Enzymatic transfer of mannose from guanosine diphosphate mannose to dolichol phosphate in yeast (Hansenula holstii) A possible step in mannan synthesis

A particulate enzyme fraction isolated from yeast (Hansenula holstii) catalyzes the transfer of mannose from GDPmannose to endogenous lipid acceptors. Kinetic studies are presented which suggest that one of the mannolipids is a precursor to cell wall mannan. The solubility and chromatographic properties, the stability to mild alkali, and the release of mannose by mild acid hydrolysis are characteristic of polyisoprenyl phosphoryl mannose. Addition of dolichol phosphate to the enzyme system stimulates the synthesis of a mannolipid with properties similar to that synthesized from endogenous lipid. That the exogenous dolichol phosphate was acting as a mannosyl acceptor was demonstrated by showing that dolichol [³²P]phosphate was converted to dolichol [³²P]phosphate mannose.     

Spatial aspects of mannosyl phosphoryl retinol formation

Rat liver microsomes catalyze the transfer of mannose from GDPmannose to both retinyl phosphate and dolichyl phosphate to form mannosylphosphorylretinol, mannosylphosphoryldolichol and GDP. The two reactions differ in term of reversibility. In fact, a 200-fold isotopic dilution of GDP[14C]mannose by unlabeled GDPmannose causes mannosylphosphoryldolichol labeling to disappear almost completely, while mannosylphosphorylretinol labeling remains at the same level. The same observation can be made if the mannose donor is removed by centrifugation and replaced by excess GDP; again mannosylphosphorylretinol is stable, but mannosylphosphoryldolichol drops down to one-third of its initial level, as expected for, respectively, a non-reversible and a reversible reaction. Placed in an aqueous medium, mannosylphosphorylretinol releases mannose 1-phosphate (beta configuration) whereas it is quite stable when kept in a membranous environment. These results strongly suggest that mannosylphosphorylretinol as soon as it is formed is segregated in such a way that it is no longer available to the back-reaction; the functional consequence of this segregation would be the possibility for mannosylphosphorylretinol to mannosylate some non-polar regions of certain protein chains.     

Formation of alpha-1,2- and alpha-1,3-linked mannose disaccharides from dolichyl mannosyl phosphate by rat liver membrane enzymes

Dolichyl mannosyl phosphate and GDPmannose were active substrates for the transfer of mannose to methyl-alpha-D-mannose, p-nitrophenyl-alpha-D-mannose, and free mannose with rat liver microsomal membranes. The products formed during dolichyl mannosyl phosphate incubation with methyl-alpha-D-mannose or with mannose were alpha-linked. The disaccharides formed by incubation of dolichyl mannosyl phosphate or GDPmannose with mannose were identified by paper chromatography and electrophoresis as mannose-alpha-1,2-mannose and mannose-alpha-1,3-mannose. synthesis of each product was dependent on the assay conditions used and was most markedly affected by the presence of detergent. Transfer of mannose from either substrate to form mannose-alpha-1,3-mannose was severely inhibited by Triton X-100.     

Formation of α-1,2- and α-1,3-linked mannose disaccharides from dolichyl mannosyl phosphate by rat liver membrane enzymes

Dolichyl mannosyl phosphate and GDPmannose were active substrates for the transfer of mannose to methyl-α-d-mannose, p-nitrophenyl-α-d-mannose, and free mannose with rat liver microsomal membranes. The products formed during dolichyl mannosyl phosphate incubation with methyl-α-d-mannose or with mannose were α-linked. The dissaccharides formed by incubation of dolichyl mannosyl phosphate or GDPmannose with mannose were identified by paper chromatography and electrophoresis as mannose-α-1,2-mannose and mannose-α-1,3-mannose. Synthesis of each product was dependent on the assay conditions used and was most markedly affected by the presence of detergent. Transfer of mannose from either substrate to form mannose-α-1,3-mannose was severely inhibited by Triton X-100.     

