Investigations of the active site of Saccharomyces cerevisiae dolichyl-phosphate-mannose synthase using fluorescent labeled dolichyl-phosphate derivatives.

Dolichol-phosphate mannose (Dol-P-Man) is a key mannosyl donor for the biosynthesis of N-linked oligosaccharides as well as for O-linked oligosaccharides on yeast glycoproteins, and for the synthesis of the glycosyl-phosphatidylinositol anchor found on many cell surface glycoproteins. It is synthesized by Dol-P-Man synthase which is the only glycosyltransferase in the dolichol pathway that has been expressed as an active protein, solubilized and purified in large enough quantities for structural investigations. Earlier studies showed that the enzyme is closely associated with membranes of endoplasmic reticulum with unique lipid requirements for its maximal activity. This potential target of antibiotic therapy is now being investigated at the molecular level to establish information about the structure of the enzyme as well as determine the nature and properties of the enzyme-phospholipid interactions. In this paper, we have determined the activities of the fluorescent labeled dolichyl-phosphate derivatives as well as the intramolecular distances between amino acid residues near the active site and/or the fluorophores of the substrate derivatives using fluorescence energy resonance transfer. These results also show that the conserved consensus sequence is not required by Dol-P-Man synthase neither for the recognition of Dol-P nor for the catalytic activity.

Mechanism of type 3 capsular polysaccharide synthesis in Streptococcus pneumoniae.

The glycosidic linkages of the type 3 capsular polysaccharide of Streptococcus pneumoniae ([3)-beta-D-GlcUA-(1-->4)-beta-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 Mn(2+) or Mg(2+) with maximal levels seen with 10-20 mM Mn(2+). 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.

Characterization of recombinant yeast dolichyl mannosyl phosphate synthase and site-directed mutagenesis of its cysteine residues.

Dolichyl mannosyl phosphate synthase is associated with membranes of the rough endoplasmic reticulum and catalyzes mannosyl transfer from GDP-mannose to the hydrophobic long-chain acceptor dolichyl-phosphate. The gene for the yeast enzyme encodes a protein with a molecular mass of 30.36 kDa containing three cysteine residues, at positions 93, 172 and 259 [Orlean, P., Albright, C. & Robbins, P. W. (1988) J. Biol. Chem. 263, 17499-17507]. Inhibition of the synthase by thiol-specific reagents, including N-ethylmaleimide, p-hydroxymercuribenzoate, 5,5'-dithiobis(2-nitrobenzoic acid) (Nbs2), and lucifer yellow iodoacetamide (LYI), suggests that sulfhydryl groups might play a role in the catalytic mechanism of the enzyme. Titration of the synthase with Nbs2 or LYI indicated that 1 mol sulfhydryl/mol protein was accessible to these reagents, and that saturation of this site completely inhibited enzyme activity. To ascertain the reactive group and its possible function in enzyme catalysis, each of the cysteine residues was replaced individually by site-directed mutagenesis. The mutant enzymes had specific activities comparable to that of the wild-type enzyme, demonstrating that none of the cysteine residues were essential for catalytic activity. All of the mutant proteins except those containing a substitution at Cys93 were inhibited by thiol-blocking reagents, indicating that Cys93 might be physically located near the catalytic site of the enzyme. GDP-mannose, dolichyl phosphate and substrate analogs were found to protect against Nbs2 inactivation, further suggesting that Cys93 was physically near, or within, the substrate-binding site of the enzyme.

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.

Isolation and expression of murine and rabbit cDNAs encoding an alpha 1,2-mannosidase involved in the processing of asparagine-linked oligosaccharides.

We have isolated a full-length cDNA clone encoding a murine alpha 1,2-mannosidase involved in the processing of mammalian Asn-linked oligosaccharides. Oligonucleotide primers were designed based on peptide sequences derived from the purified rabbit liver enzyme and were used to generate a 1011-base pair probe using the polymerase chain reaction. This probe was used to isolate clones from rabbit and mouse cDNA libraries. The full-length murine cDNA clone encodes a 655-amino acid type II transmembrane protein with a 43-amino acid cytoplasmic tail, a single transmembrane domain, and a large COOH-terminal catalytic domain containing two potential N-glycosylation sites. Stable transfection of the murine alpha 1,2-mannosidase cDNA into mouse L cells resulted in a approximately 22-fold overexpression of alpha 1,2-mannosidase activity. Three transcripts were detected in rabbit tissues, whereas two were found in rat and mouse tissues. The sequences of the rabbit and mouse cDNA clones indicate that the multiple transcripts differ in the length of their 3' sequences as a result of the use of multiple polyadenylation signals. Immunolocalization detected cross-reactive material in a juxtanuclear pattern consistent with the Golgi complex. The catalytic portion of the murine alpha 1,2-mannosidase was found to bear a strong similarity to the processing alpha 1,2-mannosidase from Saccharomyces cerevisiae.

