Jeffrey S. Rush

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.

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.

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.

Recycling of Dolichyl Monophosphate to the Cytoplasmic Leaflet of the Endoplasmic Reticulum after the Cleavage of Dolichyl Pyrophosphate on the Lumenal Monolayer

During protein N-glycosylation, dolichyl pyrophosphate (Dol-P-P) is discharged in the lumenal monolayer of the endoplasmic reticulum (ER). Dol-P-P is then cleaved to Dol-P by Dol-P-P phosphatase (DPPase). Studies with the yeast mutant cwh8Δ, lacking DPPase activity, indicate that recycling of Dol-P produced by DPPase contributes significantly to the pool of Dol-P utilized for lipid intermediate biosynthesis on the cytoplasmic leaflet. Whether Dol-P formed in the lumen diffuses directly back to the cytoplasmic leaflet or is first dephosphorylated to dolichol has not been determined. Incubation of sealed ER vesicles from calf brain with acetyl-Asn-Tyr-Thr-NH2, an N-glycosylatable peptide, to generate Dol-P-P in the lumenal monolayer produced corresponding increases in the rates of Man-P-Dol, Glc-P-Dol, and GlcNAc-P-P-Dol synthesis in the absence of CTP. No changes in dolichol kinase activity were observed. When streptolysin-O permeabilized CHO cells were incubated with an acceptor peptide, N-glycopeptide synthesis, requiring multiple cycles of the dolichol pathway, occurred in the absence of CTP. The results obtained with sealed microsomes and CHO cells indicate that Dol-P, formed from Dol-P-P, returns to the cytoplasmic leaflet where it can be reutilized for lipid intermediate biosynthesis, and dolichol kinase is not required for recycling. It is possible that the flip-flopping of the carrier lipid is mediated by a flippase, which would provide a mechanism for the recycling of Dol-P derived from Man-P-Dol-mediated reactions in N-, O-, and C-mannosylation of proteins, GPI anchor assembly, and the three Glc-P-Dol-mediated reactions in Glc3Man9GlcNAc2-P-P-Dol (DLO) biosynthesis.

Inhibitors of protein kinase C block activation of B lymphocytes by bacterial lipopolysaccharide

Activation of murine splenic B lymphocytes (B cells) by bacterial lipopolysaccharide (LPS) was found to be markedly inhibited by 1-(5-isoquinolinylsulfonyl)-2-methylpiperazine (H-7) and N-[2-(methylamino)ethyl]-5-isoquinolinesulfonamide (H-8), two potent inhibitors of protein kinases. The higher sensitivity of DNA synthesis, RNA synthesis and protein N-glycosylation activity to H-7, relative to H-8, strongly supports the proposal that protein kinase C plays a critical role in the activation of B cells. A kinetic study on the time of addition of H-7 indicated that protein kinase C promoted the activation process continuously after the addition of LPS.

Expression of functional bacterial undecaprenyl pyrophosphate synthase in the yeast rer2Δ mutant and CHO cells

During evolution the average chain length of polyisoprenoid glycosyl carrier lipids increased from C55 (prokaryotes) to C75 (yeast) to C95 (mammalian cells). In this study, the ability of the E. coli enzyme, undecaprenyl pyrophosphate synthase (UPPS), to complement the loss of the yeast cis-isoprenyltransferase in the rer2Δ mutant was tested to determine if (55)dolichyl phosphate (Dol-P) could functionally substitute in the protein N-glycosylation pathway for (75)Dol-P, the normal isoprenologue synthesized in S. cerevisiae. First, expression of UPPS in the yeast mutant was found to complement the growth and the hypoglycosylation of carboxypeptidase Y defects suggesting that the (55)polyprenyl-P-P intermediate was converted to (55)Dol-P and that (55)Dol-P could effectively substitute for (75)Dol-P in the biosynthesis and function of Man-P-Dol, Glc-P-Dol and Glc(3)Man(9)GlcNAc(2)-P-P-Dol (mature DLO) in the protein N-glycosylation pathway and glycosylphosphatidylinositol anchor assembly. In support of this conclusion, mutant cells expressing UPPS (1) synthesized (55)Dol-P based on MS analysis, (2) utilized (55)Dol-P to form Man-P-(55)Dol in vitro and in vivo, and (3) synthesized N-linked glycoproteins at virtually normal rates as assessed by metabolic labeling with [(3)H]mannose. In addition, an N-terminal GFP-tagged construct of UPPS was shown to localize to the endoplasmic reticulum of Chinese hamster ovary cells. Consistent with the synthesis of (55)Dol-P by the transfected cells, microsomes from the transfected cells synthesized the [(14)C](55)polyprenyl-P-P intermediate when incubated with [(14)C]isopentenyl pyrophosphate and [(3)H]Man-P-(55)Dol when incubated with GDP-[(3)H]Man. These results indicate that (C55)polyisoprenoid chains, significantly shorter than the natural glycosyl carrier lipid, can function in the transbilayer movement of DLOs in the endoplasmic reticulum of yeast and mammalian cells, and that conserved sequences in the cis-isoprenyltransferases are recognized by, yet to be identified, binding partners in the endoplasmic reticulum of mammalian cells.

ALG1-CDG: Clinical and Molecular Characterization of 39 Unreported Patients.

Congenital disorders of glycosylation (CDG) arise from pathogenic mutations in over 100 genes leading to impaired protein or lipid glycosylation. ALG1 encodes a β1,4 mannosyltransferase that catalyzes the addition of the first of nine mannose moieties to form a dolichol‐lipid linked oligosaccharide intermediate required for proper N‐linked glycosylation. ALG1 mutations cause a rare autosomal recessive disorder termed ALG1‐CDG. To date 13 mutations in 18 patients from 14 families have been described with varying degrees of clinical severity. We identified and characterized 39 previously unreported cases of ALG1‐CDG from 32 families and add 26 new mutations. Pathogenicity of each mutation was confirmed based on its inability to rescue impaired growth or hypoglycosylation of a standard biomarker in an alg1‐deficient yeast strain. Using this approach we could not establish a rank order comparison of biomarker glycosylation and patient phenotype, but we identified mutations with a lethal outcome in the first two years of life. The recently identified protein‐linked xeno‐tetrasaccharide biomarker, NeuAc‐Gal‐GlcNAc2, was seen in all 27 patients tested. Our study triples the number of known patients and expands the molecular and clinical correlates of this disorder.