Dimannosyldiacylglycerol serves as a lipid anchor precursor in the assembly of the membrane-associated lipomannan in Micrococcus luteus

Based on recent analytical and enzymological studies, a topological model for the role of alpha-D-mannosyl-(1-->3)-alpha-D-mannosyl-(1-->3)-diacylglycerol (Man(2)-DAG) as a lipid anchor precursor and mannosylphosphorylundecaprenol (Man-P-Und) as a mannosyl donor in the assembly of a membrane-associated lipomannan (LM) in Micrococcus luteus has been proposed. In this study, a [(3)H]mannose-suicide selection procedure has been used to identify temperature-sensitive (ts) mutants defective in LM assembly. Two micrococcal mutants with abnormal levels of Man(2)-DAG and LM at the nonpermissive temperature (37 degrees C), mms1 and mms2, have been isolated and characterized. In vivo and in vitro biochemical assays indicate that mms1 cells have a defect in the mannosyltransferase catalyzing the conversion of Man-DAG to Man(2)-DAG, and mms2 has a temperature-sensitive defect in the synthesis of Man-P-Und. Because mms1 cells are depleted of endogenous Man(2)-DAG, membranes from this mutant efficiently converted purified, exogenous [(3)H]Man(2)-DAG to [(3)H]LM by a Man-P-Und-dependent process. An obligatory role for Man-P-Und as a mannosyl donor in the elongation process was also demonstrated by showing that the conversion of exogenous [(3)H]Man(2)-DAG to [(3)H]LM by membranes from mms1 cells in the presence of GDP-Man was inhibited by amphomycin. In addition, consistent with Man(2)-DAG serving as a lipid anchor precursor for LM assembly, endogenous, prelabeled [(3)H]Man(2)-DAG was converted to [(3)H]LM when membranes from mms2 cells were incubated with purified, exogenous Man-P-Und. These studies provide the first direct proof for the role of Man(2)-DAG as the lipid anchor precursor for LM, and suggest that Man(2)-DAG may be essential for the normal growth of M. luteus cells.     

Involvement of Escherichia coli K-12 recombination functions in elimination of multicopy plasmid R6K by DNA-damaging agents

Abstract A neutral lipomannan has been isolated from the membranes of Micrococcus agilis . In contrast to the lipomannans from 2 strains of Micrococcus luteus , which contained succinic acid ranging from 5.1%–8.0%, the M. agilis lipomannan had no detectable succinyl residues and exhibited neutral behaviour on Concanavalin A-agarose rocket electrophoresis. As with the M. luteus lipomannans, mannose was the only sugar detectable (as alditol acetate) by GLC analysis in the purified M. agilis lipomannan. Fatty acids accounted for 2% of the M. agilis lipomannan and were predominantly C₁₅ branched-chain acids, with higher amounts of C₁₆ iso and C₁₇ anteiso than that found in the M. luteus polymers. Neither conditions of growth of the organism nor the method of membrane preparation appeared to be responsible for the absence of succinyl residues. This appears to be the first report of a neutral membrane amphiphile.     

Mannose supplementation corrects GDP-mannose deficiency in cultured fibroblasts from some patients with Congenital Disorders of Glycosylation (CDG)

