Biosynthesis of glycoproteins in Candida albicans: activity of mannosyl and glucosyl transferases

The enzymes dolichol phosphate glucose synthase and dolichol phosphate mannose synthase (DPMS), which catalyze essential steps in glycoprotein biosynthesis, were solubilized and partially characterized in Candida albicans. Sequential incubation of a mixed membrane fraction with increasing concentrations of Nonidet P-40 released a soluble fraction that transferred glucose from UDP-Glc to dolichol phosphate glucose and minor amounts of glucoproteins in the absence of exogenous dolichol phosphate. Studies with the soluble fraction revealed that some properties were different from those previously determined for the membrane-bound enzyme. Accordingly, the soluble enzyme exhibited a twofold higher affinity for UDP-Glc and a sixfold higher affinity over the competitive inhibitor UMP, and the transfer reaction was fourfold more sensitive to inhibition by amphomycin. On the other hand, a previously described protocol for the solubilization of mannosyl transferases that rendered a fraction exhibiting both DPMS and protein mannosyl transferase (PMT) activities operating in a functionally coupled reaction was modified by increasing the concentration of Nonidet P-40. This resulted in a solubilized preparation enriched with DPMS and nearly free of PMT activity which remained membrane bound. DPMS solubilized in this manner exhibited an absolute dependence on exogenous Dol-P. Uncoupling of these enzyme activities was a fundamental prerequisite for future individual analysis of these transferases.     

Biosynthesis of mannoproteins by cell-free extracts fromPhycomyces blakesleeanus

Most of the manosyl transferase activity inPhycomyces blakesleeanus was found associated with a crude membrane fraction sedimenting at 48,400g (R_av). Triton X-100 and Nonidet NP-40 inhibited 95% of the enzyme activity. Digitonin caused 47% of inhibition and when removed, the membrane-bound enzymatic activity increased by about 35%; no activity was detected in supernatant. The rate of mannosyl transfer increased in the presence of 4 or 8 mM Mg²⁺ ions. Several compounds, including glycoproteins, mucoran, and mucoric acid, failed to act as acceptors of mannosyl residues. Guanosine diphosphate and guanosine monophosphate inhibited the transfer of mannosyl residues by 60 and 19%, respectively. Mannosyl transfer involves participation of lipid intermediates.β elimination of the product synthesizedin vitro revealed the presence of mannose, mannobiose, and mannotriose, suggesting that they are bound to protein viaO-glycosidic linkages. The alkaline-resistant carbohydrate part of the glycoproteins consisted mainly of mannose residues that were probably connected to the protein moiety throughN-glycosidic bonds.     

Biosynthesis of glycoproteins in Candida albicans: Solubilization and partial characterization of dolichol phosphate mannose synthase and protein mannosyl transferases

Incubation of a mixed membrane fraction isolated from C. albicans yeast cells with Nonidet P-40 at a detergent/protein ratio as low of 0.025 (0.016–0.019%, w/v) yielded a soluble fraction that catalyzed the transfer of mannose from GDP-[¹⁴C] Man into dolichol phosphate mannose and from this intermediate into mannoproteins. Over 95% of the sugar in mannoproteins was O-linked as judged from its release after -elimination. Mannose was identified as the sole product after this treatment. Transfer activity did not depend on exogenous lipid acceptor indicating that the latter was solubilized along with the mannosyl transferases. Synthesis of mannolipid and mannoproteins occurred at optima temperatures of 20 °C and 37 °C, respectively, and at a pH in the range of 7.5-9.5. Mannosyl transfer into the mannolipid was stimulated by Mg²⁺and inhibited by Ca²⁺and Mn²⁺whereas mannoprotein labeling was stimulated by Mn²⁺and to a lower extent by Mg²⁺. When measured as a function of substrate concentration, the synthesis of the mannolipid was a nearly linear function of GDP-Man concentration in the range of 5 to 32 M whereas protein mannosylation exhibited hyperbolic kinetics with saturation reached at about 10 M. The solubilized preparation was able to utilize an exogenous source of mannolipid as sugar donor for protein mannosylation. Dinucleotides and, to a higher extent trinucleotides, inhibited mannosyl transfer into the mannolipid and hence into mannoproteins.     

