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Iduronidase (IDUA)-deficient mice accumulate glycosaminoglycans in cells and tissues and exhibit many of the same neuropathological symptoms of patients suffering from Mucopolysaccharidosis I. Intravenous enzyme-replacement therapy forMucopolysaccharidosis I ameliorates glycosaminoglycan storage and many of the somatic aspects of the disease but fails to treat neurological symptoms due to poor transport across the blood-brain barrier. In this study, we examined the delivery of IDUA conjugated to guanidinoneomycin (GNeo), a molecular transporter. GNeo-IDUA and IDUA injected intravenously resulted in reduced hepatic glycosaminoglycan accumulation but had no effect in the brain due to fast clearance from the circulation. In contrast, intranasally administered GNeo-IDUA entered the brain rapidly. Repetitive intranasal treatment with GNeo-IDUA reduced glycosaminoglycan storage, lysosome size and number, and neurodegenerative astrogliosis in the olfactory bulb and primary somatosensory cortex, whereas IDUA was less effective. The enhanced efficacy of GNeo-IDUA was not the result of increased nose-to-brain delivery or enzyme stability, but rather due to more efficient uptake into neurons and astrocytes. GNeo conjugation also enhanced glycosaminoglycan clearance by intranasally delivered sulfamidase to the brain of sulfamidase-deficient mice, a model of Mucopolysaccharidosis IIIA. These findings suggest the general utility of the guanidinoglycoside-based delivery system for restoring missing lysosomal enzymes in the brain.

Therapeutic development and monitoring require demonstration of effects on disease phenotype. However, due to the complexity of measuring clinically-relevant effects in rare multisystem diseases, robust biomarkers are essential. For the mucopolysaccharidoses (MPS), the measurement of glycosaminoglycan levels is relevant as glycosaminoglycan accumulation is the primary event that occurs due to reduced lysosomal enzyme activity. Traditional dye-based assays that measure total glycosaminoglycan levels have a high background, due to a normal, baseline glycosaminoglycan content in unaffected individuals. An assay that selectively detects the disease-specific non-reducing ends of heparan sulfate glycosaminoglycans that remain undegraded due to deficiency of a specific enzyme in the catabolic pathway avoids the normal background, increasing sensitivity and specificity. We evaluated glycosaminoglycan content by dye-based and non-reducing end methods using urine, serum, and cerebrospinal fluid from MPS I human samples before and after treatment with intravenous recombinant human alpha-l-iduronidase. We found that both urine total glycosaminoglycans and serum heparan sulfate derived non-reducing end levels were markedly decreased compared to baseline after 26 weeks and 52 weeks of therapy, with a significantly greater percentage reduction in serum non-reducing end (89.8% at 26 weeks and 81.3% at 52 weeks) compared to urine total glycosaminoglycans (68.3% at 26 weeks and 62.4% at 52 weeks, p

Mucopolysaccharidosis type IIIB (MPS IIIB) or Sanfilippo Syndrome type B is a lysosomal storage disease resulting from the deficiency of N-acetyl glucosaminidase (NAGLU) activity. We previously showed that intracranial adeno-associated virus (AAV)-based gene therapy results in partial improvements of several aspects of the disease. In an attempt to further correct the disease, MPS IIIB mice were treated at 2-4 days of age with intracranial AAV2/5-NAGLU (IC-AAV), intravenous lentiviral-NAGLU (IV-LENTI) or the combination of both (BOTH). The BOTH group had the most complete biochemical and histological improvements of any treatment group. Compared with untreated MPS IIIB animals, all treatments resulted in significant improvements in motor function (rotarod) and hearing (auditory-evoked brainstem response). In addition, each treatment group had a significantly increased median life span compared with the untreated group (322 days). The combination arm had the greatest increase (612 days), followed by IC-AAV (463 days) and IV-LENTI (358 days). Finally, the BOTH group had nearly normal circadian rhythm measures with improvement in time to activity onset. In summary, targeting both the systemic and central nervous system disease of MPS IIIB early in life appears to be the most efficacious approach for this inherited metabolic disorder.