Mannosylation of glycoproteins and dolichol derivatives in fibroblasts from patients with cystic fibrosis

Increased incorporation of mannose into endogenous glycoprotein fractions has been found in whole cell lysates and crude membrane preparations of cultured skin fibroblasts from patients with cystic fibrosis (1.3–2.3-times normal) when GDP[¹⁴C]mannose served as the mannosyl donor. In contrast, the incorporation of mannose from GDPmannose into lipid fractions containing dolichol phosphate and dolichol pyrophosphate oligosaccharides as well as the incorporation of mannose from dolichol phospho[³H]mannose into both glycoproteins and dolichol derivatives were not significantly different among cell preparations from patients with cystic fibrosis and normal controls. Mannosyltransferase activity toward exogenous glycoproteins as well as the activities of soluble and membranous α-mannosidase and β-mannosidase appeared to be normal and could not account for the observed differences. The altered incorporation of mannose into endogenous glycoprotein may reflect changes in glycosylation processes other than mannosylation.     

Influence of metal ions on the biosynthesis of N-acetylglucosaminyl polyprenols by the retina

The following enzymatic process was investigated, catalyzed by an enzyme preparation from the retina of the embryonic chick: UDP-GlcNAc + dolichol phosphate GDPmannose leads to metal ions GlcNAc-P-P-polyprenol + (GlcNAc)2-P-P-polyprenol + Man-(GlcNAc)2-P-P-polyprenol. These reactions were carried out in the presence of a dolichol phosphate mannose-synthesizing system, shown previously to be an activator of GlcNAc-lipid synthesis. The process was also strongly influenced by the choice of the divalent cation used during the reactions. In the presence of Mg2+, not only was the extent of incorporation of radioactivity from UDP-[3H]GlcNAc increased 4-fold into the GlcNAc lipids, as compared to Mn2+, but the relative distribution of the products was affected as well. In the presence of Mg2+ the reaction was driven mainly in the direction of the formation of the first intermediate of the dolichol pathway, GlcNAc-P-P-polyprenol. Many of the other characteristics of the GlcNAc-transferases, such as pH optimum, requirement for dolichol phosphate and specificity for stimulation by sugar nucleotides, were similar for either the Mn2+ or Mg2+ systems. Retinol phosphate could not replace the requirement for dolichol phosphate. The influence of metal ions, in addition to the stimulation by dolichol phosphate mannose, on GlcNAc-lipid synthesis may be aspects of metabolic regulation of the dolichol pathway.     

Formation and properties of retinylphosphate galactose.

Crude cell membrane fractions from a number of tissues can form acidic glycolipids. The formation of acidic galactose lipid and mannose lipid was greatly reduced in vitamin A deficiency, primarily in tissues known to be mucus-producing. Mouse mastocytoma tissue was active in forming acidic galactose lipids with UDP-galactose as substrate. One of the products was identified as retinylphosphate galactose. The synthetase reaction producing this compound exhibited an apparent pH optimum at 6.3. The presence of detergent and retinol stimulated the synthetase reaction, which exhibited an absolute requirement for Mn2+ or Mg2+. The synthetase reaction was readily reversible. Incubation of particulate enzyme with retinylphosphate galactose and UDP yielded UDP-galactose and a compound tentatively identified as retinylphosphate. The galactose lipid was isolated by column chromatography on DEAE-cellulose and silica gel. The retinylphosphate galactose was homogeneous when examined by thin layer chromatography. Mild acid hydrolysis of labeled retinylphosphate galactose yields [14C]galactose, whereas alkaline hydrolysis and hydrogenolysis produced [14C]galactose 1-phosphate. Retinylphosphate galactose bound to vitamin A-depleted, retinol-binding protein.     

