oligosaccharides, or to certain beta-glucoside acceptors, in a reaction catalyzed by phosphoglycerol transferase I, an enzyme of the inner membrane (Jackson, B. J., and Kennedy, E.P. (1983) J. Biol. Chem. 258, 2394-2398). We now report that this enzyme catalyzes the transfer of phosphoglycerol residues to arbutin (p-hydroxyphenyl-beta-D-glucoside) added to the medium with Km similar to that observed with the cell-free enzyme. The active site of the enzyme must therefore be on the periplasmic face of the inner membrane. We assayed phosphoglycerol transferase I in vivo and found that it is present and completely active even in cells growing in medium of very high osmolarity, in which the synthesis of membrane-derived oligosaccharides is severely reduced. We conclude that osmotic regulation must occur at the stage of the synthesis of oligosaccharide chains. A study of the kinetics of transfer of phosphoglycerol residues to membrane-derived oligosaccharides in vivo revealed that synthesis of the polyglucose chains must stop abruptly upon transfer of cells from medium of low to high osmolarity, inconsistent with a model postulating simple dilution of some rate-limiting enzyme during growth at the higher osmolarity.

Arbutin↗
PubMed1984

Beta-linked N-acetylgalactosamine residues present at the nonreducing termini of O-linked oligosaccharides of a cloned murine cytotoxic T lymphocyte line are absent in a Vicia villosa lectin-resistant mutant cell line.

The O-linked oligosaccharides of the cloned, murine cytotoxic T cell line B6.1.SF.1 were compared with the corresponding oligosaccharides from a Vicia villosa lectin-resistant mutant of B6.1.SF.1 called VV6 (Conzelmann, A., Pink, R., Acuto, O., Mach, J.-P., Dolivo, S., and Nabholz, M. (1980) Eur. J. Immunol. 10, 860-868). The VV6 mutant cells are deficient in binding sites for this GalNAc-specific lectin. Cells were grown in the presence of [3H]glucosamine and [3H] galactose to label the glycoproteins, and the desialyzed, alkaline borohydride-released oligosaccharides were isolated and characterized. The VV6 cells contained a series of O-linked oligosaccharides ranging in size from a disaccharide to a pentasaccharide. These were composed of galactose, N-acetylglucosamine, and N-acetylhexosaminitol, the latter sugar being derived from the reducing terminus. The predominant oligosaccharide had the partial structure Gal beta GlcNAc beta-(Gal beta)N-acetylhexosaminitol. In contrast, the analogous oligosaccharides of the parental cells contained additional beta-linked GalNAc residues located at nonreducing termini. The smallest of these had the structure GalNAc beta 1,4Gal beta-N-acetylhexosaminitol. Neither cell line contained significant amounts of terminal GalNAc linked to Ser/Thr which is the main binding site for the V. villosa B4 lectin on Tn erythrocytes (Tollefsen, S. R., and Kornfeld, R. (1983) J. Biol. Chem. 258, 5172-5176). These findings suggest that the major binding sites for the V. villosa lectin on the parental cytotoxic T cell line consist of structures containing beta 1,4-linked GalNAc residues at the nonreducing ends of conventional O-linked structures. The VV6 cells lack these beta-linked GalNAc residues, and this may account for their deficiency of V. villosa lectin-binding sites. In the following paper (Conzelmann, A., and Kornfeld, S. (1984) J. Biol. Chem. 259, 12536-12542), we demonstrate that the VV6 cells are missing the N-acetylgalactosaminyltransferase that is responsible for the synthesis of these unusual oligosaccharides.

Acetylgalactosamine↗
PubMed1983

The mod A mutant of Dictyostelium discoideum is missing the alpha 1,3-glucosidase involved in asparagine-linked oligosaccharide processing.

The recessive mutation, mod A, in the Dictyostelium discoideum strain M31 results in an alteration in the post-translational modification of lysosomal enzymes. We now report studies which indicate that mod A is deficient in glucosidase II, an enzyme which is involved in the processing of asparagine-linked oligosaccharides. [2-3H]Mannose-labeled glycopeptides were prepared from three purified mod A lysosomal enzymes and compared to the equivalent glycopeptides from parental enzymes. The mod A glycopeptides were deficient in high mannose oligosaccharides containing two phosphomannosyl residues and accumulated oligosaccharides with one phosphomannosyl residue. The phosphate was present in the form of an acid-stable phosphodiester in both instances. There was also an increase in the amount of nonphosphorylated high mannose oligosaccharides mod A and these were larger than the corresponding material from the parental enzymes. In addition, the nonphosphorylated oligosaccharides were only partially degraded by alpha-mannosidase, indicating the presence of a blocking moiety. In vitro enzyme assays demonstrated that the mod A cells cannot remove the inner 1 leads to 3-linked glucose from a glucosylated high mannose oligosaccharide. The cells are also deficient in membrane-bound neutral p-nitrophenyl-alpha-D-glucosidase activity. This activity has been attributed to glucosidase II in other systems. Removal of the outer 1 leads to 2-linked glucose from Glc3Man9Glc-NAc2 is normal, demonstrating the presence of glucosidase I activity. We conclude from these data that M31 cells are deficient in glucosidase II, the enzyme which removes the two inner glucose residues from the glucosylated oligosaccharides of newly glycosylated proteins. This defect can explain the mod A phenotype and is proposed to be the primary genetic defect in these cells.

Asparagine↗