Increasing intracellular mannose-6-phosphate (Man-6-P) was previously reported to reduce the amount of the major lipid linked oligosaccharide (LLO) precursor of N-glycans; a loss that might decrease cellular N-glycosylation. If so, providing dietary mannose supplements to glycosylation-deficient patients might further impair their glycosylation. To address this question, we studied the effects of exogenous mannose on intracellular levels of Man-6-P, LLO, and N-glycosylation in human and mouse fibroblasts. Mannose (500μM) did not increase Man-6-P pools in human fibroblasts from controls or from patients with Congenital Disorders of Glycosylation (CDG), who have 90–95% deficiencies in either phosphomannomutase (CDG-Ia) or phosphomannose isomerase (MPI) (CDG-Ib), enzymes that both use Man-6-P as a substrate. In the extreme case of fibroblasts derived from Mpi null mice (<0.001% MPI activity), intracellular Man-6-P levels greatly increased in response to exogenous mannose, and this produced a dose-dependent decrease in the steady state level of the major LLO precursor. However, LLO loss did not decrease total protein N-glycosylation or that of a hypoglycosylation indicator protein, DNaseI. These results make it very unlikely that exogenous mannose could impair N-glycosylation in glycosylation-deficient CDG patients.

PMM2-CDG patients have phosphomannomutase (Pmm2) deficiency, with developmental and N-linked glycosylation defects attributed to depletion of mannose-1-phosphate and downstream lipid-linked oligosaccharides (LLOs). This, the first PMM2-CDG zebrafish model, shows, unexpectedly, that accumulation of the Pmm2 substrate mannose-6-phosphate explains LLO deficiency.

Congenital disorder of glycosylation (PMM2-CDG) results from mutations in pmm2, which encodes the phosphomannomutase (Pmm) that converts mannose-6-phosphate (M6P) to mannose-1-phosphate (M1P). Patients have wide-spectrum clinical abnormalities associated with impaired protein N-glycosylation. Although it has been widely proposed that Pmm2 deficiency depletes M1P, a precursor of GDP-mannose, and consequently suppresses lipid-linked oligosaccharide (LLO) levels needed for N-glycosylation, these deficiencies have not been demonstrated in patients or any animal model. Here we report a morpholino-based PMM2-CDG model in zebrafish. Morphant embryos had developmental abnormalities consistent with PMM2-CDG patients, including craniofacial defects and impaired motility associated with altered motor neurogenesis within the spinal cord. Significantly, global N-linked glycosylation and LLO levels were reduced in pmm2 morphants. Although M1P and GDP-mannose were below reliable detection/quantification limits, Pmm2 depletion unexpectedly caused accumulation of M6P, shown earlier to promote LLO cleavage in vitro. In pmm2 morphants, the free glycan by-products of LLO cleavage increased nearly twofold. Suppression of the M6P-synthesizing enzyme mannose phosphate isomerase within the pmm2 background normalized M6P levels and certain aspects of the craniofacial phenotype and abrogated pmm2-dependent LLO cleavage. In summary, we report the first zebrafish model of PMM2-CDG and uncover novel cellular insights not possible with other systems, including an M6P accumulation mechanism for underglycosylation.

SUMMARY

Individuals with congenital disorders of glycosylation (CDG) have recessive mutations in genes required for protein N-glycosylation, resulting in multi-systemic disease. Despite the well-characterized biochemical consequences in these individuals, the underlying cellular defects that contribute to CDG are not well understood. Synthesis of the lipid-linked oligosaccharide (LLO), which serves as the sugar donor for the N-glycosylation of secretory proteins, requires conversion of fructose-6-phosphate to mannose-6-phosphate via the phosphomannose isomerase (MPI) enzyme. Individuals who are deficient in MPI present with bleeding, diarrhea, edema, gastrointestinal bleeding and liver fibrosis. MPI-CDG patients can be treated with oral mannose supplements, which is converted to mannose-6-phosphate through a minor complementary metabolic pathway, restoring protein glycosylation and ameliorating most symptoms, although liver disease continues to progress. Because Mpi deletion in mice causes early embryonic lethality and thus is difficult to study, we used zebrafish to establish a model of MPI-CDG. We used a morpholino to block mpi mRNA translation and established a concentration that consistently yielded 13% residual Mpi enzyme activity at 4 days post-fertilization (dpf), which is within the range of MPI activity detected in fibroblasts from MPI-CDG patients. Fluorophore-assisted carbohydrate electrophoresis detected decreased LLO and N-glycans in mpi morphants. These deficiencies resulted in 50% embryonic lethality by 4 dpf. Multi-systemic abnormalities, including small eyes, dysmorphic jaws, pericardial edema, a small liver and curled tails, occurred in 82% of the surviving larvae. Importantly, these phenotypes could be rescued with mannose supplementation. Thus, parallel processes in fish and humans contribute to the phenotypes caused by Mpi depletion. Interestingly, mannose was only effective if provided prior to 24 hpf. These data provide insight into treatment efficacy and the broader molecular and developmental abnormalities that contribute to disorders associated with defective protein glycosylation.

