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.     

Cyclic glucans produced by Agrobacterium tumefaciens are substituted with sn-1-phosphoglycerol residues

In a previous study (Miller, K.J., Kennedy, E.P. and Reinhold, V.N. (1986) Science 231, 48-51) it was reported that the biosynthesis of periplasmic cyclic beta-1,2-glucans by Agrobacterium tumefaciens is strictly osmoregulated in a pattern closely similar to that found for the membrane-derived oligosaccharides of Escherichia coli (Kennedy, E.P. (1982) Proc. Natl. Acad. Sci. USA 79, 1092-1095). In addition to the well-characterized neutral cyclic glucan, the periplasmic glucans were found to contain an anionic component not previously reported. Biosynthesis of the anionic component is osmotically regulated in a manner indistinguishable from that of the neutral cyclic beta-1,2-glucan. We now find that the anionic component consists of cyclic beta-1,2-glucans substituted with one or more sn-1-phosphoglycerol residues. The presence of sn-1-phosphoglycerol residues represents an additional, striking similarity to the membrane-derived oligosaccharides of E. coli.     

Characterization of an Escherichia coli mdoB mutant strain unable to transfer sn-1-phosphoglycerol to membrane-derived oligosaccharides

A procedure for the isolation of mutants affected in components containing glycerol derived from phospholipids yielded two mutant strains that contain membrane-derived oligosaccharides (MDO) devoid of glycerol (Rotering, H., Fiedler, W., Rollinger, W., and Braun, V. (1984) FEMS Microbiol. Lett. 22, 61-68). MDO are found in the periplasmic space of Escherichia coli and other Gram-negative bacteria, and they may comprise up to 7% of the cells dry weight. The biosynthesis of MDO is osmoregulated (Kennedy, E. P. (1982) Proc. Natl. Acad. Sci. U. S. A. 79, 1092-1095) and linked to the metabolism of phospholipids (van Golde, L. M. G., Schulman, H., and Kennedy, E. P. (1973) Proc. Natl. Acad. Sci. U. S. A. 70, 1368-1372). This leads to substitution of MDO with sn-1-phosphoglycerol and phosphoethanolamine (Kennedy, E. P., Rumley, M. K., Schulman, P., and van Golde, L. M. G. (1976) J. Biol. Chem. 251, 4208-4213). MDO also contain succinate in O-ester linkage. We now report that one mutant strain lacks phosphoglycerol transferase I activity and thus is unable to transfer sn-1-phosphoglycerol residues from phosphatidylglycerol to MDO. The mdoB gene affected in this mutant has been located at 99.2 min on the E. coli chromosome. The ethanolamine content of MDO isolated from the mutant strain is elevated, whereas the number of succinate residues is not affected. The only phenotype of mdoB mutants we found is a dramatic reduction of the diglyceride content observed in dgk mdoB double mutants when the beta-glucoside arbutin is present in the growth medium.     

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.     

Biosynthesis of membrane-derived oligosaccharides: characterization of mdoB mutants defective in phosphoglycerol transferase I activity.

Phosphoglycerol transferase I, an enzyme of the inner, cytoplasmic membrane of Escherichia coli, catalyzes the in vitro transfer of phosphoglycerol residues from phosphatidylglycerol to membrane-derived oligosaccharides or to the model substrate arbutin (p-hydroxyphenyl-beta-D-glucoside). The products are a phosphoglycerol diester derivative of membrane-derived oligosaccharides or arbutin, respectively, and sn-1,2-diglyceride (B. J. Jackson and E. P. Kennedy, J. Biol. Chem. 258:2394-2398, 1983). Because this enzyme has its active site on the outer aspect of the inner membrane, it also catalyzes the transfer of phosphoglycerol residues to arbutin added to the medium (J.-P. Bohin and E. P. Kennedy, J. Biol. Chem. 259:8388-8393, 1984). When strains bearing the dgk mutation, which are defective in the enzyme diglyceride kinase, are grown in medium containing arbutin, they accumulate large amounts of sn-1,2-diglyceride, a product of the phosphoglycerol transferase I reaction. Growth is inhibited under these conditions. A further mutation in such a dgk strain, leading to the loss of phosphoglycerol transferase I activity, should result in the phenotype of arbutin resistance. We have exploited this fact to obtain strains with such mutations, designated mdoB, that map near min 99. Such mutants lack detectable phosphoglycerol transferase I activity, cannot transfer phosphoglycerol residues to arbutin in vivo, and synthesize membrane-derived oligosaccharides devoid of phosphoglycerol residues. These findings offer strong genetic support for the function of phosphoglycerol transferase I in membrane-derived oligosaccharide biosynthesis.     

