John S. Schutzbach

Dolichol induces membrane leakage of liposomes composed of phosphatidylethanolamine and phosphatidylcholine

Dolichol promotes the leakage of membranes in liposomes composed ofphosphatidylethanolamine and phosphatidylcholine but not liposomes composed only of phosphatidylcholine. The membrane leakage was assayed by measuring the entrapment of TEMPOcholine, a cationic spin probe, in liposomes using ESR methods. The percent of membrane leakage induced by dolichol was found to be linearly proportional to the concentrations of dolichol. It is proposed that dolichol enhances the formation of non‐bilayer configurations in liposomes containing phosphatidylethanolamine, thereby inducing membrane leakage.

Mechanism of Type 3 Capsular Polysaccharide Synthesis inStreptococcus pneumoniae

The glycosidic linkages of the type 3 capsular polysaccharide of Streptococcus pneumoniae([3)-β-d-GlcUA-(1→4)-β-d-Glc-(1→] n ) are formed by the membrane-associated type 3 synthase (Cps3S), which is capable of synthesizing polymer from UDP sugar precursors. Using membrane preparations of S. pneumoniae in an in vitro assay, we observed type 3 synthase activity in the presence of either Mn2+ or Mg2+ with maximal levels seen with 10–20 mm Mn2+. High molecular weight polymer synthesized in the assay was composed of Glc and glucuronic acid and could be degraded to a low molecular weight product by a type 3-specific depolymerase from Bacillus circulans. Additionally, the polymer bound specifically to an affinity column made with a type 3 polysaccharide-specific monoclonal antibody. The polysaccharide was rapidly synthesized from smaller chains and remained associated with the enzyme-containing membrane fraction throughout its synthesis, indicating a processive mechanism of synthesis. Release of the polysaccharide was observed, however, when the level of one of the substrates became limiting. Finally, addition of sugars to the growing type 3 polysaccharide was shown to occur at the nonreducing end of the polysaccharide chain.

Effects of dolichol on membrane permeability

Small vesicles containing the tetra-anionic fluorescent probe calcein were prepared by sonication of mixtures of plant phosphatidylethanolamine, plant phosphatidylcholine, and dolichol. Following chromatography, the isolated vesicles were found to retain entrapped calcein over the temperature range of 15 to 40 degrees C. Utilizing an assay measuring the fluorescence quenching of entrapped calcein by cobalt ions, the presence of dolichol in the membranes was found to promote the permeability of the phospholipid bilayers to the divalent cation. The permeability was shown to be dependent on temperature with an increase in rate of 17-fold between 15 and 35 degrees C although the plant phospholipids used in these experiments have no known phase transition within this temperature range. The incorporated dolichol was distributed uniformly throughout the vesicle population. Similar vesicles prepared from phosphatidylethanolamine and phosphatidylcholine without added dolichol, from phosphatidylcholine alone, or with phosphatidylcholine and dolichol were far less permeable to the divalent cation under the same assay conditions. These results demonstrate that dolichols have significant effects on the permeability properties of phospholipid bilayers that contain phosphatidylethanolamine.

Bilayer membrane destabilization induced by dolichylphosphate

Small vesicles containing the fluorescent probe calcein were used to investigate the effect of dolichyl phosphate (Dol-P) on phospholipid bilayer stability. In the absence of Dol-P, phospholipid vesicles retained the fluorescent probe upon the addition of divalent cations. Small vesicles containing Dol-P, however, exhibited calcein leakage when incubated in the presence of divalent cations. This effect was observed in liposomes composed of a mixture of phosphatidylethanolamine (PE), phosphatidylcholine (PC) and Dol-P, but not in PC/Dol-P liposomes. The rate of calcein leakage was proportional to divalent cation concentration and to temperature, but was independent of vesicle concentration. These results demonstrate that Dol-P has significant effects on the stability of PE containing phospholipid bilayers. Vesicle leakage was also promoted by the addition of rat liver Dol-P-mannose synthase (EC 2.4.1.83) to intact PE/PC/Dol-P vesicles. Enzyme induced leakage from phospholipid vesicles required the presence of both unsaturated PE and Dol-P. The phospholipid composition of leaky vesicles could be correlated with the lipid matrix required for maximal transferase activity of the rat liver synthase. The destabilizing effects of Dol-P on phospholipid bilayers may therefore be involved in the translocation of activated sugars across biological membranes.