Intrathecal enzyme replacement therapy is an experimental option to treat central nervous system disease due to lysosomal storage. Previous work shows that MPS I dogs receiving enzyme replacement with recombinant human alpha-L-iduronidase into the cisterna magna showed normal brain glycosaminoglycan (GAG) storage after three or four doses. We analyzed MPS I dogs that received intrathecal enzyme in a previous study using an assay that detects only pathologic GAG (pGAG). To quantify pGAG in MPS I, the assay measures only those GAG which display terminal iduronic acid residues on their non-reducing ends. Mean cortical brain pGAG in six untreated MPS I dogs was 60.9 ± 5.93 pmol per mg wet weight, and was 3.83 ± 2.64 in eight normal or unaffected carrier animals (p

A significant need exists for improved biomarkers for differential diagnosis, prognosis and monitoring of therapeutic interventions for mucopolysaccharidoses (MPS), inherited metabolic disorders that involve lysosomal storage of glycosaminoglycans. Here, we report a simple reliable method based on the detection of abundant non-reducing ends of the glycosaminoglycans that accumulate in cells, blood, and urine of MPS patients. In this method, glycosaminoglycans were enzymatically depolymerized releasing unique mono-, di-, or trisaccharides from the non-reducing ends of the chains. The composition of the released mono- and oligosaccharides depends on the nature of the lysosomal enzyme deficiency, and therefore they serve as diagnostic biomarkers. Analysis by liquid chromatography/mass spectrometry allowed qualitative and quantitative assessment of the biomarkers in biological samples. We provide a simple conceptual scheme for diagnosing MPS in uncharacterized samples and a method to monitor efficacy of enzyme replacement therapy or other forms of treatment.

The disaccharide peracetylated GlcNAcbeta1-3Galbeta-O-naphthalenemethanol (disaccharide 1) diminishes the formation of the glycan sialyl Lewis X (Neu5Acalpha2-3Galbeta1-4(Fucalpha1-3) GlcNAc; sLe(X)) in tumor cells. Previous studies showed that the mechanism of action of disaccharide 1 involves three steps: (i) deacetylation by carboxyesterases, (ii) action as a biosynthetic intermediate for downstream enzymes involved in sLe(X) assembly, and (iii) generation of several glycans related to sLe(X). In this report, we show that GlcNAcbeta1-3Galbeta-O-naphthalenemethanol binds to the acceptor site of human beta1-4-galactosyltransferase much like the acceptor trisaccharide, GlcNAcbeta1-2Manbeta1-6Man, which is present on N-linked glycans. The 4'-deoxy analog, in which the acceptor hydroxyl group was replaced by -H, did not act as a substrate but instead acted as a competitive inhibitor of the enzyme. The acetylated form of this compound inhibited sLe(X) formation in U937 monocytic leukemia cells, suggesting that it had inhibitory activity in vivo as well. A series of synthetic acetylated analogs of 1 containing -H, -F, -N(3), -NH(2), or -OCH(3) instead of the hydroxyl groups at C-3'- and C-4'-positions of the terminal N-acetylglucosamine residue also blocked sLe(X) formation in cells. The reduction of sLe(X) by the 4'-deoxy analog also diminished experimental tumor metastasis by Lewis lung carcinoma in vivo. These data suggest that nonsubstrate disaccharides have therapeutic potential through their ability to bind to glycosyltransferases in vivo and to alter glycan-dependent pathologic processes.

In a search for small molecule antagonists of heparan sulfate, we examined the activity of bis -2-methyl-4-amino-quinolyl-6-carbamide, also known as surfen. Fluorescence-based titrations indicated that surfen bound to glycosaminoglycans, and the extent of binding increased according to charge density in the order heparin > dermatan sulfate > heparan sulfate > chondroitin sulfate. All charged groups in heparin ( N -sulfates, O -sulfates, and carboxyl groups) contributed to binding, consistent with the idea that surfen interacted electrostatically. Surfen neutralized the anticoagulant activity of both unfractionated and low molecular weight heparins and inhibited enzymatic sulfation and degradation reactions in vitro . Addition of surfen to cultured cells blocked FGF2-binding and signaling that depended on cell surface heparan sulfate and prevented both FGF2- and VEGF 165 -mediated sprouting of endothelial cells in Matrigel. Surfen also blocked heparan sulfate-mediated cell adhesion to the Hep-II domain of fibronectin and prevented infection by HSV-1 that depended on glycoprotein D interaction with heparan sulfate. These findings demonstrate the feasibility of identifying small molecule antagonists of heparan sulfate and raise the possibility of developing pharmacological agents to treat disorders that involve glycosaminoglycan–protein interactions.