Is there a "dolichol recognition sequence" in enzymes that interact with dolichols and other polyisoprenoid substrates?

Yeast dolichyl-P-mannose synthase and a number of other enzymes that interact with dolichol or dolichyl-P as substrates contain a highly conserved amino-acid sequence that has been proposed as a potential dolichol recognition sequence [Albright, C.F., Orlean, P. & Robbins, P.W. (1989) Proc. Natl. Acad. Sci. U.S.A. 86, 7366-7369]. In dolichyl-P-mannose synthase, the most highly conserved amino-acid residues of this domain were modified by site directed mutagenesis, and for one construct the sequence was completely deleted. Enzymes containing the site directed modifications, and the deletion mutant, were found to retain catalytic activity, and all of the modified enzymes had the same apparent affinity for Dol-P as wild type enzyme when assayed in a phospholipid matrix. Based on these results, the amino-acid composition and sequence of the conserved domain are not critically important for the recognition and binding of Dol-P when the synthase is reconstituted in a lipid matrix.

Human immunodeficiency virus type 1 envelope glycoprotein is modified by O-linked oligosaccharides.

The human immunodeficiency virus type 1 (HIV-1) envelope glycoprotein has been shown to be extensively modified by N-linked glycosylation; however, the presence of O-linked carbohydrates on the glycoprotein has not been firmly established. We have found that enzymatic deglycosylation of the HIV-1 envelope glycoprotein with neuraminidase and O-glycosidase results in a decrease in the apparent molecular weight of the envelope glycoprotein. This result was observed in both vaccinia virus recombinant-derived envelope glycoproteins and glycoproteins derived from the IIIB, SG3, and HXB2, strains of HIV-1. The decrease in molecular weight was also observed when the envelope glycoprotein had been deglycosylated with N-glycanase F after treatment with neuraminidase and O-glycosidase, indicating that the decrease in apparent molecular weight was not attributable to the removal of N-linked carbohydrate. Treatment with neuraminidase, O-glycosidase, and N-glycanase F was found to be necessary to remove all radiolabel from [3H]glucosamine-labelled envelope glycoprotein, a result seen for both recombinant and HIV-1-derived envelope glycoprotein. [3H]glucosamine-labelled carbohydrates liberated by O-glycosidase treatment were separated by paper chromatography and were found to be of a size consistent with O-linked oligosaccharides. We, therefore, conclude that the HIV-1 envelope glycoprotein is modified by the addition of O-linked carbohydrates.

The purification and characterization of recombinant yeast dolichyl-phosphate-mannose synthase. Site-directed mutagenesis of the putative dolichol recognition sequence.

Yeast dolichyl-phosphate-mannose synthase was purified from cultures of Escherichia coli carrying the gene for this enzyme in a high expression vector. The synthase contains a highly conserved hydrophobic amino acid sequence proposed to be involved in the recognition of dolichols (Albright, C. F., Orlean, P., and Robbins, P. W. (1989) Proc. Natl. Acad. Sci. U. S. A. 86, 7366-7369) and amino acid residues in this sequence were altered by site-directed mutagenesis. Conservative substitutions had no effect on the affinity of the enzyme for dolichyl-P. The substitution of asparagine for isoleucine at position 253 resulted in higher values for the apparent Km for Dol-P when assayed in detergent solutions, but this substitution had no effect on Km when the enzyme was reconstituted with phosphatidylethanolamine. Enzyme containing a deletion of the entire putative dolichol recognition sequence retained catalytic activity. The apparent Km for Dol-P was increased when this enzyme was assayed in detergent solution but was the same as wild type enzyme when reconstituted in phosphatidylethanolamine. These results suggest that the amino acid composition and sequence of the conserved domain are not critically important for the recognition and binding of Dol-P when the synthase is present in a lipid matrix.

Yeast dolichyl-phosphomannose synthase: reconstitution of enzyme activity with phospholipids.