A novel phosphodiesterase from Aspergillus niger and its application to the study of membrane-derived oligosaccharides and other glycerol-containing biopolymers.

A novel phosphodiesterase has been found in commercially available extracts of Aspergillus niger and has been partially purified by fractionation with acetone and chromatography on carboxymethylcellulose. The enzyme attacks glycerophosphodiester bonds with the liberation of free glycerol only. The synthetic substrate glucose 6-phospho-sn-1'(3')-glycerol is hydrolyzed with production of equivalent amounts of free glycerol and glucose 6-phosphate. Similarly, the enzymic hydrolysis of sn-glycero-3-phosphocholine liberates glycerol and phosphocholine. The hydrophilic head groups of membrane phospholipids of Escherichia coli are continuously transferred to a closely related family of oligosaccharides ("membrane-derived oligosaccharides") containing glucose as the sole sugar (van Golde, L. M. G., Schulman, H., and Kennedy, E. P. (1973) Proc. Natl. Acad. Sci. U. S. A. 70, 1368--1372). Oligosaccharide A-2 contains sn-1-glycerophosphate residues (derived from phosphatidylglycerol) in phosphodiester linkage. Treatment of this oligosaccharide with the phosphodiesterase led to the liberation of nearly all of the glycerol as free glycerol. Subsequent partial acid hydrolysis of the enzyme-treated oligosaccharide led to the recovery of glucose 6-phosphate in almost quantitative yield. The sn-1-glycerophosphate residues are therefore linked to position 6 of glucose units of the oligosaccharide. The activity of the enzyme is not restricted to glycerophosphodiesterases since it will hydrolyze phosphodiesters containing other polyols such as the synthetically prepared glucose 6-phospho-DL-1'(2'-hydroxy-3'-ethoxy)propane.     

Identification of UDP-glucose as an intermediate in the biosynthesis of the membrane-derived oligosaccharides of Escherichia coli.

The membrane-derived oligosaccharides of Escherichia coli constitute a closely related family of oligosaccharides containing approximately 9 glucose units variously substituted with sn-glycero-1-phosphate and phosphoethanolamine residues derived from the head groups of membrane phospholipids, and also with succinate in O-ester linkage (Kennedy, E.P., Rumley, M.K., Schulman, H., and van Golder, L.M.G. (1976) J. Biol. Chem. 251, 4208-4213). Studies with mutant strains defective in the synthesis of various nucleoside diphosphate sugars have now revealed that UDP-glucose is an essential intermediate in the biosynthesis of these oligosaccharides. Mutants unable to synthesize UDP-glucose do not contain significant amounts of the membrane-derived oligosaccharides. In contrast, a strain unable to synthesize ADP-glucose, the glucosyl donor for glycogen synthesis in E. coli, contained normal amounts of the membrane-derived oligosaccharides, although with a somewhat different pattern of distribution of the various subspecies. In confirmation of these genetic studies, pulse-label isotope tracer studies have been carried out with glucose of high specific activity, under conditions in which UDP-glucose comprises a large fraction of the total radioactivity in the low molecular weight pool. Subsequent "chase" experiments clearly revealed the conversion of UDP-glucose to the higher molecular weight membrane-derived oligosaccharides.     