The role of the lipid matrix in the biosynthesis of dolichyl-linked oligosaccharides

The enzymes in the dolichol pathway are membrane-proteins that utilize a combination of hydrophilic and extremely hydrophobic substrates. The enzymes in this pathway that have been purified and characterized to any extent have either been shown to be stabilized by mixed phospholipid/detergent micelles, or else require a lipid matrix for catalytic activity. Further understanding of the mechanisms of these essential enzymes may require developing methods for the reconstitution of the glycosyltransferases and their hydrophobic substrates in appropriate lipid matrices. Abbreviations: CHO, Chinese hamster ovary; Dol, dolichol; DAG, diacylglycerol; DOPC, dioleolylphosphatidylcholine; DOPE, dioleolyphosphatidylethanolamine; ER, endoplasmic reticulum; PC, phosphatidylcholine; PE, phosphatidylethanolamine; PG, phosphatidylglycerol; PI, phosphatidylinositol

Characterization of mannosyl-transfer reactions catalyzed by dolichyl-mannosyl-phosphate-synthase

Evidence suggesting that a single enzyme catalyzes mannosyl transfer from GDP-mannose to both dolichyl phosphate and to phenyl phosphate was obtained as follows: (a) The two activities were coeluted from columns of DEAE-cellulose and Sepharose CL-6B, (b) both reactions demonstrated similar kinetic constants for the glycosyl donor and for guanosine nucleoside inhibitors, (c) both reactions were sensitive to inhibition by low concentrations of nonionic detergents, and (d) both activities were found to be thermally inactivated at similar rates upon incubation at 55 degrees. The reaction conditions required for optimal mannosyl transfer by the purified enzyme preparation to the hydrophobic and water soluble acceptors, however, were found to be quite different. Whereas mannosyl transfer from GDP-mannose to dolichyl phosphate occurred at maximal rates only in the presence of specific phospholipids, the rate of mannosyl transfer to phenyl phosphate was essentially unaffected by the addition of phospholipid. These results indicate that dolichyl-mannosyl-phosphate-synthase, which has some of the properties of an intrinsic membrane protein, does not have an absolute requirement for phospholipid for catalytic activity, but rather that phospholipid is required for interaction of the enzyme with the long chain polyisoprenol substrate dolichyl phosphate.

Localization of dolichols in phospholipid membranes. An ESR spin label study.

We have used ESR methods employing spin‐labeled stearates to investigate the effects of dolichol on the motion of lipid molecules in phospholipid membranes of phosphatidylethanolamine and phosphatidylcholine. The ESR spectra show that the presence of dolichol affects the motion of the spin probes at carbon‐16, but not at carbon‐5. Similar results are obtained with phospholipid membranes comprising only phosphatidylcholine. It is suggested that dolichol molecules are present mainly in the lipid core region of phospholipid membranes.

Synthesis of 3-O-β-d-xylopyranosyl-l-serine (xylosylserine) andO-β-d-galactopyranosyl-(1-4)-O-β-d-xylopyranosyl-l-serine (galactosylxylosylserine) and use of the synthetic products for detection of galactosyltransferase I activity in rat liver