Congenital Disorders of Glycosylation (CDG) are human deficiencies in glycoprotein biosynthesis. Previous studies showed that 1 mM mannose corrects defective protein N-glycosylation in cultured fibroblasts from some CDG patients. We hypothesized that these CDG cells have limited GDP-mannose (GDP-Man) and that exogenous mannose increases the GDP-Man levels. Using a well established method to measure GDP-Man, we found that normal fibroblasts had an average of 23.5 pmol GDP-Man/10(6) cells, whereas phosphomannomutase (PMM)-deficient fibroblasts had only 2.3-2.7 pmol/10(6) cells. Adding 1 mM mannose to the culture medium increased the GDP-Man level in PMM-deficient cells to approximately 15.5 pmol/10(6) cells, but had no significant effect on GDP-Man levels in normal fibroblasts. Similarly, mannose supplementation increased GDP-Man from 4.6 pmol/10(6) cells to 24.6 pmol/10(6) cells in phosphomannose isomerase (PMI)-deficient fibroblasts. Based on the specific activity of the GDP-[(3)H]Man pool present in [2-(3)H]mannose labeled cells, mannose supplementation also partially corrected the impaired synthesis of mannosylphosphoryldolichol (Man-P-Dol) and Glc(0)(-)(3)Man(9)GlcNAc(2)-P-P-Dol. These results confirm directly that deficiencies in PMM and PMI result in lowered cellular GDP-Man levels that are corrected by the addition of mannose. In contrast to these results, GDP-Man levels in fibroblasts from a CDG-Ie patient, who is deficient in Man-P-Dol synthase, were normal and unaffected by mannose supplementation even though mannose addition was found to correct abnormal lipid intermediate synthesis in another study (Kim et al. [2000] J. Clin. Invest., 105, 191-198). The mechanism by which mannose supplementation corrects abnormal protein N-glycosylation in Man-P-Dol synthase deficient cells is unknown, but this observation suggests that the regulation of Man-P-Dol synthesis and utilization may be more complex than is currently understood.     

Hydrophobic Man-1-P derivatives correct abnormal glycosylation in Type I congenital disorder of glycosylation fibroblasts

Patients with Type I congenital disorders of glycosylation (CDG-I) make incomplete lipid-linked oligosaccharides (LLO). These glycans are poorly transferred to proteins resulting in unoccupied glycosylation sequons. Mutations in phosphomannomutase (PMM2) cause CDG-Ia by reducing the activity of PMM, which converts mannose (Man)-6-P to Man-1-P before formation of GDP-Man. These patients have reduced Man-1-P and GDP-Man. To replenish intracellular Man-1-P pools in CDG-Ia cells, we synthesized two hydrophobic, membrane permeable acylated versions of Man-1-P and determined their ability to normalize LLO size and N-glycosylation in CDG-Ia fibroblasts. Both compounds, compound I (diacetoxymethyl 2,3,4,6-tetra-O-acetyl-alpha-D-mannopyranosyl phosphate) (C-I) and compound II (diacetoxymethyl 2,3,4,6-tetra-O-ethyloxycarbonyl-alpha-D-mannopyranosyl phosphate) (C-II), contain two acetoxymethyl (CH2OAc) groups O-linked to phosphorous. C-I contains acetyl esters and C-II contains ethylcarbonate (CO2Et) esters on the Man residue. Both C-I and C-II normalized truncated LLO, but C-II was about 2-fold more efficient than C-I. C-II replenished the GDP-Man pool in CDG-Ia cells and was more efficiently incorporated into glycoproteins than exogenous Man at low concentrations (25-75 mM). In a glycosylation assay of DNaseI in CDG-Ia cells, C-II restored glycosylation to control cell levels. C-II also corrected impaired LLO biosynthesis in cells from a Dolichol (Dol)-P-Man deficient patient (CDG-Ie) and partially corrected LLO in cells from an ALG12 mannosyltransferase-deficient patient (CDG-Ig), whereas cells from an ALG3-deficient patient (CDG-Id) and from an MPDU1-deficient patient (CDG-If) were not corrected. These results validate the general concept of using pro-Man-1-P substrates as potential therapeutics for CDG-I patients.     

Further evidence for dolichyl phosphate-mediated glycosyl translocation through membranes

Abstract Mannosyl residues from GDP-[¹⁴C]Man are transferred into liposomes and onto internal GDP if the liposomal membranes contain dolichyl phosphate and the enzyme GDP-Man: Dol-P mannosyl transferase. Experiments using a mixture of GDP-[¹⁴C]Man and [³H]GDP-Man excluded the possibility that the transmembrane translocation of [¹⁴C]mannose is due to a GDP-[¹⁴C]Man_outside ag GDP_inside exchange reaction.     