Glycoprotein biosynthesis: studies on thyroid mannosyltransferases. II. Characterization of a polyisoprenyl mannosyl phosphate and evaluation of its intermediary role in the glycosylation of exogenous acceptors

The transfer of mannose from GDP-mannonse to exogenous glycopeptides and simple glycosides has been shown to be carried out by calf thyroid particles (Adamany, A. M., and Spiro, R. G. (1975) J. Biol. Chem. 250, 2830-2841). The present investigation indicates that this mannosylation process is accomplished through two sequential enzymatic reactions. The first involves the transfer of mannose from the sugar nucleotide to an endogenous acceptor to form a compound which has the properties of dolichyl mannosyl phosphate, while in the properties of dolichyl mannosyl phosphate, while in the second reaction this mannolipid serves as the glycosyl donor to exogenous acceptors. The particle-bound enzyme which catalyzed the first reaction utilized GDP-mannose (Km = 0.29 microM) as the most effective mannosyl donor, required a divalent cation, preferably manganese or calcium, and acted optimally at pH 6.3. Mannolipid synthesis was reversed by addition of GDP and a ready exchange of the mannose moiety was observed between [14C]mannolipid and unlabeled GDP-mannose. Exogenously supplied dolichyl phosphate, and to a lesser extent ficaprenyl phosphate, served as acceptors for the transfer reaction. The 14C-labeled endogenous lipid had the same chromatographic behavior as synthetic dolichyl mannosyl phosphate and enzymatically mannosylated dolichyl phosphate. The mannose component in the endogenous lipid was not susceptible to reduction with sodium borohydride and was released by mild acid hydrolysis. Alkaline treatment of the mannolipid released a phosphorylated mannose with properties consistent with that of mannose 2-phosphate. The formation of this compound which can arise from a cyclic 1,2-phosphate indicated, on the basis of steric considerations, that the mannose is present in beta linkage to the phosphate of the lipid. An intermediate role of the mannolipid in the glycosylation of exogenous acceptors was suggested by the observation that addition of dolichyl phosphate to thyroid particles resulted in a marked enhancement of mannose transfer from GDP-mannose to methyl-alpha-D-mannopyranoside acceptor while the presence of the glycoside caused a decrease in the mannolipid level. The glycosyl donor function of the polyisoprenyl mannosyl phosphate in the second reaction of the mannosylation sequence could be directly demonstrated by the transfer of [14C]mannose from purified endogenous mannolipid to either methyl-alpha-D-mannoside or dinitrophenyl unit A glycopeptides by thyroid enzyme in the presence of Triton X-100. The mannosylation of the glycoside was not inhibited by EDTA whereas the transfer of mannose to glycopeptide was cation-dependent. While dolichyl [14C]mannosyl phosphate, prepared from exogenous dolichyl phosphate, served as a donor of mannose to exogenous acceptor, this function could not be fulfilled by ficaprenyl [14C]mannosyl phosphate. The two-step reaction sequence carried out by thyroid enzymes which leads to the formation of an alpha-D-manno-pyranosyl-D-mannose linkage in exogenous acceptors by transfer of mannose from GDP-mannose through a beta-linked intermediate appears to involve a double inversion of anomeric configuration of this sugar.     

Biosynthesis of yeast mannan. Diversity of mannosyltransferases in the mannan-synthesizing enzyme system from yeast.

1. A microsomal enzyme preparation from the yeast Saccharomyces cerevisiae catalyzes the transfer of mannosyl units from GDPmannose to mannose and a number of mannose-containing oligosaccharides and glycosides whereby different glycosidic bonds are formed. 2. Of the compounds tested besides mannose, only those containing an alpha-linked mannosyl unit at the nonreducing position of their molecule were effective as acceptors. Monodeoxyanalogues of mannose as well as alpha-mannose phosphates did not serve as acceptors in the above reaction. 3. The structure of the product formed with mannose as acceptor was determined to be O-alpha-D-mannosyl-(1 leads to 2)-mannose; with alphaMan (1 leads to 6)mannose as the acceptor, the product was alphaMan(1 leads to 6)mannose and with alphaMan-(1 leads to 2)mannose the product was tentatively characterized as a mixture of alphaMan-(1 leads to 3)alphaMan(1 leads to 2)mannose and alphaMan(1 leads to 2)alphaMan(1 leads to 2)mannose. 4. The enzymes catalyzing the formation of different types of glycosidic bonds differed in their acceptor specificity, pH-activity curves and rates of heat denaturation. 5. Radioactive disaccharides were unable to enter the mannan protein molecule in the cell-free system while free radioactive mannose did incorporate into polysaccharide to a minor extent under the same conditions.     

