Mark A. Lehrman

Under normoxia, 2-deoxy-d-glucose elicits cell death in select tumor types not by inhibition of glycolysis but by interfering with N-linked glycosylation

In tumor cells growing under hypoxia, inhibiting glycolysis with 2-deoxy-d-glucose (2-DG) leads to cell death, whereas under normoxic conditions cells similarly treated survive. Surprisingly, here we find that 2-DG is toxic in select tumor cell lines growing under normal oxygen tension. In contrast, a more potent glycolytic inhibitor, 2-fluorodeoxy-d-glucose, shows little or no toxicity in these cell types, indicating that a mechanism other than inhibition of glycolysis is responsible for their sensitivity to 2-DG under normoxia. A clue to this other mechanism comes from previous studies in which it was shown that 2-DG interferes with viral N-linked glycosylation and is reversible by exogenous addition of mannose. Similarly, we find that 2-DG interferes with N-linked glycosylation more potently in the tumor cell types that are sensitive to 2-DG under normoxia, which can be reversed by exogenous mannose. Additionally, 2-DG induces an unfolded protein response, including up-regulation of GADD153 (C/EBP-homologous protein), an unfolded protein response–specific mediator of apoptosis, more effectively in 2-DG–sensitive cells. We conclude that 2-DG seems to be toxic in select tumor cell types growing under normoxia by inhibition of N-linked glycosylation and not by glycolysis. Because in a phase I study 2-DG is used in combination with an anticancer agent to target hypoxic cells, our results raise the possibility that in certain cases, 2-DG could be used as a single agent to selectively kill both the aerobic (via interference with glycosylation) and hypoxic (via inhibition of glycolysis) cells of a solid tumor. [Mol Cancer Ther 2007;6(11):3049–58]

Spliced X-box binding protein 1 couples the unfolded protein response to hexosamine biosynthetic pathway

The hexosamine biosynthetic pathway (HBP) generates uridine diphosphate N-acetylglucosamine (UDP-GlcNAc) for glycan synthesis and O-linked GlcNAc (O-GlcNAc) protein modifications. Despite the established role of the HBP in metabolism and multiple diseases, regulation of the HBP remains largely undefined. Here, we show that spliced X-box binding protein 1 (Xbp1s), the most conserved signal transducer of the unfolded protein response (UPR), is a direct transcriptional activator of the HBP. We demonstrate that the UPR triggers HBP activation via Xbp1s-dependent transcription of genes coding for key, rate-limiting enzymes. We further establish that this previously unrecognized UPR-HBP axis is triggered in a variety of stress conditions. Finally, we demonstrate a physiologic role for the UPR-HBP axis by showing that acute stimulation of Xbp1s in heart by ischemia/reperfusion confers robust cardioprotection in part through induction of the HBP. Collectively, these studies reveal that Xbp1s couples the UPR to the HBP to protect cells under stress.

A mutation in the human MPDU1 gene causes congenital disorder of glycosylation type If (CDG-If)

We describe a new congenital disorder of glycosylation, CDG-If. The patient has severe psychomotor retardation, seizures, failure to thrive, dry skin and scaling with erythroderma, and impaired vision. CDG-If is caused by a defect in the gene MPDU1, the human homologue of hamster Lec35, and is the first disorder to affect the use, rather than the biosynthesis, of donor substrates for lipid-linked oligosaccharides. This leads to the synthesis of incomplete and poorly transferred precursor oligosaccharides lacking both mannose and glucose residues. The patient has a homozygous point mutation (221T-->C, L74S) in a semiconserved amino acid of MPDU1. Chinese hamster ovary Lec35 cells lack a functional Lec35 gene and synthesize truncated lipid-linked oligosaccharides similar to the patients. They lack glucose and mannose residues donated by Glc-P-Dol and Man-P-Dol. Transfection with the normal human MPDU1 allele nearly completely restores normal glycosylation, whereas transfection with the patients MPDU1 allele only weakly restores normal glycosylation. This work provides a new clinical picture for another CDG that may involve synthesis of multiple types of glycoconjugates.

The Xbp1s/GalE axis links ER stress to postprandial hepatic metabolism

Postprandially, the liver experiences an extensive metabolic reprogramming that is required for the switch from glucose production to glucose assimilation. Upon refeeding, the unfolded protein response (UPR) is rapidly, though only transiently, activated. Activation of the UPR results in a cessation of protein translation, increased chaperone expression, and increased ER-mediated protein degradation, but it is not clear how the UPR is involved in the postprandial switch to alternate fuel sources. Activation of the inositol-requiring enzyme 1 (IRE1) branch of the UPR signaling pathway triggers expression of the transcription factor Xbp1s. Using a mouse model with liver-specific inducible Xbp1s expression, we demonstrate that Xbp1s is sufficient to provoke a metabolic switch characteristic of the postprandial state, even in the absence of caloric influx. Mechanistically, we identified UDP-galactose-4-epimerase (GalE) as a direct transcriptional target of Xbp1s and as the key mediator of this effect. Our results provide evidence that the Xbp1s/GalE pathway functions as a novel regulatory nexus connecting the UPR to the characteristic postprandial metabolic changes in hepatocytes.