Enzymatic synthesis of mannosyl retinyl phosphate from retinyl phosphate and guanosine diphosphate mannose.

A study was conducted to determine whether retinyl phosphate would act as substrate for the enzymatic synthesis of mannosyl retinyl phosphate. Retinyl phosphate, prepared chemically, supported the growth of vitamin A-deficient rats at the same rate as retinol. It also stimulated the uptake of [14C]mannose from GDP-[14C]mannose into total chloroform-methanol extractable lipid. This reaction occurred in the presence of ATP, Mn2+, detergent (Zonyl A), and a membrane-rich enzyme preparation from the livers of vitamin A-deficient rats, provided that a lipid extract of the membrane preparation of alpha-L-lecithin was also added. Total chloroform-methanol-extractable, labeled mannolipid was separated into two principal labeled mannolipids by thin-layer or column chromatography or by differential solvent extraction. The properties of these mannolipids identified them as glycophospholipids: one was identical with authentic synthetic dolichyl mannosyl phosphate, and the other was concluded to be mannosyl retinyl phosphate because of its incorporation of radioactivity from [3H]retinyl phosphate, its rapid hydrolysis by dilute acid, and the formation of substance that cochromatographed with retinol upon its acid hydrolysis. The presence of ATP or GTP was essential for the stimulation of mannolipid synthesis, probably because of their protective action on the substrates against phosphatases present in the crude enzyme fraction. A pH of 6.0-6.2 favored the formation of dolichyl mannosyl phosphate; a higher pH (6.7-7.0) that of mannosyl retinyl phosphate.     

Involvement of dolicholmonophosphate in the formation of specific mannosyl-linkages in yeast glycoproteins

A membrane fraction from Saccharomyces cerevisiae catalyzes the transfer of mannosyl residues from GDP-Man partly via dolicholmonophosphate into a heterogenous glycoprotein fraction. The pattern of radioactive products obtained after mannosylation with GDP-[¹⁴C]Man is similar to that obtained with dolicholmonophosphate-[¹⁴C]mannose. In each case more than 70% of the radioactivity can be released by β-elimination. Evidence is presented, that only the mannosyl residue directly linked to protein is incorporated via dolicholmonophosphate.     

Overexpression of GDP-mannose pyrophosphorylase in Saccharomyces cerevisiae corrects defects in dolichol-linked saccharide formation and protein glycosylation

Thermosensitive mutants of Saccharomyces cerevisiae, affected in the endoplasmic reticulum (ER) located glycosylation, i.e. in Dol-P-Man synthase (dpm1), in beta-1,4 mannosyl transferase (alg1) and in alpha-1,3 mannosyltransferase (alg2), were used to assess the role of GDP-Man availability for the synthesis of dolichol-linked saccharides. The mutants were transformed with the yeast gene MPG1 (PSA1/VIG9) encoding GDP-Man pyrophosphorylase catalyzing the final step of GDP-Man formation. We found that overexpression of MPG1 allows growth at non-permissive temperature and leads to an increase in the cellular content of GDP-Man. In the alg1 and alg2 mutants, complemented with MPG1 gene, N-glycosylation of invertase was in part restored, to a degree comparable to that of the wild-type control. In the dpm1 mutant, the glycosylation reactions that depend on the formation of Dol-P-Man, i.e. elongation of Man(5)GlcNAc(2)-PP-Dol, O-mannosylation of chitinase and synthesis of GPI anchor were normal when MPG1 was overexpressed.Our data indicate that an increased level of GDP-Man is able to correct defects in mannosylation reactions ascribed to the ER and to the Golgi.     