Role of mannosyl lipid intermediate in the synthesis of Neurospora crassa glycoproteins.

Particulate membrane preparations from Neurospora crassa incorporated mannose from GDP-[14C] mannose into endogenous lipid and particulate protein acceptors. Synthesis of the mannosyl lipid is reversible in the presence of GDP. Chemical and chromatographic characterization of the mannosyl lipid suggest that it is a mannosylphosphorylpolyisoprenol. The other endogenous acceptor was precipitated by trichloracetic acid. Gel filtration and electrophoresis studies before and after treatment with proteolytic enzymes indicate that the second acceptor is a glycoprotein(s). beta Elimination studies on the mannosyl protein formed from GDP-[14C] mannose with Mg2+ in the reaction mixture or formed from mannosyl lipid indicate thad with the peptide chain. Several lines of evidence indicate that in Neurospora crassa the mannosyl lipid is an obligatory intermediate in the in vitro mannosylation of the protein. (a) At 15 degrees C the initial formation of the mannosyl lipid is faster than the initial formation of the mannosyl protein. (b) Exogenous partially purified mannosyl lipid can function as a mannosyl donor for the synthesis of the mannosyl protein. This reaction was also dependent on a divalent metal. The rate of this reaction was optimal at a concentration of Triton X-100 which effectively inhibited the transfer of mannose from GDP-[14C] mannose to lipid and protein, indicating that GDP-mannose was not an intermediate in the transfer of mannose from lipid to protein. The mannosyl protein formed in this reaction was indistinguishable by several criteria from the mannosyl protein formed from GDP-[14C] mannose and Mg2+. (c) The effect of a chase with an excess of unlabeled GDP-mannose on the incorporation of mannose into endogenous acceptors was immediate cessation of the synthesis and subsequent turnover of the mannosyl lipid; in contrast, however, incorporation of mannose into protein continued and was proportional to the loss of mannose from the mannosyl lipid.     

Solubilization and properties of mannose and N-acetylglucosamine transferases involved in formation of polyprenyl-sugar intermediates.

A particulate fraction from porcine aorta catalyzed the incorporation of N-acetylglucosamine (GlcNAc) from UDP-[3H]GlcNAc into both GlcNAc-pyrophosphorylpolyprenol and GlcNAc-GlcNAc-pyrophosphorylpolyprenol. This transfer utilized endogenous lipid and required a divalent cation. Mn2+ was the best metal ion and was optimum at 2.3 mM. This same particulate fraction was previously shown to transfer mannose from GDP-[14C]mannose to endogenous lipid to form mannosylphosphorylpolyprenol (Chambers, J., and Elbein, A.D. (1975) J. Biol. Chem. 250, 6904-6915). Both the GlcNAc activities and the mannose activity were solubilized by treatment of the particulate fraction with the detergent Nonidet P-40. The enzymes were partially purified by chromatography on DEAE-cellulose and on Sephadex G-200. These soluble enzymes required the addition of acceptor lipid for activity. An acidic lipid fraction, isolated from pig liver and having the properties of dolichyl phosphate, was active with either the GlcNAc or the mannose transferase. Chemically synthesized dolichyl phosphate was also active with either of these enzymes. The products formed from either GlcNAc or mannose by the soluble transferases were similar to those formed by the particulate enzyme. Thus the major product formed from UDP-[3H]GlcNAc was GlcNAc-pyrophosphoryldolichol with small amounts of the disaccharide-lipid while the product formed from GDP-[14C]mannose was mannosylphosphoryldolichol.     