Purpose

Congenital disorders of glycosylation (CDG) are a heterogeneous group of disorders caused by deficient glycosylation, primarily affecting the N-linked pathway. It is estimated that over 40% of CDG patients lack a confirmatory molecular diagnosis. The purpose of this study was to improve molecular diagnosis for CDG by developing and validating a next generation sequencing (NGS) panel for comprehensive mutation detection in 24 genes known to cause CDG.

Methods

NGS validation was performed on 12 positive control CDG patients. These samples were blinded as to the disease causing mutations. Both RainDance and Fluidigm platforms were used for sequence enrichment and targeted amplification. The SOLiD platform was used for sequencing the amplified products. Bioinformatic analysis was performed using NextGENe® software.

Results

The disease causing mutations were identified by NGS for all 12 positive controls. Additional variants were also detected in three controls that are known or predicted to impair gene function and may contribute to the clinical phenotype.

Conclusions

We conclude that development of NGS panels in the diagnostic laboratory where multiple genes are implicated in a disorder is more cost-effective and will result in improved and faster patient diagnosis compared with a gene-by-gene approach. Recommendations are also provided for data analysis from the NGS-derived data in the clinical laboratory, which will be important for the widespread use of this technology.

Sugar pills are usually placebos, but Smith et al. (2002, this issue) use one to rescue designer mice unable to make GDP-Fucose. Dietary fucose enters a salvage pathway and spares the mice. Sound simple? Not so. Unknown genetic factors determine life or death.

We report the discovery and validation of a series of benzoisothiazolones as potent inhibitors of phosphomannose isomerase (PMI), an enzyme which converts mannose-6-phosphate (Man-6-P) into fructose-6-phosphate (Fru-6-P), and more importantly, competes with phosphomannomutase 2 (PMM2) for Man-6-P, diverting this substrate from critical protein glycosylation events. In Congenital Disorder of Glycosylation type Ia, PMM2 activity is compromised, thus PMI inhibition is a potential strategy for the development of therapeutics. High-throughput screening (HTS) and subsequent chemical optimization led to the identification of a novel class of benzoisothiazolones as potent PMI inhibitors having little or no PMM2 inhibition. Two complimentary synthetic routes were developed enabling the critical structural requirements for activity to be determined, and the compounds were subsequently profiled in biochemical and cellular assays to assess efficacy. The most promising compounds were also profiled for bioavailability parameters including metabolic stability, plasma stability, and permeability. The pharmacokinetic profile of a representative of this series was also assessed, demonstrating the potential of this series for in vivo efficacy when dosed orally in disease models.

Congenital disorders of glycosylation (CDGs) are metabolic deficiencies in glycoprotein biosynthesis that usually cause severe mental and psychomotor retardation. Different forms of CDGs can be recognized by altered isoelectric focusing (IEF) patterns of serum transferrin (Tf). Two patients with these symptoms and similar abnormal Tf IEF patterns were analyzed by metabolic labeling of fibroblasts with [2-3H]mannose. The patients produced a truncated dolichol-linked precursor oligosaccharide with 5 mannose residues, instead of the normal precursor with 9 mannose residues. Addition of 250 μΜ mannose to the culture medium corrected the size of the truncated oligosaccharide. Microsomes from fibroblasts of these patients were approximately 95% deficient in dolichol-phosphate-mannose (Dol-P-Man) synthase activity, with an apparent Km for GDP-Man ∼6-fold higher than normal. DPM1, the gene coding for the catalytic subunit of Dol-P-Man synthase, was altered in both patients. One patient had a point mutation, C274G, causing an R92G change in the coding sequence. The other patient also had the C274G mutation and a 13-bp deletion that presumably resulted in an unstable transcript. Defects in DPM1 define a new glycosylation disorder, CDG-Ie.