The activity of purified recombinant yeast dolichyl-phosphomannose synthase (EC 2.4.1.83) was assessed following reconstitution of the enzyme with phospholipids. The yeast synthase, similar to the mammalian enzyme, was active when reconstituted with phosphatidylethanolamine dispersions but had little (less than 5%) activity in stable phosphatidylcholine bilayers. The enzyme was activated by adding increasing amounts of diacylglycerol to phospholipid bilayers, suggesting that activity of the yeast enzyme was dependent on lipid phase properties rather than specific phospholipids. The synthase could also be reconstituted as an active enzyme in bilayers prepared with a commercial crude lipid preparation containing 40% phosphatidylcholine, but at a rate 10% of that occurring in phosphatidylethanolamine. Vesicles composed of the 40% phosphatidylcholine lipid mixture, dolichyl phosphate, and enzyme were leaky in the presence of divalent cations, and dolichyl-phosphomannose synthase did not appear to catalyze the translocation of dolichyl phosphomannose across membranes at a catalytically significant rate under the assay conditions employed.

Calcium ion activation of rabbit liver alpha 1,2-mannosidase.

Rabbit liver alpha 1,2-mannosidase is a calcium ion requiring enzyme involved in processing the asparagine-linked oligosaccharides of glycoproteins. Ca2+ activation occurs with an apparent Ka of 1.1 microM. The major effect of the metal ion activator is on Km rather than Vmax. The kinetic mechanism of the enzyme is that of an ordered equilibrium in which Ca2+ must bind before substrate and the metal ion cannot release once the substrate has added to the enzyme. Several other divalent cations including Co2+, Mn2+, and Zn2+ were competitive with Ca2+ and inhibited the enzyme. Significantly, Mg2+ had no effect on enzyme activity. 1-Deoxymannojirimycin and Tris, which inhibit glycoprotein processing in vivo, are inhibitors of the mannosidase competitive with substrate. The effect of Ca2+ on the affinity of the enzyme for substrate may be a determinant in regulation of enzyme activity in vivo.

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.

Purification and characterization of an alpha-1,2-mannosidase involved in processing asparagine-linked oligosaccharides.

A calcium-dependent alpha-1,2-mannosidase involved in the processing of asparagine-linked oligosaccharides was purified to homogeneity from rabbit liver microsomes. N-terminal amino acid analysis was consistent with the presence of a homogeneous protein. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis, under both reducing and nonreducing conditions, revealed a single protein band with an apparent molecular weight of 52,000. Gel filtration and sedimentation analysis under nondenaturing conditions suggested that the purified enzyme is a monomeric protein. The mannosidase is a glycoprotein based on the presence of protein-linked sugar and specific binding of the enzyme to concanavalin A-Sepharose. Purified mannosidase was optimally active between pH 5.0 and 6.0. The enzyme was inactive with p-nitrophenyl-alpha-D-mannopyranoside and was inhibited by deoxymannojirimycin but not by swainsonine. The enzyme was specifically activated by Ca2+, with half-maximal activation occurring at concentrations of 10 microM or less and was inhibited by Mn2+, Co2+, Ba2+, and Zn2+. Calcium ions protected the enzyme against inactivation by p-chloromercuribenzoate. Rabbit liver mannosidase hydrolyzed alpha-1,2-mannosyl-mannose linkages in a variety of substrates including methyl-2-O-alpha-D-mannopyranosyl-alpha-D-mannopyranoside (Schutzbach, J. S. (1987) Anal. Biochem. 167, 279-283), ovalbumin glycopeptide IV, and the high mannose chains of thyroglobulin and phytohemagglutinin-P. Approximately 70% of the alpha-1,2-linked mannosyl units in the oligosaccharides of thyroglobulin were accessible to rabbit liver alpha-mannosidase, whereas most of the alpha-1,2-mannosyl units in phytohemagglutinin were resistant to digestion prior to heat denaturation of the plant lectin.

Phospholipase-induced modulation of dolichyl-phosphomannose synthase activity.

