Charles J. Waechter

Biosynthesis of Mycobacterial Lipoarabinomannan

The mycobacterial lipoglycans, lipomannan (LM) and lipoarabinomannan (LAM), are potent immunomodulators in tuberculosis and leprosy. Little is known of their biosynthesis, other than being based on phosphatidylinositol (PI), and they probably originate in the phosphatidylinositol mannosides (PIMs; PIMans). A novel form of cell-free incubation involving in vitro andin situ labeling with GDP-[14C]Man of the polyprenyl-P-mannoses (C35/C50-P-Man) and the simpler PIMs of mycobacterial membranes, reisolation of the [14C]Man-labeled membranes, and in situ chase demonstrated the synthesis of a novel α(1→6)-linked linear form of LM at the expense of the C35/C50-P-Man. There was little or no synthesis under these conditions of PIMan5with its terminal α(1→2)Man unit or the mature LM or LAM with copious α(1→2)Man branching. Synthesis of the linear LM, but not of the simpler PIMan2, was susceptible to amphomycin, a lipopeptide antibiotic that specifically inhibits polyprenyl-P-requiring translocases. A mixture of P[3H]I and P[3H]IMan2 was incorporated into the linear LM, supporting other evidence that, like the PIMs, LM and LAM, it is a lipid-linked mannooligosaccharide and a new member of the mycobacterial glycosylphosphatidylinositol lipoglycan/glycolipid class. Hence, the simpler PIMs originate in PI and GDP-Man, but further growth of the linear backbone emanates from C35-/C50-P-Man and is amphomycin-sensitive. The origin of the α(1→2)Man branches of mature PIMan5, LM, and LAM is not known at this time but is probably GDP-Man.

The LPP1 and DPP1 gene products account for most of the isoprenoid phosphate phosphatase activities in Saccharomyces cerevisiae

Two genes in Saccharomyces cerevisiae, LPP1 and DPP1, with homology to a mammalian phosphatidic acid (PA) phosphatase were identified and disrupted. Neither single nor combined deletions resulted in growth or secretion phenotypes. As observed previously (Toke, D. A., Bennett, W. L., Dillon, D. A., Wu, W.-I., Chen, X., Ostrander, D. B., Oshiro, J., Cremesti, A., Voelker, D. R., Fischl, A. S., and Carman, G. M. (1998) J. Biol. Chem. 273, 3278–3284; Toke, D. A., Bennett, W. L., Oshiro, J., Wu, W.-I., Voelker, D. R., and Carman, G. M. (1998) J. Biol. Chem. 273, 14331–14338), the disruption of DPP1 and LPP1 produced profound losses of Mg2+-independent PA phosphatase activity. The coincident attenuation of hydrolytic activity against diacylglycerol pyrophosphate prompted an examination of the effects of these disruptions on hydrolysis of isoprenoid pyrophosphates. Disruption of either LPP1 or DPP1 caused respective decreases of about 25 and 75% in Mg2+-independent hydrolysis of several isoprenoid phosphates by particulate fractions isolated from these cells. The particulate and cytosolic fractions from the double disruption (lpp1Δ dpp1Δ) showed essentially complete loss of Mg2+-independent hydrolytic activity toward dolichyl phosphate (dolichyl-P), dolichyl pyrophosphate (dolichyl-P-P), farnesyl pyrophosphate (farnesyl-P-P), and geranylgeranyl pyrophosphate (geranylgeranyl-P-P). However, a modest Mg2+-stimulated activity toward PA and dolichyl-P was retained in cytosol fromlpp1Δ dpp1Δ cells. The action of Dpp1p on isoprenyl pyrophosphates was confirmed by characterization of the hydrolysis of geranylgeranyl-P-P by the purified protein. These results indicate that LPP1 and DPP1 account for most of the hydrolytic activities toward dolichyl-P-P, dolichyl-P, farnesyl-P-P, and geranylgeranyl-P-P but also suggest that yeast contain other enzymes capable of dephosphorylating these essential isoprenoid intermediates.