Biosynthesis of Glycoproteins in the Human Pathogenic Fungus Sporothrix Schenckii: Synthesis of Dolichol Phosphate Mannose and Mannoproteins by Membrane-Bound and Solubilized Mannosyl Transferases

A membrane fraction obtained from the filamentous form of Sporothrix schenckii was able to transfer mannose from GDP-Mannose into dolichol phosphate mannose and from this inTermediate into mannoproteins in coupled reactions catalyzed by dolichol phosphate mannose synthase and protein mannosyl transferase(s), respectively. Although the transfer reaction depended on exogenous dolichol monophosphate, membranes failed to use exogenous dolichol phosphate mannose for protein mannosylation to a substantial extent. Over 95% of the sugar was transferred to proteins via dolichol phosphate mannose and the reaction was stimulated several fold by Mg²⁺ and Mn²⁺. Incubation of membranes with detergents such as Brij 35 and Lubrol PX released soluble fractions that transferred the sugar from GDP-Mannose mostly into mannoproteins, which were separated by affinity chromatography on Concanavilin A–Sepharose 4B into lectin-reacting and non-reacting fractions. All proteins mannosylated in vitro eluted with the lectin-reacting proteins and analytical electrophoresis of this fraction revealed the presence of at least nine putative mannoproteins with molecular masses in the range of 26–112 kDa. The experimental approach described here can be used to identify and isolate specific glycoproteins mannosylated in vitro in studies of O-glycosylation.     

Glycoprotein biosynthesis in Chlamydomonas. A mannolipid intermediate with the properties of a short-chain alpha-saturated polyprenyl monophosphate

A crude membrane preparation of the unicellular green alga Chlamydomonas reinhardii was found to catalyse the incorporation of D-[14C]mannose from GDP-D-[14C]-mannose into a chloroform/methanol-soluble compound and into a trichloroacetic acid-insoluble polymer fraction. The labelled lipid revealed the chemical and chromatographic properties of a short-chain (about C55-C65) alpha-saturated polyprenyl mannosyl monophosphate. In the presence of detergent both long-chain (C85-C105) dolichol phosphate and alpha-unsaturated undecaprenyl phosphate (C55) were found to be effective as exogenous acceptors of D-mannose from GDP-D-[14C]mannose to yield their corresponding labelled polyprenyl mannosyl phosphates. Exogenous dolichyl phosphate stimulated the incorporation of mannose from GDP-D-[14C]mannose into the polymer fraction 5-7-fold, whereas the mannose moiety from undecaprenyl mannosyl phosphate was not further transferred. Authentic dolichyl phosphate [3H]mannose and partially purified mannolipid formed from GDP-[14C]mannose and exogenous dolichyl phosphate were found to function as direct mannosyl donors for the synthesis of labelled mannoproteins. These results clearly indicate the existence of dolichol-type glycolipids and their role as intermediates in transglycosylation reactions of this algal system. Both the saturation of the alpha-isoprene unit and the length of the polyprenyl chain may be regarded as evolutionary markers.     

Involvement of dolichol phosphates as intermediates in the mannosyl and galactosyl transferases of rat testicular germ cell Golgi apparatus membranes