The development of tuberculin skin test (TST) positivity following infection by Mycobacterium tuberculosis is not invariable and may depend on bacillary as well as host factors.First, to compare the diagnostic performance of the TST and a form of in vitro IFN-gamma release assay (IFNGRA) in the circumstances of a contact investigation prompted by an unusually severe index case of infectious pulmonary tuberculosis. Second, to investigate the ability of the strain of M. tuberculosis responsible to induce cytokine secretion from monocytes in vitro.A routine TST-based tuberculosis-contact screening procedure supplemented by the use of an "in house" IFNGRA that assays the T-cell response to the M. tuberculosis-specific antigens ESAT-6, CFP-10 (presented as a fusion protein within the inactivated adenylate cyclase of Bordetella pertussis), and purified protein derivative of M. tuberculosis. Isolation and genetic typing of the strain of M. tuberculosis responsible, and investigation of its ability to induce cytokine secretion from monocytes in vitro.TST screening suggested a low rate of transmission with just 2/75 unequivocally positive responses. By contrast, the IFNGRA suggested an infection rate of 16/75 (22%). When compared with two reference strains of M. tuberculosis (H37Rv and CDC1551), the outbreak strain induced lower levels of tumor necrosis factor-alpha and interleukin-12p40 (p0.04), cytokines associated with the development of delayed-type hypersensitivity.These data suggest that infection by M. tuberculosis can be undetected by TST, and that this may partially relate to strain differences in immunogenicity.

Crystal (Cry) proteins made by the bacterium Bacillus thuringiensis are pore-forming toxins that specifically target insects and nematodes and are used around the world to kill insect pests. To better understand how pore-forming toxins interact with their host, we have screened for Caenorhabditis elegans mutants that resist Cry protein intoxication. We find that Cry toxin resistance involves the loss of two glycosyltransferase genes, bre-2 and bre-4. These glycosyltransferases function in the intestine to confer susceptibility to toxin. Furthermore, they are required for the interaction of active toxin with intestinal cells, suggesting they make an oligosaccharide receptor for toxin. Similarly, the bre-3 resistance gene is also required for toxin interaction with intestinal cells. Cloning of the bre-3 gene indicates it is the C. elegans homologue of the Drosophila egghead (egh) gene. This identification is striking given that the previously identified bre-5 has homology to Drosophila brainiac (brn) and that egh-brn likely function as consecutive glycosyltransferases in Drosophila epithelial cells. We find that, like in Drosophila, bre-3 and bre-5 act in a single pathway in C. elegans. bre-2 and bre-4 are also part of this pathway, thereby extending it. Consistent with its homology to brn, we demonstrate that C. elegans bre-5 rescues the Drosophila brn mutant and that BRE-5 encodes the dominant UDP-GlcNAc:Man GlcNAc transferase activity in C. elegans. Resistance to Cry toxins has uncovered a four component glycosylation pathway that is functionally conserved between nematodes and insects and that provides the basis of the dominant mechanism of resistance in C. elegans.

A variety of human adenocarcinomas express sialylated, fucosylated Lewis blood group antigens on cell surface and secreted mucins. Binding of these antigens to P-selectin on platelets is thought to facilitate formation of platelet-tumor emboli in the circulation, which in turn allows sequestration of the tumor cells in the microvasculature. Here we report a pharmacologic approach for blocking these interactions through metabolic inhibition of sialylation. Peracetylated forms of Galbeta1,4GlcNAcbeta-O-naphthalenemethanol and GlcNAcbeta1,3Galbeta-O-naphthalenemethanol were taken up by LS180 human colon carcinoma cells, O-deacetylated, and utilized as biosynthetic intermediates, resulting in heterogeneous oligosaccharides. The primed oligosaccharides included sialylated, sulfated, and fucosylated products based on mass spectrometry. Assembly of free oligosaccharides on the glycosides decoyed glycosylation of cellular glycoproteins, as assessed by altered binding of lectins and carbohydrate-specific antibodies. Expression of alpha2,3-sialylated oligosaccharides on the cell surface was diminished specifically, whereas alpha2,6-sialylation and fucosylation were not. In U937 lymphoma cells, the glycosides decreased fucosylation without affecting sialylation. The differential inhibitory activities correlated inversely with fucosyltransferase and sialyltransferase activity based on enzyme assays and microarray analysis. Regardless of the mechanism, the disaccharides blocked the cells from forming selectin ligands and inhibited adhesion to immobilized selectins, suggesting that the glycosides might prove useful for interfering with tumor cell adhesion and metastasis.