Geranylgeraniol Overcomes the Block of Cell Proliferation by Lovastatin in C6 Glioma Cells

Abstract: It is well documented that 3‐hydroxy‐3‐methylglutaryl‐CoA reductase inhibitors prevent cultured mammalian cells from progressing through the cell cycle, suggesting a critical role for a mevalonate‐derived product. Recently, it has been shown that free geranylgeraniol (GG‐OH) and farnesol (F‐OH) can be utilized by C6 glioma cells for protein isoprenylation. The ability of GG‐OH and F‐OH to restore protein geranylgeranylation or farnesylation selectively has enabled us to examine the possibility that mevalonate is essential for cell proliferation because it is a precursor of farnesyl pyrophosphate or geranylgeranyl pyrophosphate, the isoprenyl donors involved in the post‐translational modification of key regulatory proteins. In this study we report that GG‐OH, as well as mevalonate, overcomes the arrest of cell proliferation of C6 glioma cells treated with lovastatin, as assessed by increased cell numbers and a stimulation in [3H]thymidine incorporation. The increase in cell number and [3H]thymidine incorporation were significantly lower when F‐OH was added. Under these conditions [3H]mevalonate and [3H]GG‐OH are actively incorporated into a set of isoprenylated proteins in the size range of small, GTP‐binding proteins (19–27 kDa) and a polypeptide with the molecular size (46 kDa) of the smaller isoform of 2′,3′‐cyclic nucleotide 3′‐phosphodiesterase. Analysis of the proteins metabolically labeled by [3H]mevalonate and [3H]GG‐OH reveals the presence of labeled proteins containing geranylgeranylated cysteinyl residues. Consistent with geranylgeranylated proteins playing a critical role in the entry of C6 cells into the cell cycle, a (phosphonoacetamido)oxy derivative of GG‐OH, a drug previously shown to interfere with protein geranylgeranylation, prevented the increase in cell number when mevalonate or GG‐OH was added to lovastatin‐treated cells. These results strongly suggest that geranylgeranylated proteins are essential for progression of C6 cells into the S phase of the cell cycle and provide the first evidence that the “salvage” pathway for the utilization of the free isoprenols is physiologically significant in the CNS.

A Novel Epimerase That Converts GlcNAc-P-P-undecaprenol to GalNAc-P-P-undecaprenol in Escherichia coli O157

Escherichia coli strain O157 produces an O-antigen with the repeating tetrasaccharide unit α-d-PerNAc-α-l-Fuc-β-d-Glc-α-d-GalNAc, preassembled on undecaprenyl pyrophosphate (Und-P-P). These studies were conducted to determine whether the biosynthesis of the lipid-linked repeating tetrasaccharide was initiated by the formation of GalNAc-P-P-Und by WecA. When membrane fractions from E. coli strains K12, O157, and PR4019, a WecA-overexpressing strain, were incubated with UDP-[3H]GalNAc, neither the enzymatic synthesis of [3H]GlcNAc-P-P-Und nor [3H]GalNAc-P-P-Und was detected. However, when membrane fractions from strain O157 were incubated with UDP-[3H]GlcNAc, two enzymatically labeled products were observed with the chemical and chromatographic properties of [3H]GlcNAc-P-P-Und and [3H]GalNAc-P-P-Und, suggesting that strain O157 contained an epimerase capable of interconverting GlcNAc-P-P-Und and GalNAc-P-P-Und. The presence of a novel epimerase was demonstrated by showing that exogenous [3H]GlcNAc-P-P-Und was converted to [3H]GalNAc-P-P-Und when incubated with membranes from strain O157. When strain O157 was metabolically labeled with [3H]GlcNAc, both [3H]GlcNAc-P-P-Und and [3H]GalNAc-P-P-Und were detected. Transformation of E. coli strain 21546 with the Z3206 gene enabled these cells to synthesize GalNAc-P-P-Und in vivo and in vitro. The reversibility of the epimerase reaction was demonstrated by showing that [3H]GlcNAc-P-P-Und was reformed when membranes from strain O157 were incubated with exogenous [3H]GalNAc-P-P-Und. The inability of Z3206 to complement the loss of the gne gene in the expression of the Campylobacter jejuni N-glycosylation system in E. coli indicated that it does not function as a UDP-GlcNAc/UDP-GalNAc epimerase. Based on these results, GalNAc-P-P-Und is synthesized reversibly by a novel GlcNAc-P-P-Und epimerase after the formation of GlcNAc-P-P-Und by WecA in E. coli O157.

Does Rft1 flip an N-glycan lipid precursor?