Isolated Golgi apparatus membranes from the germinal elements (spermatocytes and early spermatids) of rat testis were examined for their ability to incorporate [14C]mannose and [14C]galactose into glycolipid and glycoprotein fractions. Transfer of mannose from GDP-[14C]mannose into a Lipid I fractions (GPD:MPP mannosyl transferase activity), identified as mannosyl phosphoryl dolichol, showed optimal activity at 1.5 mM manganese and at pH 7.5. Low concentrations of Triton X-100 (0.1%) stimulated transferase activity in the presence of exogenous dolichol phosphate (Dol-P); however, inhibition occurred at Triton X-100 concentrations greater than 0.1%. Maximal activity of this GDP:MPP mannosyl transferase occurred at 25 microM Dol-P. Activity using endogenous acceptor was 2.34 pmole/min/mg, whereas in the presence of 25 microM Dol-P the specific activity was 284 pmole/min/mg, a stimulation of 125-fold. Incorporation of mannose into a Lipid II (oligosaccharide pyrophosphoryl dolichol) and a glycoprotein fraction was also examined. In the absence of exogenous Dol-P, rapid incorporation into Lipid I occurred with a subsequent rise in Lipid II and glycoprotein fractions suggesting precursor-product relationships. Addition of exogenous Dol-P to galactosyl transferase assays showed only a minor stimulation, less than twofold, in all fractions. Over the concentration range of 9.4 to 62.5 micrograms/ml Dol-P, only 1% of radioactive product accumulated in the combined lipid fractions. These observations suggest that the mannose transfer involves Dol-P intermediates and also that spermatocyte Golgi membranes may be involved in formation of the oligosaccharide core as well as in terminal glycosylations.     

A mannosyl-carrier lipid of bovine adrenal meddulla and rat parotid.

1. The transfer of mannose from GDP-(U-14-C)mannose into endogenous acceptors of bovine adrenal medullla and rat parotid was studied. The rapidly labelled product, a glycolipid, was partially purified and characterized. 2. It was stable to mild alkaline hydrolysis but yielded (14-C)mannose on mild acid hydrolysis. It co-chromatographed with mannosyl phosphoryl dolichol in four t.l.c. systems and on DEAE-cellulose acetate. Addition of dolichol phosphate or a dolichol phosphate-enriched fraction prepared from pig liver stimulated mannolipid synthesis. 3. The formation of mammolipid appeared reversible, since addition of GDP to a system synthesizing the mannolipid caused a rapid loss of label from the mannolipid. UDP-N-acetylglucosamine did not inhibit mannolipid synthesis except at high concentrations (2 mM), even though in the absence of GDP-mannose, N-acetylglucosamine was incorporated into a lipid having the properties of a glycosylated polyprenyl phosphate. 4. Mannose from GDP-mannose was also incorporated into two other acceptors, (2y being insoluble in chloroform-methanol (2:1, v/v) but soluble in choloroform-methanol-water (10:10:3, by vol.) and (ii) protein. These are formed much more slowly than the mannolipid. 5. Exogenous mannolipid served as a mannose donor for acceptors (i) and (ii), and it is suggested that transfer of mannose from GDP-mannose to mannosylated protein occurs via two intermediates, the mannolipid and acceptor (i).     

Synthesis of the mannosyl-O-serine (threonine) linkage of glycoproteins from polyisoprenylphosphate mannose in yeast (Hansenula holstii)

The mannolipid synthesized from GDP-mannose and lipid acceptors in a particulate enzyme preparation from the yeast Hansenula holstii (R. K. Bretthauer, S. Wu, and W. E. Irwin, (1973) Biochim. Biophys. Acta, 304, 736–747) has the properties of dolicholmonophosphate mannose. Transfer of [¹⁴C]mannose from exogenously supplied, purified mannolipid to endogenous protein acceptors of the particulate enzyme fraction has now been demonstrated. The synthesis of radioactive products which are insoluble in chloroform-methanol and water is dependent upon time and concentrations of substrate, particulate fraction protein, and detergent. Addition of MgCl₂ or MnCl₂ to incubation mixtures prepared in the absence of these ions had only small stimulatory effects (20–25%), suggesting that the reaction is not dependent upon metal ions. Relatively high concentrations (0.005 m-0.05 m) of EDTA did partially inhibit the reaction, but this is considered to be due to secondary effects.Seventy percent of the radioactivity in the chloroform-methanol insoluble residue was solubilized with hot, neutral citrate buffer. The Chromatographic properties of this material on Sephadex gels and on DEAE-Sephadex were very similar to the properties of glycoprotein products derived from GDP-[¹⁴C]mannose. The chloroform-methanol insoluble products were also solubilized with Pronase which subsequently resulted in the isolation of a radioactive glycopeptide that contained 25% of the radioactivity transferred from mannolipid. The radioactive component of this glycopeptide was shown by β-elmination experiments and by amino acid analyses to be [¹⁴C]mannose residues linked O-glycosidically to serine and threonine residues. It was concluded, therefore, that one function of the mannolipid is to serve as mannosyl donor in the synthesis of the mannosyl-O-serine (threonine) linkage region of glycoproteins which may be part of the cell wall mannan-protein complex. Other mannose-containing products may also be synthesized from the mannolipid, as β-elimination of the chloroform-methanol insoluble fraction or of the Pronase soluble fraction did not result in recovery of all of the radioactivity as [¹⁴C]mannose.     