Structures of the sialylated oligosaccharide chains in swine tracheal mucin glycoproteins

The structures of the major sialylated oligosaccharide chains in swine tracheal mucin glycoprotein were established. The oligosaccharide chains were released by treatment with alkaline borohydride and isolated by gel filtration on Bio-Gel P6 columns and chromatography on DEAE-cellulose. The neutral oligosaccharide chains in this glycoprotein have been characterized in previous studies (Rana, S.S., Chandrasekaran, E.V., Kennedy, J., and Mendicino, J. (1984) J. Biol. Chem. 259, 12899-12907; Chandrasekaran, E.V., Rana, S.S., Davila, M., and Mendicino, J. (1984) J. Biol. Chem. 259, 12908-12914). The present study reports the isolation of four monosialylated chains ranging in length from 6 to 14 sugar units, two disialylated chains containing 6 and 12 sugar units, and one trisialylated chain containing 9 sugar units. The structure of the sialylated oligosaccharides was determined by permethylation analysis and sequential hydrolysis with specific exoglycosidases. The following structures (where GalNAcol is N-acetylgalactosaminitol) were assigned to these oligosaccharides.     

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.     

Transfer of phosphoethanolamine residues from phosphatidylethanolamine to the membrane-derived oligosaccharides of Escherichia coli.

The membrane-derived oligosaccharides (MDO) of Escherichia coli are periplasmic constituents composed of glucose residues linked by beta-1,2 and beta-1,6 glycosidic bonds. MDO are substituted with phosphoglycerol, phosphoethanolamine, and succinic acid moieties. The phosphoglycerol residues present on MDO are derived from phosphatidylglycerol (B. J. Jackson and E. P. Kennedy, J. Biol. Chem. 258:2394-2398, 1983), but evidence as to the source of the phosphoethanolamine residues has been lacking. We now report that phosphatidylethanolamine, exogenously added to intact cells of E. coli, provides a source of phosphoethanolamine residues that are transferred to MDO. The biosynthesis of phosphoethanolamine-labeled MDO is osmotically regulated, with maximum synthesis occurring during growth in medium of low osmolarity.     

Isolation and sequencing of a cDNA clone encoding lysosomal membrane glycoprotein mouse LAMP-1. Sequence similarity to proteins bearing onco-differentiation antigens

We have isolated and sequenced a cDNA clone encoding the mouse LAMP-1 (mLAMP-1) major lysosomal membrane glycoprotein. The deduced protein sequence, which included the NH2-terminal portion of the mLAMP-1 molecule, consisted of 382 amino acids (Mr 41,509). The predicted structure of this protein included an NH2-terminal intralumenal domain consisting of two homology units of approximately 160 residues each separated by a proline-rich hinge region. Each homology unit contained four cysteine residues with two intercysteine intervals of 36-38 residues and one of 68 or 76 residues. The molecule also contained 20 asparagine-linked glycosylation sites within residues 1-287, a membrane-spanning region from residues 347 to 370, and a carboxyl-terminal cytoplasmic domain of 12 residues. The biochemical properties and amino acid sequence of mLAMP-1 were highly similar to those of two other molecules that have been studied as cell surface onco-differentiation antigens: a highly sialylated polylactosaminoglycan-containing glycoprotein isolated from human chronic myelogenous leukemia cells (Viitala, J., Carlsson, S. R., Siebert, P. D., and Fukuda, M. (1988) Proc. Natl. Acad. Sci. U.S.A. 85, in press) and the mouse gp130 (P2B) glycoprotein, in which an increase in beta 1-6 branching of asparagine-linked oligosaccharides has been correlated with metastatic potential in certain tumor cells (Dennis, J.W., Laferte, S., Waghorne, C., Breitman, M.L., and Kerbel, R.S. (1987) Science 236, 582-585).     