Arising from: J. Helenius et al. 415, 447–450 (2002)10.1038/415447a; Helenius et al. replyProtein N-glycosylation requires flipping of the glycolipid Man5GlcNAc2-diphosphate dolichol (Man5GlcNAc2-PP-Dol) across the endoplasmic reticulum (ER). Helenius et al. report genetic evidence suggesting that Rft1, an essential ER membrane protein in yeast, is required directly to translocate Man5GlcNAc2-PP-Dol. We now show that a specific ER protein(s), but not Rft1, is required to flip Man5GlcNAc2-PP-Dol in reconstituted vesicles. Rft1 may have a critical accessory role in translocating Man5GlcNAc2-PP-Dol in vivo, but the Man5GlcNAc2-PP-Dol flippase itself remains to be identified.

Differential regulation of the N-methyl transferases responsible for phosphatidylcholine synthesis in Saccharomyces cerevisiae

Abstract The enzymes catalyzing the conversion of phosphatidylethanolamine to phosphatidylcholine were assayed by measuring the incorporation of label from [ 14 C-CH 3 ]- S -adenosyl-methionine into the endogenous phospholipids of particulate, cell-free preparations from S. cerevisiae grown in the presence of N -methylethanolamine, N,N -dimethylethanolamine, or choline. The results indicate that each base in the growth medium results in reduced levels of all the N -methyltransferase activity involved in the formation of the phosphatidyl ester of the given base. By following the conversion of exogenous [ 32 P]-phosphatidyldimethylethanolamine to [ 32 P]-phosphatidylcholine it has been shown that the activity of the third methyl transfer is 90% lower in particles prepared from choline grown cells than in particles prepared from cells grown without choline. The results suggest that there are at least two enzymes involved in the conversion of phosphatidylethanolamine to phosphatidylcholine and that their levels can be regulated individually. Supplementing the growth medium with any of the three methylated aminoethanols results in markedly increased cellular levels of their corresponding phosphatidyl esters and decreased levels of the precursor phosphatidyl esters. The fatty acid composition of phosphatidylcholine also changes when the medium is supplemented with choline suggesting that the proportions of the molecular species of this phosphatide depends on whether synthesis is via methylation of phosphatidylethanolamino or from the supplemented aminoethanol.

Nogo‐B receptor is necessary for cellular dolichol biosynthesis and protein N‐glycosylation

Dolichol monophosphate (Dol‐P) functions as an obligate glycosyl carrier lipid in protein glycosylation reactions. Dol‐P is synthesized by the successive condensation of isopentenyl diphosphate (IPP), with farnesyl diphosphate catalysed by a cis‐isoprenyltransferase (cis‐IPTase) activity. Despite the recognition of cis‐IPTase activity 40 years ago and the molecular cloning of the human cDNA encoding the mammalian enzyme, the molecular machinery responsible for regulating this activity remains incompletely understood. Here, we identify Nogo‐B receptor (NgBR) as an essential component of the Dol‐P biosynthetic machinery. Loss of NgBR results in a robust deficit in cis‐IPTase activity and Dol‐P production, leading to diminished levels of dolichol‐linked oligosaccharides and a broad reduction in protein N‐glycosylation. NgBR interacts with the previously identified cis‐IPTase hCIT, enhances hCIT protein stability, and promotes Dol‐P production. Identification of NgBR as a component of the cis‐IPTase machinery yields insights into the regulation of dolichol biosynthesis.

Congenital disorder of glycosylation due to DPM1 mutations presenting with dystroglycanopathy-type congenital muscular dystrophy.

Congenital disorders of glycosylation (CDG) are rare genetic defects mainly in the post-translational modification of proteins via attachment of carbohydrate chains. We describe an infant with the phenotype of a congenital muscular dystrophy, with borderline microcephaly, hypotonia, camptodactyly, severe motor delay, and elevated creatine kinase. Muscle biopsy showed muscular dystrophy and reduced α-dystroglycan immunostaining with glycoepitope-specific antibodies in a pattern diagnostic of dystroglycanopathy. Carbohydrate deficient transferrin testing showed a pattern pointing to a CDG type I. Sanger sequencing of DPM1 (dolichol-P-mannose synthase subunit 1) revealed a novel Gly > Val change c.455G > T missense mutation resulting in p.Gly152Val) of unknown pathogenicity and deletion/duplication analysis revealed an intragenic deletion from exons 3 to 7 on the other allele. DPM1 activity in fibroblasts was reduced by 80%, while affinity for the substrate was not depressed, suggesting a decrease in the amount of active enzyme. Transfected cells expressing tagged versions of wild type and the p.Gly152Val mutant displayed reduced binding to DPM3, an essential, non-catalytic subunit of the DPM complex, suggesting a mechanism for pathogenicity. The present case is the first individual described with DPM1-CDG (CDG-Ie) to also have clinical and muscle biopsy findings consistent with dystroglycanopathy.