The participation of lipid-linked oligosaccharide in synthesis of membrane glycoproteins.

Membrane preparations from hen oviduct catalyze the transfer of mannose from GDP-mannose into three components: mannosyl phosphoryl polyisoprenol, oligosaccharide-lipid, and glycoprotein. Eivence that mannosyl phosphoryl polyisoprenol serves as a mannosyl donor for synthesis of both oligosaccharide-lipid and glycoproteins was previously reported (Waechter, C.J., Lucas, J.J., and Lennarz, W.J. (1973) J. Biol. Chem. 248, 7570-7579). In this study the oligosaccharide-lipid has been isolated, and the oligosaccharide has been partially characterized. Based on paper chromatography the oligosaccharide chain contains 7 to 9 glycose units. The glycose at the reducing terminus is N-acetylglucosamine, whereas mannose is found at the nonreducing end. When UDP-N-acetyl[14C]glucosamine is incubated with oviduct membranes in the absence of GDP-mannose, a 14C-labeled chitobiosyl lipid, but little oligosaccharide-lipid is synthesized. When GDP-mannose is also present in the incubation mixture an oligosaccharide-lipid is formed containing N-acetyl[14C]glucosaminyl residues. This oligosaccharide-lipid is chromatographically identical with the [14C]mannose-containing oligosaccharide-lipid isolated in the earlier study cited above. When the N-acetyl[14C]glucosamine-oligosaccharide released from the oligosaccharide-lipid by mild acid is treated with partially purified alpha-mannosidase the major radioactive product is [14C]chitobiose. Evidence that the [14C]mannose-containing oligosaccharide-lipid serves as an oligosaccharide donor for glycoprotein synthesis was obtained by incubation of partially purified oligosaccharide-lipid with the membranes. The products of this incubation were shown to be glycoproteins on the basis of their sensitivity to pronase, as determined by both gel filtration and paper electrophoresis. Similar experiments, using oligosaccharide-lipid doubly labeled with [14C]mannose and N-acetyl[3H]glucosamine, provided evidence that the oligosaccharide chain of the oligosaccharide-lipid is transferred en bloc to glycoprotein s.     

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.     

Purification and characterization of dolichyl-P-mannose:Man5(GlcNAc)2-PP-dolichol mannosyltransferase