Structural basis of the heterogeneity of asparagine-linked oligosaccharides of a Waldenstrom's macroglobulinemia immunoglobulin M

The extent of glycans heterogeneity in a pathological human immunoglobulin M ZAJ has been studied on oligosaccharides released by hydrazinolysis from the purified glycoprotein. After reduction with NaB3H4, asparagine-linked carbohydrate chains were separated by affinity chromatography on concanavalin A-Sepharose into oligomannosidic and N-acetyllactosaminic types. Glycans of the oligomannosidic type were further fractionated by HPLC and those of the N-acetyllactosamine type by preparative high-voltage electrophoresis. The primary structure of the main oligosaccharides was investigated on the basis of micro-methylation analysis, mass spectrometry and sequential exo-glycosidase digestion. Glycans of the oligomannosidic type varied in size from Man5GlcNAc2 to Man9GlcNAc2. N-Acetyllactosaminic glycans were found of the biantennary, bisected-biantennary and triantennary types. They presented a higher degree of heterogeneity due to the presence of a variable number of NeuAc and fucose residues. The new structures we report here were in addition to the major biantennary one we previously described on the basis of methylation analysis and 500 MHz 1H-NMR spectroscopy (Cahour, A., Debeire, P., Hartmann, L., Montreuil, J., Van Halbeek, H. and Vliegenthart, J.F.G. (1984) FEBS Lett. 170, 343-349): NeuAc(alpha 2-6)Gal(beta 1-4)GlcNAc(beta 1-2)Man(alpha 1-3)[Gal(beta 1-4)Glc-NAc(beta 1-2)Man(alpha 1-6)]Man(beta 1-4)]Glc-NAc(beta 1-4) [Fuc(alpha 1-6)]GlcNAc.     

Diglyceride Kinase Mutants of Escherichia coli: Inner Membrane Association of 1,2-Diglyceride and Its Relation to Synthesis of Membrane-Derived Oligosaccharides

Mutants of Escherichia coli defective in diglyceride kinase contain 10 to 20 times more sn-1,2-diglyceride than normal cells. This material constitutes about 8% of the total lipid in such strains. We now report that this excess diglyceride is recovered in the particulate fraction, primarily in association with the inner, cytoplasmic membrane. The diglyceride kinase of wild-type cells was recovered in the same inner membrane fractions. The conditions employed for the preparation of the membranes did not appear to cause significant redistribution of lipids and proteins. The biochemical reactions leading to the formation of diglyceride in E. coli are not known. To determine whether diglyceride formation requires concurrent synthesis of the membrane-derived oligosaccharides (H. Schulman and E. P. Kennedy, J. Biol. Chem. 252:4250-4255, 1977), we have constructed a double mutant defective in both the kinase (dgk) and phosphoglucose isomerase (pgi). When oligosaccharide synthesis was inhibited in this organism by growing the cells on amino acids as the sole carbon source, the diglyceride was no longer present in large amounts. When glucose was also added to the medium, the pgi mutation was bypassed, oligosaccharide synthesis resumed, and diglyceride again accumulated. These findings suggest that diglyceride may arise during the transfer of the sn-glycero-1-P moiety from phosphatidylglycerol (and possibly cardiolipin) to the oligosaccharides. In wild-type cells the kinase permits the cyclical reutilization of diglyceride molecules for phospholipid biosynthesis.     

Effects of mannoprotein mutations on Saccharomyces cerevisiae core oligosaccharide structure

By the combined actions of an endo-alpha-1 leads to 6-mannanase and an endo-beta-N-acetylglucosaminidase, the core oligosaccharides can be released from Saccharomyces cerevisiae X2180 mnn2 mannoproteins. The effects of various mannoprotein mutations were evaluated by structural comparison of these core oligosaccharides with those prepared from double mutant strains with the genotypes mnn1 mnn2, mnn2 mnn3, mnn2 mnn4, and mnn2 mnn5. The results indicate that only the mnn1 lesion has a major effect on the mannoprotein core structure. Whereas the mnn2 mannoprotein yields a core composed of 6 fragments that differ in size from each other by single mannose units, only the two smallest species predominate in the mnn1 mnn2 preparation. This change is correlated with a loss of terminal alpha 1 leads to 3-mannosyl residues, an effect on the mnn1 lesion that is found also in the polysaccharide outer chain and hydroxyamino acid-linked mannooligosaccharides. The mnn3 and mnn5 mutations also had slight effects on the core size, but clear differences in linkage composition were not apparent. The results suggest that core oligosaccharides have an average composition of Man11GlcNAc, whereas Man9GlcNAc is the major oligosaccharide in strains containing the mnn1 defect. These values are 2 to 3 sugars less than those estimated previously (Nakajima, T., and Ballou, C. E. (1975) Biochem. Biophys. Res. Commun. 66, 870-879). Detailed analysis of the major core oligosaccharide from the mnn1 mnn2 mutant revealed that the two mannoses in alpha 1 leads to 3 linkage to the backbone were adjacent to each other and that the oligosacccharide is nearly identical with one isolated from chinese hamster ovary cell membranes (Li, E., and Kornfeld, S. (1979) J. Biol. Chem. 254, 1600-1605). This finding provides strong evidence for the evolutionary conservation of this structural feature of the high mannose core oligosaccharides.     