Biosynthesis of lipid-linked oligosaccharides in yeast: the ALG3 gene encodes the Dol-P-Man:Man5GlcNAc2-PP-Dol mannosyltransferase

The formation of N-glycosidic linkages of glycoproteins involves the ordered assembly of the common Glc3Man9GlcNAc2 core-oligosaccharide on the lipid carrier dolichyl pyrophosphate. Whereas early mannosylation steps occur on the cytoplasmic side of the endoplasmic reticulum with GDP-Man as donor, the final reactions from Man5GlcNAc2-PP-Dol to Man9GlcNAc2-PP-Dol on the lumenal side use Dol-P-Man. We have investigated these later stages in vitro using a detergent-solubilized enzyme extract from yeast membranes. Mannosyltransfer from Dol-P-Man to [3H]Man5GlcNAc2-PP-Dol with formation of all intermediates up to Man9GlcNAc2-PP-Dol occured in a rapid, time- and protein-dependent fashion. We find that the initial reaction from Man5GlcNAc2-PP-Dol to Man6GlcNAc2-PP-Dol is independent of metal ions, but further elongations need Mn2+ that can be partly replaced by Mg2+ or Ca2+. Zn2+ or Cd2+ ions were found to inhibit formation of Man(7-9)GlcNAc2-PP-Dol, but do not affect synthesis of Man6GlcNAc2-PP-Dol. Extension did not occur when the acceptor was added as a free Man5GlcNAc2 oligosaccharide or when GDP-Man was used as mannosyl donor. The alg3 mutant was described to accumulate Man5GlcNAc2-PP-Dol. We expressed a functional active HA-epitope tagged ALG3 fusion and succeeded to selectively immunoprecipitate the Dol-P-Man:Man5GlcNAc2-PP-Dol mannosyltransferase activity from the other enzymes of the detergent extract involved in the subsequent mannosylation reactions. This demonstrates that Alg3p represents the mannosyltransferase itself and not an accessory protein involved in the reaction.     

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 α-linked mannosyl unit at the nonreducing position of their moleculae were effective as receptors. Monodeoxyanalogues of mannose as well as α-mannose phosphates did not serve as receptors in the above reaction.3. The structure of the product formed with mannose as receptor was determined to be O-α-D-mannosyl-(1→2)-mannose; with αMan(1→Man(1→6)mannose as the acceptor, the product was αMan(1→6)αMan(1→6)mannose and with αMan-(1→2)mannose the product was tentatively characterized as a mixture of αMan-(1→3)αMan(1→2)mannose and αMan(1→2)αMan(1→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 disaccharids were unable to enter the mannan protein molecule in the cell-free system while free radioactive mannose did incorporate into polysacchride to a minor extent under the same conditions.     

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.     

Galactoglucomannan from the secondary cell wall of Picea abies L. Karst

The fine structural features of alkali-extracted galactoglucomannan composed of D-galactose, D-glucose and D-mannose in a 1:8:33 mole proportion from the secondary cell walls of Picea abies L. Karst have been determined. Compositional and methylation analyses of the polymer, partial acid hydrolysis, as well as 1H and 13C NMR measurements of the polymer and products of partial acid hydrolysis confirmed a beta-(1-->4)-linked backbone of galactoglucomannan containing the segments of mannosyl residues (Man2, Man3, Man4, etc.) interrupted with the segments having both mannose and glucose residues, as well as the segments in which D-Glcp units can be adjacent to each other (Glc2). Further, the low content of branching points (approximately 3%) at the positions of 0-6, 0-3 and 0-2 of mannosyl and 0-6 and 0-3 of glucosyl residues, but preferably of mannosyl ones, indicates the presence of short side-chains terminated at position 0-6 predominantly by D-galactose units as single stubs.     

Structural studies and mechanism of Saccharomyces cerevisiae dolichyl-phosphate-mannose synthase: insights into the initial step of synthesis of dolichyl-phosphate-linked oligosaccharide chains in membranes of endoplasmic reticulum