The dolichyl-P-mannose:dolichyl-PP-heptasaccharide alpha-mannosyltransferase (2.4.1.130), which catalyzes the transfer of mannose from dolichyl-P-mannose to the Man5(GlcNAc)2-PP-dolichol acceptor glycolipid, was solubilized from pig aorta microsomes with 0.5% NP-40 and purified 985-fold by a variety of conventional methods. The partially purified enzyme had a pH optimum of 6.5 and required Ca2+, at an optimum concentration of 8-10 mM, for activity. Mn2+ was only 20% as effective as Ca2+, and Mg2+ was inhibitory. The mannosyltransferase activity was also inhibited by the addition of EDTA to the enzyme, but this inhibition was fully reversible by the addition of Ca2+. The enzyme was quite specific for dolichyl-P-mannose as the mannosyl donor and Man5(GlcNAc)2-PP-dolichol as the mannosyl acceptor. The Km values for dolichyl-P-mannose and the acceptor lipid Man5(GlcNAc)2-PP-dolichol were 1.8 and 1.6 microM. On Bio-Gel P-4 columns and by HPLC, the radiolabeled oligosaccharide formed during incubation of dolichyl-P-[14C]mannose and unlabeled Man5(GlcNAc)2-PP-dolichol with the purified enzyme behaved like Man6(GlcNAc)2. This octasaccharide was susceptible to digestion by endoglucosaminidase H, indicating that the newly added mannose was attached to the 6-linked mannose in an alpha 1,3-linkage. This linkage was further confirmed by acetolysis of the oligosaccharide product [i.e., Man6(GlcNAc)2], which gave a labeled disaccharide as the major product (greater than 90%).     

Purification and properties of UDP-GlcNAc:dolichyl-phosphate GlcNAc-1-phosphate transferase. Activation and inhibition of the enzyme

The GlcNAc-1-P transferase was solubilized from pig aorta microsomal fractions using 0.5% Nonidet P-40. The activity of the solubilized enzyme was stimulated by exogeneously added phospholipids in the order phosphatidylglycerol greater than phosphatidylinositol greater than phosphatidylserine. When the enzyme was stored in 20% glycerol containing 20 micrograms of phosphatidylglycerol/mg of protein, more than 80% of the activity remained after storage for 6 days at 0-4 degrees C. On the other hand, in the absence of the stabilizers, the enzyme lost most of its activity within 24 h. The transferase was purified about 68-fold using ammonium sulfate and DEAE-cellulose fractionation. The DEAE-cellulose chromatography separated a heat-stable factor from the enzyme, which when added back to the partially purified enzyme stimulated about 5-fold. With this partially purified enzyme, the Km for UDP-GlcNAc was found to be 1 X 10(-7) M, and that for dolichyl-P about 1 X 10(-6) M. The stimulatory factor increased the Vmax for both UDP-GlcNAc and dolichyl-P 5-10-fold, but the Km values remained the same. The pH optimum for the enzyme was between 7.4 and 7.6, and either Mn2+ (1 mM) or Mg2+ (10 mM) was required for optimum activity. The GlcNAc-1-P transferase was also stimulated by the addition of GDP-mannose (or other purine sugar nucleotides) or dolichyl-phosphoryl-mannose to the incubation mixtures. These two compounds acted in different ways on the enzyme since their stimulatory effects were additive. The effect of GDP-mannose was found to be due to protection of the substrate, UDP-GlcNAc, from degradation, but the effect of dolichyl-P-mannose remains to be established. In addition, the stimulations shown by phosphatidylglycerol, GDP-mannose, and factor, or phosphatidylglycerol, dolichyl-P-mannose, and factor, were all additive, indicating that they were acting at different sites on the enzyme. The transferase was quite sensitive to the action of sulfhydryl reagents such as N-ethylmaleimide or p-chloromercuribenzene sulfonate, and was rapidly inactivated in their presence. The enzyme could be protected to the extent of about 50% when all of the substrates (UDP-GlcNAc, dolichyl-P, Mn2+) were added before the addition of the sulfhydryl reagents.     

Biosynthesis of carbohydrate-containing polymers in plants. I. Products formed with enzyme preparation from clover seedlings. Characterization of polyprenylphosphosugars

Membrane preparations from clover seedlings catalyzed the incorporation of monosaccharide residues from GDPMan, UDPGlc and UDPGal into glycolipids, lipid-oligosaccharides and polymers. The lipid-oligosaccharides were shown to be alpha-dihydropolyprenyl pyrophosphate derivatives. Incorporation of mannose residues into the lipid-oligosaccharides and the polymers was significantly stimulated by addition of UDPGlc, GDPGlc, UDPGal but not by UDPGlcNAc. Exogenic polyprenyl phosphates also stimulated the process; the formation of moraprenyl pyrophosphate oligosaccharides was demonstrated after addition of moraprenyl phosphate. The lipid-oligosaccharides were precursors of the polymers which were shown to be mainly glycoproteins. A solubilized preparation of mannosyl transferase from clover membranes was obtained and some properties of the enzyme were studied.     