Purpose

Redundant receptor tyrosine kinase (RTK) signaling is a mechanism for therapeutic resistance to EGFR inhibition. A strategy to reduce parallel signaling by co-expressed RTKs is inhibition of N-linked glycosylation (NLG), an endoplasmic reticulum (ER) co-translational protein modification required for receptor maturation and cell surface expression. We therefore investigated the feasibility of blocking NLG in vivo to reduce over-expression of RTKs.

Experimental design

We developed a model system to dynamically monitor NLG in vitro and in vivo using bioluminescent imaging techniques. Functional imaging of NLG is accomplished with a luciferase reporter (ER-LucT) modified for ER-translation and glycosylation. After in vitro validation, this reporter was integrated with D54 glioma xenografts to perform non-invasive imaging of tumors, and inhibition of NLG was correlated with RTK protein levels and tumor growth.

Results

The ER-LucT reporter demonstrates the ability to sensitively and specifically detect NLG inhibition. Using this molecular imaging approach we performed serial imaging studies to determine safe and efficacious in vivo dosing of the GlcNAc-1-phosphotransferase inhibitor, tunicamycin, which blocks N-glycan precursor biosynthesis. Molecular analyses of tunicamycin treated tumors showed reduced levels of EGFR and Met, two RTKs over-expressed in gliomas. Furthermore, D54 and U87MG glioma xenograft tumor experiments demonstrated significant reductions in tumor growth following NLG inhibition and radiation therapy, consistent with an enhancement in tumor radiosensitivity.

Conclusions

This study suggests NLG inhibition is a novel therapeutic strategy for targeting EGFR and RTK signaling in both gliomas and other malignant tumors.

Mutations in the Conserved Oligomeric Golgi (COG) complex give rise to type II congenital disorders of glycosylation (CDG). Thus far, mutations have been identified in 6 of the 8 COG subunits. Here we present data identifying a previously reported CDG-IIx case from Singapore as a new COG4 patient with 2 novel mutations leading to p.E233X and p.L773R; with p.E233X being a de novo mutation. As a result, COG4 protein expression was dramatically reduced, while expression of the other subunits remained unaffected. Analysis of serum N-glycans revealed deficiencies in both sialylation and galactosylation. Furthermore, patient fibroblasts have impaired O-glycosylation. Importantly, patient fibroblasts exhibited a delay in Brefeldin A (BFA) induced retrograde transport, a common characteristic seen in COG deficiencies.

The enrichment of phosphatidylinositol-4-phosphate (PI(4)P) at the trans-Golgi-network (TGN) is instrumental for proper protein and lipid sorting, yet how the restricted distribution of PI(4)P is achieved remains unknown. Here we show that lipid phosphatase SAC1 is crucial for the spatial regulation of Golgi PI(4)P. Ultrastructural analysis revealed that SAC1 is predominantly located at cisternal Golgi membranes but is absent from the TGN, thus confining PI(4)P to the TGN. RNAi-mediated knockdown of SAC1 caused changes in Golgi morphology and mislocalization of Golgi enzymes. Enzymes involved in glycan processing such as mannosidase II (Man-II) and N-acetylglucosamine transferase I (GnT-I) redistributed to aberrant intracellular structures and to the cell surface in SAC1 knockdown cells. SAC1 depletion also induced a unique pattern of Golgi-specific defects in N- and O-linked glycosylation. These results indicate that SAC1 organizes PI(4)P distribution between the Golgi complex and the TGN, which is instrumental for resident enzyme partitioning and Golgi morphology.

RAGE, the Receptor for Advanced Glycation End Products, is a signaling receptor protein of the immunoglobulin superfamily implicated in multiple pathologies. It binds a diverse repertoire of ligands, but the structural basis for the interaction of different ligands is not well understood. We earlier showed that carboxylated glycans on the V-domain of RAGE promote the binding of HMGB1 and S100A8/A9. Here we study the role of these glycans on the binding and intracellular signaling mediated by another RAGE ligand, S100A12. S100A12 binds carboxylated glycans, and a subpopulation of RAGE enriched for carboxylated glycans shows more than ten fold higher binding potential for S100A12 than total RAGE. When expressed in mammalian cells, RAGE is modified by complex glycans predominantly at the first glycosylation site (N25IT) that retains S100A12 binding. Glycosylation of RAGE and maximum binding sites for S100A12 on RAGE are also cell type dependent. Carboxylated glycan-enriched population of RAGE forms higher order multimeric complexes with S100A12, and this ability to multimerize is reduced upon deglycosylation or by using non-glycosylated sRAGE expressed in E.coli. mAbGB3.1, an antibody against carboxylated glycans, blocks S100A12 mediated NF-κB signaling in HeLa cells expressing full length RAGE. These results demonstrate that carboxylated N-glycans on RAGE enhance binding potential and promote receptor clustering and subsequent signaling events following oligomeric S100A12 binding.