Comparative study of the oligosaccharides released from baby hamster kidney cells and their polyoma transformant by hydrazinolysis

The asparagine-linked sugar chains of the membrane of baby hamster kidney cells and their polyoma transformant were quantitatively released as oligosaccharides by hydrazinolysis and labeled by NaB3H4 reduction. The radioactive oligosaccharides thus obtained were fractionated by paper electrophoresis. The neutral oligosaccharides of both cells were exclusively of high mannose type. The acidic oligosaccharides were bi-, tri-, and tetraantennary complex-type sugar chains with Man alpha 1----6 (Man alpha 1----3) Man beta 1----4 GlcNAc beta 1----4 (+/- Fuc alpha 1----6) GlcNAc as their cores and Gal beta 1----4 GlcNAc and various lengths of Gal beta 1----4 GlcNAc repeating chains in their outer-chain moieties. Prominent features of these acidic oligosaccharides are that all sialic acid residues were N-acetylneuraminic acid and were linked exclusively at C-3 of the nonreducing terminal galactose residues of the outer chains. Comparative study of oligosaccharides of the two cells by Bio-Gel P-4 column chromatography revealed that transformation of baby hamster kidney cells leads to a reduction in high mannose-type oligosaccharides and an increase in tetraantennary oligosaccharides. Increase of the outer chains linked at C-6 of the Man alpha 1----6 residue of the core is the cause of increase in the relative amount of highly branched oligosaccharides in the polyoma transformant.     

Structures of the asparagine-linked sugar chains of human chorionic gonadotropin.

The asparagine-linked sugar chains of human chorionic gonadotropin were released from the polypeptide moiety by hydrazinolysis followed by N-acetylation and NaB3H4 reduction. More than 90% of the released radioactive oligosaccharides contained N-acetylneuraminic acid residues. After removal of N-acetylneuraminic acid residues by sialidase treatment, two neutral oligosaccharide fractions were obtained by paper chromatography. Sequential exoglycosidase digestion revealed that one of them was a mixture of two neutral oligosaccharides. The complete structures of the three oligosaccharides were elucidated by methylation analysis. It was confirmed that all the N-acetylneuraminic acid residues of the asparagine-linked sugar chains of human chorionic gonadotropin occur as NeuAc alpha 2 leads to 3Gal groupings by comparing the methylation analysis data for the acidic oligosaccharide mixture before and after sialidase treatment. Based on these results, the structures of the asparagine-linked sugar chains of human chorionic gonadotropin were confirmed to be +/- NeuAc alpha 2 leads to 3Gal beta 1 leads to 4GlcNAc beta 1 leads to 2Man alpha 1 leads to 6(NeuAc alpha 2 leads to 3Gal beta 1 leads to 4GlcNAc beta 1 leads to 2Man alpha 1 leads to 3)Man beta 1 leads to 4GlcNAc beta 1 leads to 4(+/- Fuc alpha 1 leads to 6)GlcNAc and Man alpha 1 leads to 6(NeuAc alpha 2 leads to 3 Gal beta 1 leads to 4 GlcNAc beta 1 leads to Man alpha 1 leads to 3)Man beta 1 leads to 4 GlcNAc beta 1 leads to 4GlcNAc.     