In mutants defective in any of eight Caenorhabditis elegans sqv (squashed vulva) genes, the vulval extracellular space fails to expand during vulval morphogenesis. Strong sqv mutations result in maternal-effect lethality, caused in part by the failure of the progeny of homozygous mutants to initiate cytokinesis and associated with the failure to form an extracellular space between the egg and the eggshell. Recent studies have implicated glycosaminoglycans in these processes. Here we report the cloning and characterization of sqv-2 and sqv-6. sqv-6 encodes a protein similar to human xylosyltransferases. Transfection of sqv-6 restored xylosyltransferase activity to and rescued the glycosaminoglycan biosynthesis defect of a xylosyltransferase mutant hamster cell line. sqv-2 encodes a protein similar to human galactosyltransferase II. A recombinant SQV-2 fusion protein had galactosyltransferase II activity with substrate specificity similar to that of human galactosyltransferase II. We conclude that C. elegans SQV-6 and SQV-2 likely act in concert with other SQV proteins to catalyze the stepwise formation of the proteoglycan core protein linkage tetrasaccharide GlcAbeta1,3Galbeta1, 3Galbeta1,4Xylbeta-O-(Ser), which is common to the two major types of glycosaminoglycans in vertebrates, chondroitin and heparan sulfate. Our results strongly support a model in which C. elegans vulval morphogenesis and zygotic cytokinesis depend on the expression of glycosaminoglycans.

Mucopolysaccharidosis type IIIB (MPS IIIB, Sanfilippo syndrome type B) is a lysosomal storage disease characterized by profound intellectual disability, dementia, and a lifespan of about two decades. The cause is mutation in the gene encoding α–N-acetylglucosaminidase (NAGLU), deficiency of NAGLU, and accumulation of heparan sulfate. Impediments to enzyme replacement therapy are the absence of mannose 6-phosphate on recombinant human NAGLU and the blood–brain barrier. To overcome the first impediment, a fusion protein of recombinant NAGLU and a fragment of insulin-like growth factor II (IGFII) was prepared for endocytosis by the mannose 6-phosphate/IGFII receptor. To bypass the blood–brain barrier, the fusion protein (“enzyme”) in artificial cerebrospinal fluid (“vehicle”) was administered intracerebroventricularly to the brain of adult MPS IIIB mice, four times over 2 wk. The brains were analyzed 1–28 d later and compared with brains of MPS IIIB mice that received vehicle alone or control (heterozygous) mice that received vehicle. There was marked uptake of the administered enzyme in many parts of the brain, where it persisted with a half-life of approximately 10 d. Heparan sulfate, and especially disease-specific heparan sulfate, was reduced to control level. A number of secondary accumulations in neurons [β-hexosaminidase, LAMP1(lysosome-associated membrane protein 1), SCMAS (subunit c of mitochondrial ATP synthase), glypican 5, β-amyloid, P-tau] were reduced almost to control level. CD68, a microglial protein, was reduced halfway. A large amount of enzyme also appeared in liver cells, where it reduced heparan sulfate and β-hexosaminidase accumulation to control levels. These results suggest the feasibility of enzyme replacement therapy for MPS IIIB.

Cells depend on polyamines for growth and their depletion represents a strategy for the treatment of cancer. Polyamines assemblede novothrough a pathway sensitive to the inhibitor, α-difluoromethylornithine (DFMO). However, the presence of cell-surface heparan sulfate proteoglycans may provide a salvage pathway for uptake of circulating polyamines, thereby sparing cells from the cytostatic effect of DFMO. Here we show that genetic or pharmacologic manipulation of proteoglycan synthesis in the presence of DFMO inhibits cell proliferationin vitroandin vivo. In cell culture, mutant cells lacking heparan sulfate were more sensitive to the growth inhibitory effects of DFMO than wild-type cells or mutant cells transfected with the cDNA for the missing biosynthetic enzyme. Moreover, extracellular polyamines did not restore growth of mutant cells, but completely reversed the inhibitory effect of DFMO in wild-type cells. In a mouse model of experimental metastasis, DFMO provided in the water supply also dramatically diminished seeding and growth of tumor foci in the lungs by heparan sulfate-deficient mutant cells compared with the controls. Wild-type cells also formed tumors less efficiently in mice fed both DFMO and a xylose-based inhibitor of heparan sulfate proteoglycan assembly. The effect seemed to be specific for heparan sulfate, because a different xyloside known to affect only chondroitin sulfate did not inhibit tumor growth. Hence, combined inhibition of heparan sulfate assembly and polyamine synthesis may represent an additional strategy for cancer therapy.