Dolichyl-phosphate-mannose (Dol-P-Man) synthase catalyzes the reversible formation of a key intermediate that is involved as a mannosyl donor in at least three different pathways for the synthesis of glycoconjugates important for eukaryotic development and viability. The enzyme is found associated with membranes of the endoplasmic reticulum (ER), where it transfers mannose from the water soluble cytoplasmic donor, guanosine 5'-diphosphate (GDP)-Man, to the membrane-bound, extremely hydrophobic, and long-chain polyisoprenoid acceptor, dolichyl-phosphate (Dol-P). The enzyme from Saccharomyces cerevisiae has been utilized to investigate the structure and activity of the protein and interactions of the enzyme with Dol-P and synthetic Dol-P analogs containing fluorescent probes. These interactions have been explored utilizing fluorescence resonance energy transfer (FRET) to establish intramolecular distances within the protein molecule as well as intermolecular distances to determine the localization of the active site and the hydrophobic substrate on the enzyme's surface. A three-dimensional (3D) model of the enzyme was produced with bound substrates, Dol-P, GDP-Man, and divalent cations to delineate the binding sites for these substrates as well as the catalytic site. The FRET analysis was used to characterize the functional properties of the enzyme and to evaluate its modeled structure. The data allowed for proposing a molecular mechanism of catalysis as an inverting mechanism of mannosyl residue transfer.     

Transmembrane movement of a water-soluble analogue of mannosylphosphoryldolichol is mediated by an endoplasmic reticulum protein

Based on topological studies mannosylphosphoryldolichol (Man-P-Dol) is synthesized on the cytoplasmic face of the RER, but functions as a mannosyl donor in Glc3Man9GlcNAc2-P-P-dolichol biosynthesis after the mannosyl-phosphoryl headgroup diffuses transversely to the luminal compartment. The transport of mannosylphosphorylcitronellol (Man-P- Cit), a water-soluble analogue of Man-P-Dol, by microsomal vesicles from mouse liver, has been investigated as a potential experimental approach to determine if a membrane protein(s) mediates the transbilayer movement of Man-P-Dol. For these studies beta-[3H]Man-P- Cit was synthesized enzymatically with a partially purified preparation of Man-P-undecaprenol synthase from Micrococcus luteus. The uptake of the radiolabeled water-soluble analogue was found to be (a) time dependent; (b) stereoselective; (c) dependent on an intact permeability barrier; (d) saturable; (e) protease-sensitive; and (f) highest in ER- enriched vesicles relative to Golgi complex-enriched vesicles and intact mitochondria. Consistent with the involvement of a membrane protein, the analogue did not enter synthetic phosphatidylcholine- liposomes. [3H]Man-P-Cit also was not transported by human erythrocytes. These results indicate that the transport of Man-P-Cit by sealed microsomal vesicles from mouse liver is mediated by a membrane protein transport system. It is possible that the same membrane protein(s) participates in the transbilayer movement of Man-P-Dol in the ER.     

Syntheses of two trimannose analogs each containing C-mannosyl or 5-thio-C-mannosyl residue: their affinities to concanavalin A

C-Mannosyl residue-containing trimannose ManC alpha(1,6)[Man alpha(1,3)Man] (2) and 5-thio-C mannosyl residue-containing trimannose 5SManC alpha(1,6)[Man alpha(1,3)Man] (3) were synthesized via a glycosyl radical addition to enone derivative of mannose (6). Dissociation constants for the binding of these trisaccharides to concanavalin A (ConA) were determined by a fluorescence anisotropy inhibition assay: Kd = 198 and 31 microM, respectively. The unexpectedly large Kd value for the compound 2 compared with the compound 3 and the natural trimannose 1 demonstrates a characteristic of C-glycoside.     

Transient glucosylation of protein-bound Man9GlcNAc2, Man8GlcNAc2, and Man7GlcNAc2 in calf thyroid cells. A possible recognition signal in the processing of glycoproteins