Suppression of Rft1 Expression Does Not Impair the Transbilayer Movement of Man5GlcNAc2-P-P-Dolichol in Sealed Microsomes from Yeast

To further evaluate the role of Rft1 in the transbilayer movement of Man5GlcNAc2-P-P-dolichol (M5-DLO), a series of experiments was conducted with intact cells and sealed microsomal vesicles. First, an unexpectedly large accumulation (37-fold) of M5-DLO was observed in Rft1-depleted cells (YG1137) relative to Glc3Man9GlcNAc2-P-P-Dol in wild type (SS328) cells when glycolipid levels were compared by fluorophore-assisted carbohydrate electrophoresis analysis. When sealed microsomes from wild type cells and cells depleted of Rft1 were incubated with GDP-[3H]mannose or UDP-[3H]GlcNAc in the presence of unlabeled GDP-Man, no difference was observed in the rate of synthesis of [3H]Man9GlcNAc2-P-P-dolichol or Man9[3H]GlcNAc2-P-P-dolichol, respectively. In addition, no difference was seen in the level of M5-DLO flippase activity in sealed wild type and Rft1-depleted microsomal vesicles when the activity was assessed by the transport of GlcNAc2-P-P-Dol15, a water-soluble analogue. The entry of the analogue into the lumenal compartment was confirmed by demonstrating that [3H]chitobiosyl units were transferred to endogenous peptide acceptors via the yeast oligosaccharyltransferase when sealed vesicles were incubated with [3H]GlcNAc2-P-P-Dol15 in the presence of an exogenously supplied acceptor peptide. In addition, several enzymes involved in Dol-P and lipid intermediate biosynthesis were found to be up-regulated in Rft1-depleted cells. All of these results indicate that although Rft1 may play a critical role in vivo, depletion of this protein does not impair the transbilayer movement of M5-DLO in sealed microsomal fractions prepared from disrupted cells.

Long‐Chain os‐Isoprenyltransferase Activity Is Induced Early in the Developmental Program for Protein N‐Glycosylation in Embryonic Rat Brain Cells

A large developmental increase in Glc3Man9‐ GlcNAc2‐P‐P‐dolichol (Oligo‐P‐P‐Dol) synthesis and protein W‐glycosylation in primary cultures of embryonic rat brain cells has been reported previously. In vitro enzyme studies and metabolic labeling experiments now show that there is a coordinate induction of long‐chain c/s‐iso‐ prenyltransferase (IPTase) activity, an activity required for the chain‐elongation stage of dolichyl monophosphate (Dol‐P) biosynthesis de novo, and Oligo‐P‐P‐Dol biosynthesis in embryonic rat brain. Different developmental patterns were observed for IPTase and |8‐hydroxy‐/3‐methyl‐ glutaryl‐CoA (HMG‐CoA) reductase activity as well as Dol‐ P and cholesterol biosynthesis, indicating that these pathways are regulated independently in rat brain. Three separate experimental approaches provide evidence that the amount of Dol‐P available in the rough endoplasmic reticulum (RER) is a rate‐limiting factor in the expression of the lipid intermediate pathway. First, metabolic labeling experiments show that the biosynthesis of Dol‐P is induced at the same time or just prior to the induction of Oligo‐P‐P‐Dol biosynthesis. Second, the time of induction and rate of Oligo‐P‐P‐Dol synthesis are accelerated when Dol‐P is supplemented in the culture medium. Third, in vitro assays of mannosylphosphoryldolichol synthase and A/‐acetylglucosaminylpyrophosphoryldolichol synthase indicate that there are only minor increases in the levels of these enzymes during development, but the amount of endogenous Dol‐P in the RER that is accessible to the glycosyltransferases increases when IPTase activity is induced. In summary, the current studies with embryonic rat brain cells document the coordinate induction of IPTase activity and Oligo‐P‐P‐Dol synthesis, support the hypothesis that the availability of Dol‐P in the RER is one rate‐limiting factor in Oligo‐P‐P‐Dol synthesis, and strongly suggest that increases in IPTase activity and the rate of de novo Dol‐P biosynthesis enhance the capacity of embryonic rat brain cells for lipid intermediate synthesis early in the developmental program for N‐linked glycoprotein biosynthesis.