A family of five beta1,3-galactosyltransferases has been characterized that catalyze the formation of Galbeta1,3GlcNAcbeta and Galbeta1,3GalNAcbeta linkages present in glycoproteins and glycolipids (beta3GalT1, -2, -3, -4, and -5). We now report a new member of the family (beta3GalT6), involved in glycosaminoglycan biosynthesis. The human and mouse genes were located on chromosomes 1p36.3 and 4E2, respectively, and homologs are found in Drosophila melanogaster and Caenorhabditis elegans. Unlike other members of the family, beta3GalT6 showed a broad mRNA expression pattern by Northern blot analysis. Although a high degree of homology across several subdomains exists among other members of the beta3-galactosyltransferase family, recombinant enzyme did not utilize glucosamine- or galactosamine-containing acceptors. Instead, the enzyme transferred galactose from UDP-galactose to acceptors containing a terminal beta-linked galactose residue. This product, Galbeta1,3Galbeta is found in the linkage region of heparan sulfate and chondroitin sulfate (GlcAbeta1,3Galbeta1,3Galbeta1,4Xylbeta-O-Ser), indicating that beta3GalT6 is the so-called galactosyltransferase II involved in glycosaminoglycan biosynthesis. Its identity was confirmed in vivo by siRNA-mediated inhibition of glycosaminoglycan synthesis in HeLa S3 cells. Its localization in the medial Golgi indicates that this is the major site for assembly of the linkage region.

We report the characterization of two Chinese hamster ovary cell lines that produce large amounts of sulfated N-linked oligosaccharides. Clones 26 and 489 were derived by stable transfection of the glycosaminoglycan-deficient cell mutant pgsA-745 with a cDNA library prepared from wild-type cells. Peptide:N-glycanase F released nearly all of the sulfate label, indicating that sulfation had occurred selectively on the Asn-linked glycans. Hydrazinolysis followed by nitrous acid treatment at pH 4 and borohydride reduction yielded reduced sulfated disaccharides that comigrated with standard Gal3SO4beta1-4anhydromannitol. The disaccharides were resistant to periodate oxidation but became sensitive after the sulfate group was removed by methanolysis, indicating that the sulfate was located at C3 of the galactose residues. Maackia amurensis lectin bound to the sulfated glycopeptides on the cell surface and in free form, even after sialidase treatment. This finding indicates that the lectin requires only a charged group at C3 of the galactose unit and not an intact sialic acid. Growth of cells with chlorate restored sialidase sensitivity to lectin binding, indicating that sulfation and sialylation occurred largely at the same sites. The enhanced sulfation was due to elevated sulfotransferase activity that catalyzed transfer of sulfate from phosphoadenosine-5'-phosphosulfate to Galbeta1-4(3)GlcNAcbeta-O-naphthalenemethanol.

このレビューは、グリコシル化反応の阻害剤と糖鎖の代謝経路においてあたかも囮のように挙動する化合物に焦点をあてている。これらの化合物は、代謝過程の中間体に似ているため、糖鎖の生合成や、内因性の複合糖質の糖鎖生合成経路を変えたりするプライマーとして作用する。したがって、グリコシド誘導体は、複合糖質の生成を阻害したり、生体や細胞内での糖鎖の機能を研究するための道具となる。そのようなプライマーは、いろいろな病気において、糖鎖が原因となる病気の発症過程を変える機能を有する治療薬になるであろう。

Ahead of Print article withdrawn by publisher. At request of the authors, this article will be published in the journal Cancer Biomarkers (ISSN 1574-0153).