Antiangiogenic activity of 2-deoxy-D-glucose

Background During tumor angiogenesis, endothelial cells (ECs) are engaged in a number of energy consuming biological processes, such as proliferation, migration, and capillary formation. Since glucose uptake and metabolism are increased to meet this energy need, the effects of the glycolytic inhibitor 2-deoxy-D-glucose (2-DG) on in vitro and in vivo angiogenesis were investigated. Methodology/Principal Findings In cell culture, 2-DG inhibited EC growth, induced cytotoxicity, blocked migration, and inhibited actively forming but not established endothelial capillaries. Surprisingly, 2-DG was a better inhibitor of these EC properties than two more efficacious glycolytic inhibitors, 2-fluorodeoxy-D-glucose and oxamate. As an alternative to a glycolytic inhibitory mechanism, we considered 2-DGs ability to interfere with endothelial N-linked glycosylation. 2-DGs effects were reversed by mannose, an N-linked glycosylation precursor, and at relevant concentrations 2-DG also inhibited synthesis of the lipid linked oligosaccharide (LLO) N-glycosylation donor in a mannose-reversible manner. Inhibition of LLO synthesis activated the unfolded protein response (UPR), which resulted in induction of GADD153/CHOP and EC apoptosis (TUNEL assay). Thus, 2-DGs effects on ECs appeared primarily due to inhibition of LLOs synthesis, not glycolysis. 2-DG was then evaluated in two mouse models, inhibiting angiogenesis in both the matrigel plug assay and the LHBETATAG transgenic retinoblastoma model. Conclusions/Significance In conclusion, 2-DG inhibits endothelial cell angiogenesis in vitro and in vivo, at concentrations below those affecting tumor cells directly, most likely by interfering with N-linked glycosylation rather than glycolysis. Our data underscore the importance of glucose metabolism on neovascularization, and demonstrate a novel approach for anti-angiogenic strategies.

Oligosaccharide-based information in endoplasmic reticulum quality control and other biological systems.

Oligosaccharides as Information Carriers Glycosidically linked sugar polymers are well known as nutritional and structural molecules, and it is now clear that they also have essential roles as carriers of biological information. The constituent sugar residues contain multiple hydroxyl groups capable of forming complex arrangements of hydrogen bonds. Sugars are often modified with amino, N-acetyl, carboxyl, phosphate, and sulfate groups, permitting more varied interactions than those achieved with hydroxyls. Many different sugars occur in nature, and these can be coupled in numerous ways through a or b glycosidic linkages of their hydroxyls to form oligosaccharides (with relatively few sugars) and polysaccharides (with many sugars) (1). For example, there are eight different ways to couple the anomeric carbon (no. 1) of one residue of glucose to the nonanomeric carbons (no. 2, 3, 4, or 6) of another. Sugar polymers are also distinguished from other biological polymers by facile formation of both linear and branched structures. For example, the b1,4-linked mannose residue in asparagine (N)-linked oligosaccharides is always linked to at least three other sugars as indicated in Fig. 1. Oligosaccharides that carry information are usually coupled to chemically distinct units termed aglycones that themselves are not carbohydrates, but typically are proteins or lipids, and whose biological properties can be dramatically changed by the oligosaccharide. The purpose of this minireview is to explore the roles of oligosaccharides as carriers of intraand intercellular information with emphasis on the relationships between oligosaccharide metabolism, quality control, and stress responses of the endoplasmic reticulum (ER).

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.

Hamster UDP-N-Acetylglucosamine:Dolichol-P N-Acetylglucosamine-1-P Transferase Has Multiple Transmembrane Spans and a Critical Cytosolic Loop

UDP-GlcNAc:dolichol-P GlcNAc-1-P transferase (GPT) is an endoplasmic reticulum (ER) enzyme responsible for synthesis of GlcNAc-P-P-dolichol, the committed step of dolichol-P-P-oligosaccharide synthesis. The sequence of hamster GPT predicted multiple transmembrane segments (Zhu, X., and Lehrman, M. A. (1990) J. Biol. Chem. 265, 14250-14255). GPT has also been predicted to act on the cytosolic face of the ER membrane, based on topological studies of its substrates and products. In this report we test these predictions by: (i) immunofluorescence microscopy with antibodies specific for native GPT sequences or epitope tags inserted into GPT, after selective permeabilization of the plasma membrane with digitonin; (ii) insertion of Factor Xa cleavage sites; (iii) in vitro translation of GPT; and (iv) site-directed mutagenesis. The loops between the 1st and 2nd and between the 9th and 10th predicted transmembrane spans of GPT were found to be cytosolic. In contrast, the loop between the 6th and 7th transmembrane spans, as well as the carboxyl terminus, were lumenal. Thus, hamster GPT must cross the ER membrane at least three times, consistent with previous computer-assisted predictions. There was no apparent N-glycosylation or signal sequence cleavage detected by in vitro translation. The cytosolic loop between the 9th and 10th transmembrane spans is the largest hydrophilic segment in GPT and, as judged by site-directed mutagenesis, has a number of conserved residues essential for activity. Hence, these results directly support the hypothesis that dolichol-P-P-oligosaccharide assembly is initiated in the cytosol and that a downstream intermediate must translocate to the lumenal face of the ER membrane.

Non-radioactive analysis of lipid-linked oligosaccharide compositions by fluorophore-assisted carbohydrate electrophoresis.

Lipid-linked oligosaccharides (LLOs) are the donors of glycans that modify newly synthesized proteins in the endoplasmic reticulum (ER) of eukaryotes, resulting in formation of N-linked glycoproteins. The vast majority of LLO analyses have relied on metabolic labeling with radioactive sugar precursors, but these approaches have technical limitations resulting in many important questions about LLO synthesis being left unanswered. Here we describe the application of a facile non-radioactive technique, fluorophore-assisted carbohydrate electrophoresis (FACE), which circumvents these limitations. With FACE, steady-state LLO compositions can be determined quantitatively from cell cultures and animal tissues. We also present FACE methods for analysis of phosphosugars and nucleotide sugars, which are metabolic precursors of LLOs.

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.