Abstract3-O-β-d-Xylopyranosyl-l-serine (xylosylserine) was synthesized by the following three-step procedure: 1) 2,3,4-tri-O-benzoyl-α-d-xylopyranosyl bromide (benzobromoxylose) was condensed withN-carbobenzoxy-l-serine benzyl ester using the silver triflate-collidine complex as promoter; 2) theN-carbobenzoxy and benzyl ester groups in the resultant glycoside were cleaved by transfer hydrogenation with palladium black as catalyst and ammonium formate as hydrogen donor; and 3) the benzoyl groups were removed with methanolic ammonia. Xylosylserine was obtained in an overall yield of 70%. O-β-d-Galactopyranosyl-(1-4)-O-β-d-xylopyranosyl-(1-3)-l-serine (galactosylxylosylserine) was also synthesized by this methodology and was characterized by 2-dimensional (2D) NMR spectroscopy techniques. The two serine glycosides (xylosylserine and galactosylxylosylserine) were used in detection and partial purification of galactosyltransferase I (UDP-d-galactose:d-xylose galactosyltransferase) from adult rat liver.

The 24th Meeting of the Society for Glycobiology was held in Boston, Massachusetts from 23–26 November 1996. The chairman of the organizing committee was Dr Vernon Reinhold.

The meeting was initiated on Saturday evening with a Plenary Lecture by S. Kornfeld on the Regulation of Mannose Phosphorylation of Lysosomal and Secretory Glycoproteins. He presented the results of his intensive research on the substrate specificity of the committed enzyme, the GlcNAc-P transferase, which synthesizes the recognition marker directing lysosomal enzymes to their proper intracellular compartment. It is now apparent that the substrate recognition site for the GlcNAc-P transferase is quite complex and involves far more than primary peptide sequence. The substrate recognition site appears to involve a large portion of the lysosomal enzyme surface, but exact details of the complete substrate recognition site have yet to be resolved. Each morning session then began with two Perspective Lectures followed by Salient Topic Symposia. The Perspective lecture speakers included P. Robbins on Oligosaccharide Signaling, D. Harn on Oligosaccharide Modulation of the Immune Response, H.P. Hauri on Golgi Recycling Pathways, D. Williams on Calnexin, R. Jefferis on The Effects of Antibody Glycosylation and M. Brenner on Antigen-specific Recognition of Glycolipids by T lymphocytes. The Salient Topics covered a wide range of subject matter from control of glycosyltransferase gene expression and localization (N. Shaper, K. Colley), early processing pathways of N-linked oligosaccharides (R. Spiro), the functions of glycolipids and glycosylphosphatidylinositol (GPI) anchors (C. Lingwood, T. Seyfried, M. Ferguson), therapeutic glycoconjugates, inhibitors of carbohydrate function and bacterial binding to carbohydrate receptors (E. Tuomanen, L. Kiessling), carbohydrates and glycosyltransferases modulating cell adhesion (I. Stamenkovic, S. Hakomori, J. Lowe) and the role of sugars on IgG (R. Dwek). The wide range of topics and large number of speakers precludes a detailed description of each talk; the overall theme was generally directed to demonstrating the importance of carbohydrates as recognition markers for biological processes. The Perspective Lectures were then followed by a number of symposia on specific topics including; biosynthesis of proteoglycans, glycosylation of proteins in insect cells,

A fluorescence assay for α-1,2-mannosidases involved in glycoprotein processing reactions

Abstract A highly specific, sensitive, and conveient fluorescence assay for α-1,2-mannosidases involved in glycoprotein processing reactions is described. The assay utilizes a coupled enzyme system to determine the amount of free mannose liberated from the disaccharide O -methyl-2- O -α- d -mannopyranosyl-α- d -mannopyranoside by the α-1,2-mannosidase. The assay was used to determine the substrate specificity of a calcium ion-activated α-1,2-mannosidase purified from rabbit liver microsomes. The microsomal mannosidase was specific for hydrolysis of the α-1,2 linkage. The mannosyl linkages in α-1,3- and α-1,6-linked methyl-disacchardes, in methyl-α- d -mannopyranoside, and in yeast mannan were hydrolyzed at rates of 2% or less than that noted with the α-1,2-linked disaccharide. Mannosidase activity was linear with time and was proportional to enzyme concentration. The K m for the α-1,2-linked methyl-disaccharide is 0.5 m m .