Rat liver dolichyl-phosphomannose synthase is optimally active when the enzyme is reconstituted with lipids that prefer a nonlamellar macroscopic organization in isolation, such as phosphatidylethanolamine (PE), but the enzyme is only negligibly active in the presence of lipids that normally form stable bilayers, such as phosphatidylcholine (PC) [Jensen, J.W., & Schutzbach, J.S. (1985) Eur. J. Biochem. 153, 41-48]. We now report that the activity of the synthase can be modulated by incorporating diacylglycerol and lysophosphatidylcholine into the lipid matrix. Enzyme activity in PC bilayers was stimulated by the presence of diacylglycerol, a lipid that has a conical dynamic molecular shape and disrupts bilayer stability. In PC/diacylglycerol mixtures the apparent Km for dolichyl-P was 30-fold lower than the apparent Km for the polyprenol acceptor in PC membranes. Enzyme activity was also stimulated when diacylglycerol was generated in situ by incubation of PC vesicles with phospholipase C. In contrast, the activity of enzyme reconstituted in PE dispersions, or in PE/PC bilayers, was markedly inhibited by the presence of lysophospholipids. Enzyme activity was also reduced by the in situ generation of lysophospholipids in PE/PC vesicles by incubation with phospholipase A2. Since lysophospholipids and diacylglycerols arise in vivo as products of phospholipid metabolism, modulation of enzyme activity by these compounds may represent a potential regulatory mechanism for the synthesis of oligosaccharide lipids.

Modulation of dolichyl-phosphomannose synthase activity by changes in the lipid environment of the enzyme.

Rat liver dolichyl-phosphomannose synthase (GDP mannose-dolicholphosphate mannosyltransferase; EC 2.4.1.83) was previously shown to catalyze optimal rates of mannosyl transfer to dolichyl-P when the polyprenol acceptor was incorporated into a phosphatidylethanolamine (PE) matrix that has a tendency to adopt a nonbilayer (hexagonal HII) phase [Jensen, J. W., & Schutzbach, J. S. (1985) Eur. J. Biochem. 153, 41-48]. The present investigations now further define the properties of the lipid environment that are essential for mannosyltransferase activity. Monogalactosyl diglyceride (MGDG), a glycoglycerolipid that prefers a nonbilayer-phase organization in isolation, was shown to provide a suitable lipid matrix for synthase activity. By comparison, the enzyme was not activated by digalactosyl diglyceride (DGDG), which forms stable bilayer structures upon hydration. Enzyme activity in MGDG/DGDG mixtures decreased as the proportion of DGDG in the dispersion was increased. Although bilayer-forming phospholipids supported low rates of mannosyl transfer, enzyme activity was stimulated by the addition of MGDG to either phosphatidylcholine (PC) or PE/PC (1:1) membranes. The incorporation of agents known to destabilize bilayer structures including dolichols, ubiquinone, dodecane, and cholesterol into PE/PC (1:1) membranes also increased the rate of mannosyl transfer. Enzyme activity in PC membranes was stimulated by the presence of gramicidin and also by greatly increased concentrations of the substrate, dolichyl-P. The results demonstrate that the enzyme does not have a requirement for PE and suggest that the physical state of the lipid matrix is an important determinant for reconstitution of the synthase and polyprenol phosphate substrate in a productive complex. The formation of an enzyme/lipid complex was demonstrated by sucrose density gradient centrifugation and could be correlated with the lipid requirements for enzyme activity.

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

A highly specific, sensitive, and convenient fluorescence assay for alpha-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-alpha-D-mannopyranosyl-alpha-D-mannopyranoside by the alpha-1,2-mannosidase. The assay was used to determine the substrate specificity of a calcium ion-activated alpha-1,2-mannosidase purified from rabbit liver microsomes. The microsomal mannosidase was specific for hydrolysis of the alpha-1,2 linkage. The mannosyl linkages in alpha-1,3- and alpha-1,6-linked methyl-disaccharides, in methyl-alpha-D-mannopyranoside, and in yeast mannan were hydrolyzed at rates of 2% or less than that noted with the alpha-1,2-linked disaccharide. Mannosidase activity was linear with time and was proportional to enzyme concentration. The Km for the alpha-1,2-linked methyl-disaccharide is 0.5 mM.

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.

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.

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.

Activation of dolichyl-phospho-mannose synthase by phospholipids.

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 beta-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, 115-1119]. 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 microM and the apparent Km for dolichyl-P was 0.3 microM. GMP, GDP, and GTP inhibited mannosyl transfer 50% at concentrations of 16 microM, 1.3 microM and 3 microM respectively.

Dolichol induces membrane leakage of liposomes composed of phosphatidylethanolamine and phosphatidylcholine.

Dolichol promotes the leakage of membranes in liposomes composed of phosphatidylethanolamine 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 membrane leakage.