Appearance of monoglyceride and triglyceride in the cell envelope of Escherichia coli mutants defective in diglyceride kinase

Diglyceride kinase mutants of Escherichia coli contain about 50- to 100-fold more 1,2-diglyceride than wild type cells. We now report that monoglyceride and triglyceride also accumulate in these strains. In mutant RZ60 (dgk-6) these compounds represent about 1 and 0.2%, respectively, of the total lipid fraction, while diglyceride represents 5-8% under most conditions. Monoglyceride accumulates predominantly in the outer membrane, while triglyceride builds up together with diglyceride in the cytoplasmic membrane. Under typical growth conditions about two-thirds of the diglyceride in E. coli arises in conjunction with synthesis of the membrane-derived oligosaccharides (Raetz, C.R.H., and Newman, K.F. (1979) J. Bacteriol. 137, 860-868). Inhibition of membrane-derived oligosaccharides (MDO) synthesis also curtails the accumulation of monoglyceride and triglyceride. However, there appears to be at least one other MDO-independent source of diglyceride and related metabolites. Since MDO synthesis is suppressed by high osmolarity (Kennedy, E.P. (1982) Proc. Natl. Acad. Sci. U.S. A. 79, 1092-1095), we have examined the effects of osmolarity on diglyceride accumulation in RZ60 (dgk-6). As expected, if MDO synthesis and diglyceride formation are coupled, the diglyceride level in RZ60 is higher at low osmolarity, while at high osmolarity the level of diglyceride is reduced to that observed in double mutants defective both in MDO synthesis and diglyceride kinase. Since dgk mutants do not grow at very low osmolarity, we have isolated several spontaneous phenotypic revertants that do. One class regains diglyceride kinase and has low diglyceride levels under all conditions. The other class remains defective in diglyceride kinase but tolerates higher diglyceride levels which amount to 13% of the total lipid during maximal induction of MDO synthesis at low osmolarity.     

Oligosaccharide structures of mucins secreted by the human colonic cancer cell line CL.16E.

Cl.16E, a stably differentiated clonal derivative of the human colonic cancer cell line HT29, was used to investigate the structure of oligosaccharide chains of mucins in colonic cancer. Secretory mucins were purified by equilibrium density gradient centrifugation in CsCl. Oligosaccharide side chains were isolated after beta-elimination. Compositional analysis of oligosaccharide-alditols performed after purification by gel filtration on a Bio-gel P-6 column showed 1) that GalNAc residues were located exclusively at the reducing ends of the chains, and 2) that fucose was absent from the preparation. Oligosaccharide-alditols were separated by high performance liquid chromatography (HPLC) on quaternary amine packings into a minor neutral fraction representing about 6.5% by weight of released oligosaccharides and four acidic fractions. Two acidic fractions, namely FI and FII encompassing mono- and disialylated structures, respectively, and containing 78% of total oligosaccharide alditols, were separated by HPLC. Structural determinations were carried out using methylation analysis, 1H NMR spectroscopy, and fast atom bombardment-mass spectrometry. Twelve oligosaccharide structures were determined which ranged in size from 3 to 8 residues. These oligosaccharides were based on core types 1, 2, and 4. Elongation of oligosaccharide chains was terminated by addition of sialic acid in alpha 2-3 linkage to Gal beta 1-3R and to Gal beta 1-4R residues. The predominant structure was a hexasaccharide (fraction FII-4). This contrasts with normal colonic mucins whose oligosaccharides were previously found to be based on core 3 structures and carry sialic acids in alpha (2-6) linkage to Gal beta 1-3R, to Gal beta 1-4R, and to GalNAc alpha-R (Podolsky, D.K. (1985) J. Biol. Chem. 260, 8262-8271; Podolsky, D.K. (1985) J. Biol. Chem. 260, 15510-15515). Collectively our findings suggest that Cl.16E colon cancer cells are able to synthesize mucin oligosaccharides of gastric type whose elongation is truncated by premature sialylation.     

Oxidatively fragmented phosphatidylcholines activate human neutrophils through the receptor for platelet-activating factor.