Biosynthesis of mannose-containing lipid-linked oligosaccharides by solubilized enzyme preparation from cultured soybean cells

Glycosyl transferases that participate in the assembly of the lipid-linked oligosaccharide intermediates were solubilized from cultured soybean cells using 0.3% Nonidet P-40 (NP-40) in the presence of 10% glycerol. The solubilized enzyme preparation was reasonably stable and 50% of the activity still remained after storage at −10°C for 1 month. The solubilized enzyme synthesized [¹⁴C]Man₃GlcNAc₂-pyrophosphoryl-polyprenol and [¹⁴C]Man₅GlcNAc₂-pyrophosphoryl-polyprenol when incubated with GDP-[¹⁴C]mannose plus a partially purified acceptor lipid isolated from calf liver. The formation of these lipid-linked oligosaccharides did not require the addition of dolichyl-phosphate or metal ions. In fact, the addition of 5 to 10 millimolar ethylenediaminetetraacetate stimulated the incorporation of mannose into lipid-linked oligosaccharides 2- to 3-fold. Since little or no dolichyl-phosphoryl-mannose is formed in the presence of ethylenediaminetetraacetate, the results suggest that the mannosyl residues added to form Man₃GlcNAc₂-lipid and Man₅GlcNAc₂-lipid come directly from GDP-mannose without the participation of dolichyl-phosphoryl-mannose. On the other hand, the formation of significant amounts of Man₆GlcNAc₂-lipid, Man₇GlcNAc₂-lipid, and Man₈GlcNAc₂-lipid occurred when the above incubations were supplemented with dolichyl-phosphate and metal ions. Based on various time course studies and supplementation studies with various additions, it appears likely that the first five mannose residues to form Man₅GlcNAc₂-lipid come directly from GDP-mannose, whereas other mannose units to form larger oligosaccharide-lipids come from dolichyl-phosphoryl-mannose.     

Biosynthesis of man-β-G1cNAc-G1cNAc-pyrophosphoryl-polyprenol by a solubilized enzyme from aorta

The particulate enzyme fraction from pig aorta was treated with Triton X-100 or Nonidet P-40 to yield a soluble enzyme preparation. This solubilized enzyme catalyzed the transfer of mannose from GDP-[¹⁴C]mannose, but not from [¹⁴C]mannosyl-phosphoryl-polyprenol, to G1cNAc-G1cNAc-pyrophosphoryl-polyprenol to form the trisaccharide-lipid, Man-β-GlcNAc-GlcNAc-pyrophosphoryl-polyprenol. The trisaccharide-lipid formed in these reactions was isolated by solvent fractionation and was subjected to mild acid hydrolysis to release the [¹⁴C]trisaccharide. Essentially all of the radioactivity was released from this trisaccharide as mannose upon treatment with β-mannosidase while α-mannosidase had no effect.     

Rat liver Golgi galactosyltransferases. Distinct enzymes for glycolipid and glycoprotein acceptor substrates.