Evidence for multiple enzymes in the dolichol utilizing pathway of glycoprotein biosynthesis.

A comparison has been made of the enzymes catalyzing the transfer of mannose, glucose and N-acetylglucosamine from, respectively, GDPmannose, UDP-glucose and UDP-N-acetylglucosamine to endogenous dolichol phosphate (Dol-P) in liver Golgi membranes. Evidence is presented with suggests that all three reactions utilize the same pool of Dol-P. The transfer of mannose from GDP-Man to Dol-P is not inhibited by 0.1 mM UDP or UMP; 0.1 mM GDP did block the accumulation of mannose in Dol-P-Man. The net transfer of glucose and N-acetylglucosamine to Dol-P is prevented by 0.1 mM UDP but not 0.1 mM GDP. UDPglucose inhibits the reverse of the glucose transfer reaction but not the reverse of the N-acetylglucosamine or mannose trasfer reaction. On the basis of this, and other data, it is concluded that the three sugar transfer reactions utilize separate enzymes.     

Glycoprotein mannosylation in rat liver nuclei

Nuclei and non-nuclear membranes were tested for their ability to transfer in vitro (14C)mannose from GDP-(14C)mannose to endogenous glycoprotein acceptors in the presence and in the absence of exogenous retinyl-phosphate. Electrophoretic analysis shows that retinylphosphate is responsible for the labeling of a few endogenous acceptors only in the non-nuclear membranes; in the nuclei the mannosylation reaction is not retinylphosphate dependent and the electrophoretic profile of the labeled protein acceptors is different from that of the non-nuclear membranes.     

Incorporation of mannose and glucose into prenylphosphate sugars in isolated human platelet membranes.

Isolated platelet membranes synthesize mannosylretinyl phosphate and dolichylmannosyl phosphate from GDP-[14C]mannose, but only dolichylglucosyl-phosphate is synthesized from UDP-[14C]glucose. Addition of exogenous retinylphosphate specifically stimulates the biosynthesis of mannosylretinylphosphate.     

Long - lasting synchrony of the division of enteric bacteria

Recent finding of α-N-acetylglucosamine(1)phospho(6)mannose diesters in lysosomal enzymes suggested that formation of mannose 6-phosphate residues involves transfer of N-acetylglucosamine 1-phosphate to mannose. Using dephosphorylated β-hexosaminidase as acceptor and [β-³²P]UDP-N-acetylglucosamine as donor for the phosphate group, phosphorylation of β-hexosaminidase by microsomes from rat liver, human placenta and human skin fibroblasts was achieved. The reaction was not affected by tunicamycin. Acid hydrolysis released mannose 6-[³²P]phosphate from the phosphorylated β-hexosaminidase. Our results suggest that lysosomal enzymes are phosphorylated by transfer of N-acetylglucosamine 1-phosphate from UDP-N-acetylglucosamine. The transferase activity was deficient in fibroblasts from patients affected with l-cell disease. This deficiency is proposed to be the primary enzyme defect in l-cell disease.     

Incorporation of mannose and glucose into prenylphosphate sugars in isolated human platelet membranes

Isolated platelet membranes synthesize mannosylretinyl phosphate and dolichylmannosyl phosphate from GDP-[¹⁴C]mannose, but only dolichylglucosyl-phosphate is synthesized from UDP-[¹⁴C]glucose.Addition of exogenous retinylphosphate specifically stimulates the biosynthesis of mannosylretinylphosphate.     