SUMMARY

Phosphomannomutaste (PMM2, Mannose-6-P→ Mannose-1-P) deficiency is the most frequent glycosylation disorder affecting the N-glycosylation pathway. There is no therapy for the hundreds of patients who suffer from this disorder. This review describes previous attempts at therapeutic interventions and introduces perspectives emerging from the drawing boards. Two approaches aim to increase Mannose-1-P: small membrane permeable molecules that increase the availability or/and metabolic flux of precursors into the impaired glycosylation pathway; and, phosphomannomutase enhancement and/or replacement therapy. Glycosylation-deficient cell and animal models are needed to determine which individual or combined approaches improve glycosylation and may be suitable for preclinical evaluation.

The glycan symbol nomenclature proposed by Harvey et al. in these pages has relative advantages and disadvantages. The use of symbols to depict glycans originated from Kornfeld in 1978, was systematized in the First Edition of “Essentials of Glycobiology” and updated for the second edition, with input from relevant organizations such as the Consortium for Functional Glycomics. We also note that >200 illustrations in the second edition have already been published using our nomenclature and are available for download at PubMed.

Chronic inflammation is a complex process that promotes carcinogenesis and tumor progression; however, the mechanisms by which specific inflammatory mediators contribute to tumor growth remain unclear. We and others recently demonstrated that the inflammatory mediators IL-1β, IL-6, and PGE2 induce accumulation of myeloid-derived suppressor cells (MDSC) in tumor-bearing individuals. MDSC impair tumor immunity and thereby facilitate carcinogenesis and tumor progression by inhibiting T and NK cell activation, and by polarizing immunity toward a tumor-promoting type 2 phenotype. We now show that this population of immature myeloid cells induced by a given tumor share a common phenotype regardless of their in vivo location (bone marrow, spleen, blood, or tumor site), and that Gr1highCD11bhighF4/80−CD80+IL4Rα+/−Arginase+ MDSC are induced by the proinflammatory proteins S100A8/A9. S100A8/A9 proteins bind to carboxylated N-glycans expressed on the receptor for advanced glycation end-products and other cell surface glycoprotein receptors on MDSC, signal through the NF-κB pathway, and promote MDSC migration. MDSC also synthesize and secrete S100A8/A9 proteins that accumulate in the serum of tumor-bearing mice, and in vivo blocking of S100A8/A9 binding to MDSC using an anti-carboxylated glycan Ab reduces MDSC levels in blood and secondary lymphoid organs in mice with metastatic disease. Therefore, the S100 family of inflammatory mediators serves as an autocrine feedback loop that sustains accumulation of MDSC. Since S100A8/A9 activation of MDSC is through the NF-κB signaling pathway, drugs that target this pathway may reduce MDSC levels and be useful therapeutic agents in conjunction with active immunotherapy in cancer patients.

Inflammatory mediators play important roles in the development and progression of cancer. Cellular stress, damage, inflammation, and necrotic cell death cause release of endogenous damage-associated molecular pattern (DAMP) molecules or alarmins, which alert the host of danger by triggering immune responses and activating repair mechanisms through their interaction with pattern recognition receptors. Recent studies show that abnormal persistence of these molecules in chronic inflammation and in tumor microenvironments underlies carcinogenesis and tumor progression, indicating that DAMP molecules and their receptors could provide novel targets for therapy. This review focuses on the role of DAMP molecules high-mobility group box 1 and S100 proteins in inflammation, tumor growth, and early metastatic events.

Patients with inflammatory bowel diseases are at increased risk for colorectal cancer, but the molecular mechanisms linking inflammation and cancer are not well defined. We earlier showed that carboxylated N-glycans expressed on receptor for advanced glycation end products (RAGE) and other glycoproteins mediate colitis through activation of nuclear factor kappa B (NF-κB). Because NF-κB signaling plays a critical role in the molecular pathogenesis of colitis-associated cancer (CAC), we reasoned that carboxylated glycans, RAGE and its ligands might promote CAC. Carboxylated glycans are expressed on a subpopulation of RAGE on colon cancer cells and mediate S100A8/A9 binding to RAGE. Colon tumor cells express binding sites for S100A8/A9 and binding leads to activation of NF-κB and tumor cell proliferation. Binding, downstream signaling and tumor cell proliferation are blocked by mAbGB3.1, an anti-carboxylate glycan antibody, and by anti-RAGE. In human colon tumor tissues and in a mouse model of CAC, we found that myeloid progenitors expressing S100A8 and S100A9 infiltrate regions of dysplasia and adenoma. mAbGB3.1 administration markedly reduces chronic inflammation and tumorigenesis in the mouse model of CAC and RAGE-deficient mice are resistant to the onset of CAC. These findings show that RAGE, carboxylated glycans and S100A8/A9 play essential roles in tumor–stromal interactions, leading to inflammation-associated colon carcinogenesis.