Background: Neurodegenerative metabolic disorders such as mucopolysaccharidosis IIIB (MPSIIIB or Sanfilippo disease) accumulate undegraded substrates in the brain and are often unresponsive to enzyme replacement treatments due to the impermeability of the blood brain barrier to enzyme. MPSIIIB is characterised by behavioural difficulties, cognitive and later motor decline, with death in the second decade of life. Most of these neurodegenerative lysosomal storage diseases lack effective treatments. We recently described significant reductions of accumulated heparan sulphate substrate in liver of a mouse model of MPSIIIB using the tyrosine kinase inhibitor genistein. Methodology/Principal Findings: We report here that high doses of genistein aglycone, given continuously over a 9 month period to MPSIIIB mice, significantly reduce lysosomal storage, heparan sulphate substrate and neuroinflammation in the cerebral cortex and hippocampus, resulting in correction of the behavioural defects observed. Improvements in synaptic vesicle protein expression and secondary storage in the cerebral cortex were also observed. Conclusions/Significance: Genistein may prove useful as a substrate reduction agent to delay clinical onset of MPSIIIB and, due to its multimodal action, may provide a treatment adjunct for several other neurodegenerative metabolic diseases. © 2010 Malinowska et al.

Hepatic clearance of triglyceride-rich lipoproteins depends on heparan sulfate and low density lipoprotein receptors expressed on the basal membrane of hepatocytes. Binding and uptake of the lipoproteins by way of heparan sulfate depends on the degree of sulfation of the chains based on accumulation of plasma triglycerides and delayed clearance of triglyceride-rich lipoproteins in mice bearing a hepatocyte-specific alteration of N-acetylglucosamine (GlcNAc) N-deacetylase-N-sulfotransferase 1 (Ndst1) (MacArthur, J. M., Bishop, J. R., Stanford, K. I., Wang, L., Bensadoun, A., Witztum, J. L., and Esko, J. D. (2007) J. Clin. Invest. 117, 153-164). Inactivation of Ndst1 led to decreased overall sulfation of heparan sulfate due to coupling of uronyl 2-O-sulfation and glucosaminyl 6-O-sulfation to initial N-deacetylation and N-sulfation of GlcNAc residues. To determine whether lipoprotein clearance depends on 2-O-and 6-O-sulfation, we evaluated plasma triglyceride levels in mice containing loxP-flanked conditional alleles of uronyl 2-O-sulfotransferase (Hs2st(f/f)) and glucosaminyl 6-O-sulfotransferase-1 (Hs6st1(f/f)) and the bacterial Cre recombinase expressed in hepatocytes from the rat albumin (Alb) promoter. We show that Hs2st(f/f)AlbCre(+) mice accumulated plasma triglycerides and exhibited delayed clearance of intestinally derived chylomicrons and injected human very low density lipoproteins to the same extent as observed in Ndst1(f/f)AlbCre(+) mice. In contrast, Hs6st1(f/f)AlbCre(+) mice did not exhibit any changes in plasma triglycerides. Chemically modified heparins lacking N-sulfate and 2-O-sulfate groups did not block very low density lipoprotein binding and uptake in isolated hepatocytes, whereas heparin lacking 6-O-sulfate groups was as active as unaltered heparin. Our findings show that plasma lipoprotein clearance depends on specific subclasses of sulfate groups and not on overall charge of the chains.

Purpose: The binding of hematogenously borne malignant cells that express the carbohydrate sialyl Lewis X (sLeX) to selectin adhesion receptors on leukocytes, platelets, and endothelial cells facilitates metastasis. The glycosylation inhibitor, per-O-acetylated GlcNAcβ1,3Galβ-O-naphthalenemethanol (AcGnG-NM), inhibits the biosynthesis of sLeX in tumor cells. To evaluate the efficacy of AcGnG-NM as an antimetastatic agent, we examined its effect on experimental metastasis and on spontaneous hematogenous dissemination of murine Lewis lung carcinoma and B16BL6 melanoma cells. Experimental Design: Tumor cells were treated in vitro with AcGnG-NM, and the degree of selectin ligand inhibition and experimental metastasis was analyzed in wild-type and P-selectin-deficient mice. Conditions were developed for systemic administration of AcGnG-NM, and the presence of tumor cells in the lungs was assessed using bromodeoxyuridine labeling in vivo. The effect of AcGnG-NM on inflammation was examined using an acute peritonitis model. Results: In vitro treatment of Lewis lung carcinoma cells with AcGnG-NM reduced expression of sLeX- and P-selectin-dependent cell adhesion to plates coated with P-selectin. Treatment also reduced formation of lung foci when cells were injected into syngeneic mice. Systemic administration of the disaccharide significantly inhibited spontaneous dissemination of the cells to the lungs from a primary s.c. tumor, whereas an acetylated disaccharide not related to sLeX in structure had no effect. AcGnG-NM did not alter the level of circulating leukocytes or platelets, the expression of P-selectin ligands on neutrophils, or sLeX-dependent inflammation. Conclusion: Taken together, these data show that AcGnG-NM provides a targeted glycoside-based therapy for the treatment of hematogenous dissemination of tumor cells.