Platelet-activating factor (PAF, 1-O-alkyl-2-acetyl-sn-glycero-3-phosphocholine) activates neutrophils (polymorphonuclear leukocytes, PMN) through a receptor that specifically recognizes short sn-2 residues. We oxidized synthetic [2-arachidonoyl]phosphatidylcholine to fragment and shorten the sn-2 residue, and then examined the phospholipid products for the ability to stimulate PMN. 1-Palmitoyl-2-arachidonoyl-sn-glycero-3-phosphocholine was fragmented by ozonolysis to 1-palmitoyl-2-(5-oxovaleroyl)-sn-glycero-3-phosphocholine. This phospholipid activated human neutrophils at submicromolar concentrations, and is effects were inhibited by specific PAF receptor antagonists WEB2086, L659,989, and CV3988. 1-Palmitoyl-2-arachidonoyl-sn-glycero-3-phosphocholine next was fragmented by an uncontrolled free radical-catalyzed reaction: it was treated with soybean lipoxygenase to form its sn-2 15-hydroperoxy derivative (which did not activate neutrophils) and then allowed to oxidize under air. The secondary oxidation resulted in the formation of numerous fragmented phospholipids (Stremler, K. E., Stafforini, D. M., Prescott, S. M., and McIntyre, T. M. (1991) J. Biol. Chem. 266, 11095-11103), some of which activated PMN. Hydrolysis of sn-2 residues with phospholipase A2 destroyed biologic activity, as did hydrolysis with PAF acetylhydrolase. PAF acetylhydrolase is specific for short or intermediate length sn-2 residues and does not hydrolyze the starting material (Stremler, K. E., Stafforini, D. M., Prescott, S. M., and McIntyre, T. M. (1991) J. Biol. Chem. 266, 11095-11103). Neutrophil activation was completely blocked by L659,989, a specific PAF receptor antagonist. We conclude that diacylphosphatidylcholines containing an sn-2 polyunsaturated fatty acyl residue can be oxidatively fragmented to species with sn-2 residues short enough to activate the PAF receptor of neutrophils. This suggests a new mechanism for the appearance of biologically active phospholipids, and shows that PAF receptor antagonists block the action of both PAF and these PAF-like lipids.     

beta1-4Galactosyltransferase activity of mouse brain as revealed by analysis of brain-specific complex-type N-linked sugar chains

We previously reported two brain-specific agalactobiantennary N-linked sugar chains with bisecting GlcNAc and alpha1-6Fuc residues, (GlcNAcbeta1-2)(0)(or)(1)Manalpha1-3(GlcNAcbeta1-2M analpha1-6)(GlcNA cbeta1-4)Manbeta1-4GlcNAcbeta1-4(Fucalpha1-6)Glc NAc [Shimizu, H., Ochiai, K., Ikenaka, K., Mikoshiba, K., and Hase, S. (1993) J. Biochem. 114, 334-338]. Here, the reason for the absence of Gal on the sugar chains was analyzed through the detection of other complex type sugar chains. Analysis of N-linked sugar chains revealed the absence of Sia-Gal and Gal on the GlcNAc residues of brain-specific agalactobiantennary N-linked sugar chains. We therefore investigated the substrate specificity of galactosyltransferase activities in brain using pyridylamino derivatives of agalactobiantennary sugar chains with structural variations in the bisecting GlcNAc and alpha1-6Fuc residues as acceptor substrates. While the beta1-4galactosyltransferases in liver and kidney could utilize all four oligosaccharides as substrates, the beta1-4galactosyltransferase(s) in brain could not utilize the agalactobiantennary sugar chain with both bisecting GlcNAc and Fuc residues, but could utilize the other three acceptors. Similar results were obtained using glycopeptides with agalactobiantennary sugar chains and bisecting GlcNAc and alpha1-6Fuc residues as substrates. The beta1-4galactosyltransferase activity of adult mouse brain thus appears to be responsible for producing the brain-specific sugar chains and to be different from beta1-4galactosyltransferase-I. The agalactobiantennary sugar chain with bisecting GlcNAc and alpha1-6Fuc residues acts as an inhibitor against "brain type" beta1-4galactosyltransferase with a K(i) value of 0.29 mM.