Evidence for multiple enzymes in the dolichol utilizing pathway of glycoprotein biosynthesis

A comparison has been made of the enzymes catalyzing the transfer of manose, glucose and N-acetylglucosamine from, respectively, GDPmannose, UDP-glucose and UDP-N-acetylglucosamine to endogenous dolichol phosphate (Dol-P) in liver Golgi membranes. Evidence is presented which suggests that all three reactions utilize the same pool of Dol-P. The transfer of mannose from GDP-Man to Dol-P is not inhibited by 0.1 mM UDP or UMP; 0.1 mM GDP did block the accumulation of mannose in Dol-P-Man. The net transfer of glucose and N-acetylglucosamine to Dol-P is prevented by 0.1 mM UDP but not 0.1 mM GDP. UDPglucose inhibits the reverse of the glucose transfer reaction but not reverse of the N-acetylglucosamine or mannose transfer reaction. On the basis of this, and other data, it is concluded that the three sugar transfer reactions utilize separate enzymes.     

GDPmannose dolicholphosphate mannosyltransferase of chicken liver mitochondria

Chicken liver mitochondria contain enzymes for the dolichol cycle. GDPmannose dolicholphosphate mannosyltransferase has been solubilized with Emulgen 909 and purified. The purified enzyme was not homogeneous, but highly specific for GDPmannose and dolichyl phosphate. The enzyme activity was stimulated by MgCl2 (3 mM optimum) and exhibited a pH optimum at around 7.2. Bisubstrate kinetic analysis indicated that the enzyme follows a sequential mechanism. The Km values for GDPmannose and dolichyl phosphate were 0.43 and 14.3 microM, respectively. The purified enzyme was labile and lost its activity on storage at 0 degree C overnight or incubation at 30 degrees C or higher temperature. Inactivation could be prevented by the addition of heat-denatured mitochondrial extract. Further investigation revealed that phospholipids and dolichyl phosphate are responsible for the stabilization. Single addition of either phospholipid or dolichyl phosphate showed little activity, but the combination of these lipids enhanced the stabilizing activity greatly. Eight naturally occurring phospholipids were tested and found to be effective in combination with dolichyl phosphate. Among these, sphingomyelin was the most effective. Dolichol could partially substitute dolichyl phosphate but worked at higher concentrations.     

Dolichylphosphate-dependent Glycosyl Transfer Reactions in the Endoplasmic Reticulum of Castor Bean Endosperm

In the endosperm of Ricinus communis (castor bean) a number of glycosyl transferases were found to be present during germination. They catalyze the incorporation of mannose from guanosine diphosphate mannose and of N-acetylglucosamine from uridine diphosphate N-acetylglucosamine into a glycolipid fraction, which had all of the properties of dolichylphosphate and pyrophosphate sugars, respectively. The sugar moiety of dolichylphosphate mannose is transferred to a lipid-oligosaccharide, containing more than 6 hexose units. When the membranes are preincubated with nonradioactive guanosine diphosphate mannose and uridine diphosphate N-acetylglucosamine, radioactivity from dolichylphosphate [¹⁴C]mannose is also transferred to a glycopolymer. In addition, the formation of radioactive glycoproteins from guanosine diphosphate [¹⁴C]mannose has been demonstrated using a combination of sodium dodecyl sulfate-polyacrylamide gel electrophoresis and autofluorography.     

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%).     

Rat liver microsomes catalyse mannosyl transfer from GDP-d-mannose to retinyl phosphate with high efficiency in the absence of detergents