Mycobacterium tuberculosis is a significant threat to global health. Mycobacterium bovis BCG vaccine provides only partial protection, and the skin test reagent used to aid diagnosis of both active and latent tuberculosis, purified protein derivative (PPD), lacks specificity and sensitivity. The use of genetically detoxified Bordetella pertussis adenylate cyclase toxin (CyaA) as a delivery system for two immunodominant proteins of M. tuberculosis that are of greater specificity than PPD, early-secreted antigenic target 6-kDa protein (ESAT-6) and culture filtrate protein 10 (CFP-10), was therefore investigated. CyaA toxoids incorporating these antigens were able to restimulate T cells from more than 91% tuberculosis patients and healthy sensitized donors. Delivery of antigen by CyaA decreased by 10-fold the amount of ESAT-6 and CFP-10 required to restimulate T cells, and in low responders, the overall frequency of gamma interferon-producing cells detected by enzyme-linked immunospot assay was increased ( P < 0.01 for both antigens). Delivery of ESAT-6 and CFP-10 by CyaA enabled the detection of both CD4 + and CD8 + T cells: these responses could be blocked by inhibition of major histocompatibility complex class II or class I, respectively. Covalent linkage of antigen to the CyaA vector was required for enhancement to occur, as a mixture of mock CyaA toxoid plus recombinant ESAT-6 did not lead to enhancement. In a simplified whole-blood model to detect tuberculosis infection, the frequency of positive responses to CFP-10 was increased by CyaA delivery, a potentially important attribute that could facilitate the identification of latent infection.

The importance of heparan sulfate proteoglycans has been highlighted by a number of human genetic disorders associated with mutations in genes encoding for heparan sulfate proteoglycan protein cores or biosynthetic enzymes required for heparan sulfate (HS) assembly. To study the functional role of HS in Caenorhabditis elegans development cosmid sequence C34F6.4 was identified as the C. elegans ortholog of vertebrate heparan 2- O -sulfotransferase (HS2ST) and the gene named hst-2 . HS2ST activity is present in C. elegans and is completely absent in a deletion mutant of hst-2 , ok595 , and specifically reduced by hst-2 RNA interference. Expression of hst-2 in CHO cells deficient in HS2ST rescues enzyme activity and binding of FGF2 to cell surface HS. hst-2 expression is found in the hypodermis, muscle, distal tip cells (DTCs), and in neurons. A null mutation in hst-2 causes cell migration defects. This work demonstrates sulfotransferase activity in C. elegans and indicates that specific 2- O -sulfate modifications are critical for normal HS functions in controlling cell migration.

Clustered presentation of sialyl Lewis X (sLe(X)) on tumor cell mucins is thought to facilitate metastasis through binding to selectin adhesion receptors expressed on platelets, leukocytes and endothelial cells. Thus, interfering with sLe(X) assembly might provide a chemotherapeutic method for treating metastatic disease. Prior studies have shown that peracetylated disaccharides can act in cells as substrates for the assembly of oligosaccharides related to sLe(X) synthesis, and the assembly of oligosaccharides on the disaccharides diverts the assembly of sLe(X) from endogenous cell surface glycoconjugates. Here, we show that treatment of cultured human adenocarcinoma cells with micromolar concentrations of the peracetylated disaccharide, (Ac)(6)GlcNAcbeta1,3Galbeta-O-naphthalenemethanol (AcGnG-NM) reduces the expression of sLe(X) and diminishes binding in vitro to selectin-coated dishes, thrombin-activated platelets, and tumor necrosis factor alpha-activated endothelial cells. Altering glycosylation in this way significantly reduced the ability of tumor cells to distribute to the lungs of wild-type mice over a 3-h period after i.v. injection. No significant difference in biodistribution was noted after the injection of AcGnG-NM-treated tumor cells into P-selectin deficient mice, although the extent of lung seeding was reduced compared with that in wild-type mice. In vitro, we demonstrate that normal mouse platelets, but not P-selectin-deficient platelets, bound to control tumor cells and protected them from leukocyte-mediated cytolysis. Conversely, treatment of tumor cells with disaccharide markedly reduced the ability of normal platelets to protect them from cytolysis. Finally, in an experimental metastasis model, we show that treatment of tumor cells with the disaccharide markedly reduced their lung colonization potential after injection into severe combined immunodeficient mice. These findings suggest that this compound may represent a novel class of chemotherapeutic agents for prevention and treatment of metastatic disease.