Two enzymes that catalyse the transfer of galactose from UDP-galactose to GM2 ganglioside were partially purified from rat liver Golgi membranes. These preparations, designated enzyme I (basic) and enzyme II (acidic), utilized as acceptors GM2 ganglioside and asialo GM2 ganglioside as well as ovalbumin, desialodegalactofetuin, desialodegalacto-orosomucoid, desialo bovine submaxillary mucin and GM2 oligosaccharide. Enzyme II catalysed disaccharide synthesis in the presence of the monosaccharide acceptors N-acetylglucosamine and N-acetylgalactosamine. The affinity adsorbent alpha-lactalbumin-agarose, which did not retard GM2 ganglioside galactosyltransferase, was used to remove most or all of galactosyltransferase activity towards glycoprotein and monosaccharide acceptors from the extracted Golgi preparation. After treatment of the extracted Golgi preparation with alpha-lactalbumin-agarose, enzyme I and enzyme II GM2 ganglioside galactosyltransferase activities, prepared by using DEAE-Sepharose chromatography, were distinguishable from transferase activity towards GM2 oligosaccharide and glycoproteins by the criterion of thermolability. This residual galactosyltransferase activity towards glycoprotein substrates was also shown to be distinct from GM2 ganglioside galactosyltransferase in both enzyme preparations I and II by the absence of competition between the two acceptor substrates. The two types of transferase activities could be further distinguished by their response to the presence of the protein effector alpha-lactalbumin. GM2 ganglioside galactosyltransferase was stimulated in the presence of alpha-lactalbumin, whereas the transferase activity towards desialodegalactofetuin was inhibited in the presence of this protein. The results of purification studies, comparison of thermolability properties and competition analysis suggested the presence of a minimum of five galactosyltransferase species in the Golgi extract. Five peaks of galactosyltransferase activity were resolved by isoelectric focusing. Two of these peaks (pI 8.6 and 6.3) catalysed transfer of galactose to GM2 ganglioside, and three peaks (pI 8.1, 6.8 and 6.3) catalysed transfer to glycoprotein acceptors.     

A possible role of Golgi membrane-associated galactosyltransferase in the formation of zymogen granule glycoproteins

A galactosyltransferase activity in smooth microsomes and Golgi membrane-rich fractions from rat pancreas glycosylated endogenous acceptors during incubation with UDP-[¹⁴C]galactose in the absence of exogenous glycoproteins. To evaluate the role of this activity in secretion, the endogenous products were partially characterized. Galactose-labeled fractions were sequentially extracted in 0.2 m NaHCO₃ and 0.25 m NaBr to prepare membranes and soluble acceptors. Bound radioactivity was equally distributed between these two fractions. Analysis by polyacrylamide gel electrophoresis in sodium dodecyl sulfate indicated that the particulate galactose-labeled polypeptides were distinct from the soluble galactose acceptors. Rabbit antisera against highly purified zymogen granule membranes precipitated approximately 40% of the radioactivity of the particulate fraction when solubilized in nonionic detergents. In polyacrylamide gels, the galactose-labeled species of the immunoprecipitate migrated with zymogen granule membrane glycoproteins. Rabbit antisera against secretory proteins cross-reacted with less than 5% of the galactose-labeled soluble acceptors. Mature zymogen granule membranes neither contained detectable galactosyltransferase activity nor served as galactosyltransferase acceptors. These results suggest that galactosyltransferase activity associated with membranes derived from the Golgi complex glycosylated zymogen granule membrane precursors. Analysis of [¹⁴C]galactolipids did not implicate lipid intermediates in this process.     

Enzymatic transfer of mannose from guanosine diphosphate mannose to yeast mannan-protein complexes

The radioactive products derived from transfer of [¹⁴C]mannose residues from GDP-[¹⁴C]mannose to endogenous acceptors of a Hansenula holstii particulate enzyme preparation have been solubilized by Pronase digestion. From this soluble mixture, glycopeptides containing [¹⁴C]mannose have been purified and have been shown by β-elimination-reduction experiments to contain radioactive mannose and oligosaccharides of mannose linked to serine and threonine residues. Radioactive macromolecular complexes of mannan-protein were extracted from the particulate enzyme fraction with hot, neutral citrate buffer. These components contained variable quantities of protein, mannose, and phosphate. The more neutral components were reduced in size by Pronase digestion and yielded glycopeptides similar to those obtained by direct Pronase digestion of the particulate fraction.