In the absence of detergent, the transfer of mannose from GDP-mannose to rat liver microsomal vesicles was highly stimulated by exogenous retinyl phosphate in incubations containing bovine serum albumin, as measured in a filter binding assay. Under these conditions 65% of mannose 6-phosphatase activity was latent. The transfer process was linear with time up to 5min and with protein concentration up to 1.5mg/0.2ml. It was also temperature-dependent. The microsomal uptake of mannose was highly dependent on retinyl phosphate and was saturable against increasing amounts of retinyl phosphate, a concentration of 15mum giving half-maximal transfer. The uptake system was also saturated by increasing concentrations of GDP-mannose, with an apparent K(m) of 18mum. Neither exogenous dolichyl phosphate nor non-phosphorylated retinoids were active in this process in the absence of detergent. Phosphatidylethanolamine and synthetic dipalmitoylglycerophosphocholine were also without activity. Several water-soluble organic phosphates (1.5mm), such as phenyl phosphate, 4-nitrophenyl phosphate, phosphoserine and phosphocholine, did not inhibit the retinyl phosphate-stimulated mannosyl transfer to microsomes. This mannosyl-transfer activity was highest in microsomes and marginal in mitochondria, plasma and nuclear membranes. It was specific for mannose residues from GDP-mannose and did not occur with UDP-[(3)H]galactose, UDP- or GDP-[(14)C]glucose, UDP-N-acetyl[(14)C]-glucosamine and UDP-N-acetyl[(14)C]galactosamine, all at 24mum. The mannosyl transfer was inhibited 85% by 3mm-EDTA and 93% by 0.8mm-amphomycin. At 2min, 90% of the radioactivity retained on the filter could be extracted with chloroform/methanol (2:1, v/v) and mainly co-migrated with retinyl phosphate mannose by t.l.c. This mannolipid was shown to bind to immunoglobulin G fraction of anti-(vitamin A) serum and was displaced by a large excess of retinoic acid, thus confirming the presence of the beta-ionone ring in the mannolipid. The amount of retinyl phosphate mannose formed in the bovine serum albumin/retinyl phosphate incubation is about 100-fold greater than in incubations containing 0.5% Triton X-100. In contrast with the lack of activity as a mannosyl acceptor for exogenous dolichyl phosphate in the present assay system, endogenous dolichyl phosphate clearly functions as an acceptor. Moreover in the same incubations a mannolipid with chromatographic properties of retinyl phosphate mannose was also synthesized from endogenous lipid acceptor. The biosynthesis of this mannolipid (retinyl phosphate mannose) was optimal at MnCl(2) concentrations between 5 and 10mm and could not be detected below 0.6mm-MnCl(2), when synthesis of dolichyl phosphate mannose from endogenous dolichyl phosphate was about 80% of optimal synthesis. Under optimal conditions (5mm-MnCl(2)) endogenous retinyl phosphate mannose represented about 20% of dolichyl phosphate mannose at 15min of incubation at 37 degrees C.     

Inhibition of lipid-linked saccharide synthesis by bacitracin.

The antibiotic bacitracin was found to inhibit the incorporation of mannose and GlcNAc from their respective sugar nucleotides into lipid-linked saccharides. The inhibition of both systems was apparent in the aorta particulate enzyme system but it was much more pronounced with the solubilized enzyme system. In both cases, GlcNAc incorporation into Dol-P-P-GlcNAc was more sensitive than mannose incorporation into Dol-P-Man, with 50% inhibition being seen at about 0.1–0.2 mm antibiotic. Bacitracin inhibition of mannose incorporation appeared to be overcome at high concentrations of dolichyl phosphate but, in these cases, an unexplained stimulation was observed. However, GlcNAc inhibition could not be overcome by high concentrations of dolichol phosphate, metal ion, or both together. Thus, the mechanism of inhibition by bacitracin is not clear. Bacitracin also inhibited the transfer of mannose from GDP-mannose to lipid-linked oligosaccharides and to glycoprotein in the particulate enzyme, as well as the transfer of radioactivity from Dol-P-Man or from lipid-linked oligosaccharides to glycoprotein. Thus, bacitracin apparently blocks each of the steps in the lipid-linked pathway. In yeast spheroplasts, bacitracin inhibited the incorporation of [¹⁴C]mannose into Dol-P-Man, into lipid-linked oligosaccharides, and into glycoprotein. However, in this case, the antibiotic also blocked the incorporation of leucine into protein. Bacitracin also inhibited the cell-free synthesis of mannosyl-phosphoryl-decaprenol in Mycobacterium smegmatis with 50% inhibition being observed at a concentration of about 0.5 mm.