A series of hydrophobic mannosides were synthesized and tested for their ability to act as acceptor substrates for mannosyltransferases in a Trypanosoma brucei cell-free system. The thiooctyl alpha-mannosides and octyl alpha-mannosides all accepted single mannose residues in alpha-linkage, as judged by thin layer chromatography of the products before and after jack bean alpha-mannosidase digestion. The mannosylation reactions were inhibited by amphomycin, suggesting that the immediate donor was dolichol-phosphate-mannose (Dol-P-Man) in all cases. The transferred alpha-mannose residues were shown to be both alpha 1-2 and alpha 1-6 linked by Aspergillus phoenicis alpha-mannosidase and acetolysis treatments, respectively. These data suggest that the compounds can act as acceptor substrates for the Dol-P-Man dependent alpha 1-2 and alpha 1-6 mannosyltransferases of the GPI biosynthetic pathway and/or the dolichol-cycle of protein N-glycosylation. One of the compounds, Man alpha 1-6 Man alpha 1-O-(CH2)7CH3, inhibited endogenous GPI biosynthesis in the cell-free system, suggesting that it could be a substrate for the trypanosome Dol-P-Man:Man2GlcN-Pl alpha 1-2 mannosyltransferase.

Neural crest derived tumors express high levels of a unique class of lipid linked glycan known as gangliosides. Gangliosides are involved in growth factor signaling by regulating complexes in lipid rafts. Genetic studies show that through aberrant expression of gangliosides, these tumors acquire aggressive growth properties. Prior to this research, the only ganglioside inhibitors identified were non-specific and broadly blocked virtually all glycolipid classes. These non-specific glycolipid inhibitors demonstrated anti-cancer activity in animal models of neural crest tumors. However, due to substantial off-target dose-limiting toxicity from lack of specificity for the ganglioside sub-class, they effectively cannot be used in humans for cancer treatment. Selective inhibition of gangliosides without affecting other glycan classes could potentially avoid these problems and provide an effective treatment for neural crest and other ganglioside-dependent tumors. To identify the first known selective inhibitors of gangliosides, we developed a novel molecular screening strategy for identifying selective small-molecule ganglioside inhibitors. This platform identified the first drug-like selective inhibitors of gangliosides. ZP10395, a lead compound, selectively and dose-dependently reduces gangliosides in multiple tumor cell lines and is 10-15 fold more potent than the existing non-specific inhibitors. Importantly, it does not inhibit other glycolipid classes associated with dose-limiting toxicity. Administering ZP10395 to a mouse xenograph melanoma model significantly reduced ganglioside production and slowed tumor growth in the presence of a reduced T-cell response. These results demonstrate the potential utility of specific ganglioside inhibitors for treating ganglioside dependent tumors. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 2934. doi:10.1158/1538-7445.AM2011-2934

African sleeping sickness is a debilitating and often fatal disease caused by tsetse fly transmitted African trypanosomes. These extracellular protozoan parasites survive in the human bloodstream by virtue of a dense cell surface coat made of variant surface glycoprotein. The parasites have a repertoire of several hundred immunologically distinct variant surface glycoproteins and they evade the host immune response by antigenic variation. All variant surface glycoproteins are anchored to the plasma membrane via glycosylphosphatidylinositol membrane anchors and compounds that inhibit the assembly or transfer of these anchors could have trypanocidal potential. This article compares glycosylphosphatidylinositol biosynthesis in African trypanosomes and mammalian cells and identifies several steps that could be targets for the development of parasite-specific therapeutic agents.