Mutations in the N-linked glycosylation pathway cause rare autosomal recessive defects known as Congenital Disorders of Glycosylation (CDG). A previously reported mutation in the Conserved Oligomeric Golgi complex gene, COG7, defined a new subtype of CDG in a Tunisian family. The mutation disrupted the hetero-octomeric COG complex and altered both N- and O- linked glycosylation. Here we present clinical and biochemical data from a second family with the same mutation.

Patients with protein-losing enteropathy (PLE) fail to maintain intestinal epithelial barrier function and develop an excessive and potentially fatal efflux of plasma proteins. PLE occurs in ostensibly unrelated diseases, but emerging commonalities in clinical observations recently led us to identify key players in PLE pathogenesis. These include elevated IFN-γ, TNF-α, venous hypertension, and the specific loss of heparan sulfate proteoglycans from the basolateral surface of intestinal epithelial cells during PLE episodes. Here we show that heparan sulfate and syndecan-1, the predominant intestinal epithelial heparan sulfate proteoglycan, are essential in maintaining intestinal epithelial barrier function. Heparan sulfate– or syndecan-1–deficient mice and mice with intestinal-specific loss of heparan sulfate had increased basal protein leakage and were far more susceptible to protein loss induced by combinations of IFN-γ, TNF-α, and increased venous pressure. Similarly, knockdown of syndecan-1 in human epithelial cells resulted in increased basal and cytokine-induced protein leakage. Clinical application of heparin has been known to alleviate PLE in some patients but its unknown mechanism and severe side effects due to its anticoagulant activity limit its usefulness. We demonstrate here that non-anticoagulant 2,3-de-O-sulfated heparin could prevent intestinal protein leakage in syndecan-deficient mice, suggesting that this may be a safe and effective therapy for PLE patients.

Carbohydrate modification of proteins includes N-linked and O-linked glycosylation, proteoglycan formation, glycosylphosphatidylinositol anchor synthesis, and O-GlcNAc modification. Each of these modifications requires the sugar nucleotide UDP-GlcNAc, which is produced via the hexosamine biosynthesis pathway. A key step in this pathway is the interconversion of GlcNAc-6-phosphate (GlcNAc-6-P) and GlcNAc-1-P, catalyzed by phosphoglucomutase 3 (Pgm3). In this paper, we describe two hypomorphic alleles of mouse Pgm3 and show there are specific physiological consequences of a graded reduction in Pgm3 activity and global UDP-GlcNAc levels. Whereas mice lacking Pgm3 die prior to implantation, animals with less severe reductions in enzyme activity are sterile, exhibit changes in pancreatic architecture, and are anemic, leukopenic, and thrombocytopenic. These phenotypes are accompanied by specific rather than wholesale changes in protein glycosylation, suggesting that while universally required, the functions of certain proteins and, as a consequence, certain cell types are especially sensitive to reductions in Pgm3 activity.

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 patient’s. 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 patient’s 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.

SUMMARY

N-linked glycosylation is the most frequent modification of secreted and membrane-bound proteins in eukaryotic cells, disruption of which is the basis of the Congenital Disorders of Glycosylation (CDG). We describe a new type of CDG caused by mutations in the steroid 5α-reductase type 3 (SRD5A3) gene. Patients have mental retardation, ophthalmologic and cerebellar defects. We found that SRD5A3 is necessary for the reduction of the alpha-isoprene unit of polyprenols to form dolichols, required for synthesis of dolichol-linked monosaccharides and the oligosaccharide precursor used for N-glycosylation. The presence of residual dolichol in cells depleted for this enzyme suggests the existence of an unexpected alternative pathway for dolichol de novo biosynthesis. Our results thus suggest that SRD5A3 is likely to be the long-sought polyprenol reductase and reveal the genetic basis of one of the earliest steps in protein N-linked glycosylation.