Calf thyroid slices incubated with [U-14C]glucose synthesized protein-bound Glc3Man9GlcNAc2, Glc2-Man9GlcNAc2, Glc1Man9GlcNAc2, Glc1Man8GlcNAc2, and Glc1Man7GlcNAc2. Although label in the glucose residues of the last three compounds could be detected within 5 min of incubation, appearance of radioactivity in the mannose residues of the alpha-mannosidase-resistant cores of Glc1Man8GlcNAc2 and Glc1Man7GlcNAc2 took more than 30 and 60 min, respectively, to appear after label was detected in the same mannose residues of Glc1Man9GlcNAc2. The glucose residues were removed upon chasing the slices with unlabeled glucose. The last compound to disappear was Glc1Man9GlcNAc2. Calf thyroid microsomes incubated with UDP-[U-14C]Glc synthesized the five protein-bound oligosaccharides mentioned above. Although addition to GDP-Man to the incubation mixtures greatly diminished the formation of Glc3Man9GlcNAc2 bound either to dolichol-P-P or to protein, labeling of Glc1Man9GlcNAc2, Glc1Man8GlcNAc2, and Glc1Man7GlcNAc2 was not affected. Addition of kojibiose prevented deglucosylation of protein-bound Glc3Man9GlcNAc2 without affecting the formation of Glc1Man8GlcNAc2 and Glc1Man7GlcNAc2 and only partially diminishing that of Glc1Man9GlcNAc2. These results indicate that Glc1Man8GlcNAc2 and Glc1Man7GlcNAc2 were formed by glucosylation of the unglucosylated species and not be demannosylation of Glc1Man9GlcNAc2 and that probably part of the latter compound was formed in the same way.     

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%).     

An inhibitor of mannosylation of retinyl-phosphate

The guanosine disphospate and uridine diphosphate esters of the antiviral sugar analog 2-deoxy-2-fluoro-D-glucose (GDP-FGlc and UDP-FGlc) were synthesized and tested as inhibitors of formation of lipid-linked sugars in cell-free extracts . Formation of dolichol-phosphate mannose and of dolichol-diphosphate di-N-acetylchitobiose were not inhibited by either sugar nucleotide. Formation of dolichol-phosphate glucose was inhibited by UDP-FGlc, not by GDP-FGlc. Although GDP-FGlc did not inhibit formation of dollchol-phosphate mannose, it did inhibit formation of retinol-phosphate mannose from retinol-phosphate and GDP-Man. This inhibition was not reversed by exogenous retinol-phosphate, nor was FGIc from GDP-FGlc incorporated into retinolphosphate-linked derivatives. As FGLc inhibits formation of dolichol-phosphate mannose in intact cells, but does not decrease pool sizes of GDP-Man and dolichol-phosphate (Datema et al., 1980, Eur. J. Biochem.109, 331–341), we discuss that inhibition of formation of retinol-phosphate mannose by one of the metabolites of FGlc, namely GDP-FGlc, may lead to decreased synthesis of dolichol-phosphate mannose in FGlc-treated cells. This implies a role for vitamin A in the dolichol cycle of protein glycosylation.     

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.     

Platelet-derived growth factor stimulates synthesis of 1,2-diacylglycerol from monoacylglycerol in Balb/c 3T3 cells.

1,2-Diacylglycerol (1,2,-DAG) plays an important role in the protein kinase C-mediated signal-transduction system. Several reports have shown that 1,2-DAG is generated through various pathways other than classical phospholipid hydrolysis. We observed a rapid incorporation of [3H]myristate into 1,2-DAG in platelet-derived-growth-factor (PDGF)-treated Balb/c 3T3 cells. [14C]Palmitate was similarly incorporated into 1,2-DAG. The effect of PDGF, which was inhibited by cycloheximide, became maximal after 60 min treatment with PDGF, and disappeared 300 min after removal of PDGF. Treatment with triacylglycerol lipase revealed that labelled saturated fatty acid was incorporated into the sn-1 position. PDGF barely stimulated incorporation of [3H]glycerol or [14C]glucose into 1,2-DAG. Incorporation of [3H]myristate into 1,2-DAG preceded that into triacyglycerol and phospholipids. These results suggest that synthesis of 1,2-DAG from monoacylglycerol is enhanced in PDGF-treated cells. However, there is no significant difference in the activity of monoacylglycerol acyltransferase measured in vitro in quiescent and PDGF-treated cells. The reason for this discrepancy is discussed.